Bacillus cereus spore formation, structure and germination

107 downloads 0 Views 4MB Size Report
Bacillus cereus spore formation, structure, and germination. Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus.
Bacillus cereus spore formation, structure, and germination Ynte Piet de Vries

Promotoren Prof. Dr. T. Abee Persoonlijk hoogleraar bij de leerstoelgroep Levensmiddelenmicrobiologie Wageningen Universiteit Prof. Dr. W. M. de Vos Hoogleraar Microbiologie Wageningen Universiteit Prof. Dr. Ir. M. H. Zwietering Hoogleraar Levensmiddelenmicrobiologie Wageningen Universiteit

Promotiecommissie Prof. Dr. S. Brul Universiteit van Amsterdam Prof. Dr. Ir. A. J. M. Stams Wageningen Universiteit Dr. J. Sikkema Friesland Foods, Deventer Prof. Dr. G. W. Gould Voorheen Unilever Research, Bedford, UK.

Dit onderzoek is uitgevoerd binnen de onderzoekschool VLAG

Ynte Piet de Vries

Bacillus cereus spore formation, structure, and germination

Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit Prof. Dr. M. J. Kropff in het openbaar te verdedigen op vrijdag 10 februari 2006 des namiddags te half twee in de Aula

Y. P. de Vries – Bacillus cereus spore formation, structure, and germination – 2006 Thesis Wageningen University, Wageningen, the Netherlands – with summary in Frisian and Dutch – 128 p Keywords: Bacillus cereus / sporulation / spores / germination / food preservation ISBN: 90-8504-369-7

Voorwoord Eén van mijn leermeesters heeft een promotie-onderzoek wel eens vergeleken met een jungle-tocht. Je hoopt mooie dieren en planten tegen te komen maar je kunt ook in onaangename of zelfs gevaarlijke situaties verzeild raken. Met het gereed komen van zijn proefschrift rondt Ynte de Vries zijn jungle-tocht af. Een snelle blik op de inhoudsopgave en de lijst met publicaties leert dat de tocht een aantal mooie successen heeft opgeleverd. Wat je niet meteen uit de inhoud van het proefschrift kunt afleiden, is hoe moeizaam de tocht is verlopen. Het eerste deel van de tocht verliep voorspoedig. Cum laude afgestudeerd, eigenwijs en blakend van zelfvertrouwen begon Ynte in 2001 aan zijn promotie-onderzoek bij het Wageningen Centre for Food Sciences. Zijn ambitie: een doorbraak in het onderzoek naar de ontkieming van sporen. Onder supervisie van Marjon zette hij samen met Luc een nieuwe onderzoekslijn op. Maar het eerste jaar was amper voorbij of enkele forse tegenslagen dienden zich aan. De ESRonderzoekslijn waar een half jaar tijd in was gaan zitten, bleek een doodlopende weg en werd beëindigd; drie WCFS-collega’s waaronder supervisor Marjon verlieten A&F; en de langdurige procedure om een nieuwe supervisor te vinden, leverde niet het verwachte resultaat op. Bovendien bleven de aanvankelijk beloofde B. cereus micro-arrays uit. Teleurgesteld over deze gang van zaken besloot Ynte zijn onderzoek deels bij de universiteit voort te zetten waar hij een betere ambiance voor het doen van onderzoek hoopte te vinden. Uiteindelijk bleek het gras bij de buren niet veel groener dan verwacht en was Ynte steeds vaker weer bij A&F te vinden – waar hij met nieuw elan proeven deed. De nauwere samenwerking met de collega’s bij de universiteit had een paar ideeën voor zijn onderzoek opgeleverd en nieuwe inzichten hebben ertoe geleid dat ook de ESR-lijn weer werd opgepakt. Zo brak er een nieuwe en beslissende fase aan in Ynte’s jungle-tocht. Naast een bijdrage aan onze kennis van sporen, sporulatie, en ontkieming heeft Ynte’s jungle-tocht nog een ander opmerkelijk resultaat opgeleverd: een andere, meer genuanceerde kijk op toepassingsgericht onderzoek. Hoewel Ynte het grootste deel van zijn promotie-onderzoek heeft uitgevoerd in een omgeving waar markt-gedreven, toepassingsgericht onderzoek domineert, heeft hij er in de beginfase geen geheim van gemaakt dat hij eigenlijk niet veel op had met dergelijk onderzoek. Dat ook zijn eigen onderzoek uiteindelijk bruikbare kennis zou moeten opleveren voor de bij WCFS aangesloten bedrijven, zag hij aanvankelijk niet zo zitten. Gaandeweg is er in die houding verandering gekomen – zozeer zelfs dat Ynte uiteindelijk een paar maanden geleden een baan heeft aanvaard bij Friesland Foods. Voor WCFS snijdt het mes daarmee aan twee kanten: WCFS heeft een uitstekend onderzoeker afgeleverd aan een van zijn industriële partners en het project Food Preservation & Safety heeft er een waardevolle expert voor teruggekregen. Roy Moezelaar Projectleider Food Preservation & Safety Wageningen Centre for Food Sciences

Contents

Abstract

3

Chapter 1 Introduction and outline of this thesis

5

Chapter 2 Growth and sporulation of Bacillus cereus ATCC14579 under defined conditions: temporal expression of genes for key sigma factors

33

Chapter 3 Influence of glutamate on growth, sporulation and spore properties of Bacillus cereus ATCC14579 in a defined medium

47

Chapter 4 Deletion of sigB in Bacillus cereus affects spore properties

59

Chapter 5 Water in the core of Bacillus spores is not in a glassy state: a spin-probe study

67

Chapter 6 Bacillus cereus spore germination quantified by flow-cytometry

85

Chapter 7 Germination capacity and heat resistance of spores from naturally occurring Bacillus cereus strains

93

Chapter 8 Summary, future perspectives and practical applicability of the research described in this thesis

103

Gearfetting

115

Samenvatting

119

List of publications

123

Dankwoord

125

Curriculum Vitae

127

1

2

Abstract Bacterial spores are differentiated cell types, specifically designed for the survival of adverse conditions. Their structure is highly unique and very different from the structure of normal vegetative bacterial cells. Spores cause massive problems in the food industry, because their remarkable resistance allows them to survive food processing and conservation methods. The sporeforming Bacillus cereus is an important food-borne pathogen, is famous for its ability to cause food poisoning, and is an important spoilage organism in pasteurized dairy products. The work presented in this thesis has focused on the formation, structure and germination of B. cereus spores. An easy and efficient way of producing synchronized and homogeneous B. cereus spore batches was developed, using a chemically defined medium in combination with an airlift fermenter system. This setup allowed precise monitoring and manipulation of key growth- and sporulation parameters. The conditions employed resulted in synchronous growth and sporulation, which facilitated geneexpression studies. The kinetics of expression of sigA, sigB, sigF and sigG followed the model developed for Bacillus subtilis, underscoring the conservation of sporulation mechanisms among bacilli. B. cereus was able to form spores on the chemically defined medium without glucose but with lactate as a main carbon source. Sporulation was not induced by nutrient limitation, because significant amounts of carbon and nitrogen sources were still present when the cells started to sporulate. The presence of glutamate delayed the final stages of sporulation, but not the moment of sporulation initiation. Clearly, the concentration of glutamate influenced key spore properties such as heat resistance and germination. The alternative sigma factor σB, encoded by the sigB gene, is an important stress response regulator of B. cereus. An increase in sigB transcription was observed upon glucose depletion, coinciding with the transition from exponential growth to the stationary phase. This increase was specifically associated with the depletion of glucose. Deletion of sigB had a significant impact on spore heat resistance and spore germination properties. Spore heat resistance is caused by the physicochemical structure of the spore, which protects vital spore components such as membranes, proteins, and the DNA. A spin-probe-based Electron Spin Resonance (ESR) method for measuring the internal structure of intact bacterial spores was developed and applied, and provided the first direct data on the aqueous environment in the various compartments of B. subtilis and B. cereus spores. From the results obtained, it was concluded that the core cytoplasm is not in a glassy state. Instead, a three-dimensional molecular matrix incorporating free but highly viscous water exists in the core. Notably, neither heat activation nor partial germination (the excretion of DPA but not full rehydration and enzyme activity) altered the structural properties of the core matrix significantly. Complete germination resulted in the disappearance of the structure in the core, and a decrease of the micro viscosity in the core cytoplasm to levels encountered in normal vegetative cells. For a quantitative analysis of the behavior of individual spores in a large, germinating spore-population, a flow cytometry (FCM) method was developed and applied. By using several different fluorescent dyes, distinct germination parameters such as DNA accessibility and esterase activity were quantified. Finally, spore properties from a large number of B. cereus strains, including the B. cereus laboratory model strain ATCC14579 and a number of recent isolates from environmental and industrial settings were analyzed. The strains tested showed a large variation in heat resistance, and the majority had a higher heat resistance than the laboratory model strain. With respect to germination, many of the strains were less sensitive to the nutrients tested as compared to the laboratory model strain. Heat activation and ageing enhanced germination in response to several nutrients in various isolates. The knowledge that was gained and the methods that were developed in this study are expected to contribute to progress in the spore research field, and to enhanced spore control in the food industry.

3

1. Introduction and Outline of this Thesis

ABSTRACT Bacterial spores cause massive problems in the food industry, because their remarkable resistance to a variety of stresses allows them to survive food processing and conservation methods. The resistance of bacterial spores relies on their unique structure, which keeps them metabolically dormant and, their vital components protected in a relatively dehydrated state. In order to grow and proliferate, bacterial spores return to the vegetative state through the process of germination, which involves breakdown of the spore-structure and concomitant loss of spore resistance properties. The spore-forming Bacillus cereus is an important food-borne pathogen able to cause distinct types of food poisoning. This chapter provides an introduction to bacterial spores, describing their structure and several other important and interesting aspects. Secondly, B. cereus is introduced with a focus on its ecology, phenotypic and genotypic traits, and its importance in the food industry. Finally, the general outline of this thesis is presented.

5

Chapter 1

I.

Bacterial spores

Fig. 1. Scanning electron micrograph of spores from Bacillus cereus (left) and Bacillus subtilis (right). Bars indicate 1 µm. Note the presence of an exosporium around the B. cereus spores, which is visible as a loose membrane.

1.1 Introduction: What are bacterial spores? Bacterial spores (Fig. 1) are highly specialized, differentiated cell types, designed for the survival of adverse conditions. They are formed inside the bacterial cell and hence called endospores. Specialized differentiated cell types are formed by a wide range of bacteria to survive starvation and other harsh conditions, but bacterial endospores (from now on called: spores) are the toughest among these cell types and are almost certainly the longest surviving (Errington, 2003). Their structure is very different from the structure of normal vegetative bacterial cells. Well-known spore-formers are anaerobes of the genus Clostridium and facultative aerobes of the genus Bacillus; their spores are omnipresent in the environment. Spore formation involves an unique process of asymmetric cell division, followed by engulfment of the smaller cell and eventually leading to the sacrifice of the original bacterial cell for the production of a single spore (Fig. 2). Spores are metabolically dormant (Lewis, 1969) and highly resistant to adverse conditions, such as starvation, high temperatures, ionizing radiation, mechanical abrasion, chemical solvents, detergents, hydrolytic enzymes, desiccation, pH extremes and antibiotics (reviewed in Setlow, 2000). Historically, spore research has played an important role in microbiology, and therefore this section starts with a brief account of the history of spore research (paragraph 1.2). Subsequently, the remarkable resistance properties of bacterial spores are highlighted with the examples of spores surviving space travel and multi-million-year spore longevity (paragraph 1.3). These remarkable resistance properties are based on the unique structure of bacterial spores, which is explained in paragraph 1.4, together with a brief account of spore formation and germination. 1.2 The history of spore research Research on bacterial spores started more than a century ago, and historically, spores were best known as agents of human and animal disease or food poisoning. Several workers in the earlymid 19th century have directly or indirectly described spores, such as Ehrenberg (1938), Pasteur and Tyndall. However, the earliest more detailed descriptions date from 1876, and were produced by Ferdinand Cohn and, independently, by Robert Koch during their studies on sterilization of organic material and anthrax, respectively. In the late 19th and early 20th century, spore research was conducted mainly by scientists who worked alone, rather than in organized groups, which impeded rapid progress. Solid knowledge began to accumulate in the 1940s, when several laboratories around the world started to devote most of their time to spore biology. From the 1950s till the

6

Introduction and outline of this thesis

1980s, spore formation served as the model for cellular differentiation, and enormous research efforts were put into the elucidation of the sporulation process and the structure of the bacterial spore (reviewed by Keynan and Sandler, 1983; Nicholson, 2004).

Fig 2. The life cycle of spore-forming bacteria. Adapted from Errington (2003), with permission from publisher and author.

The model spore-formers in the early days were mainly Bacillus cereus and Bacillus megaterium, because these species have large cells and produce relatively large spores that can be studied easily with a light microscope. In the 1960s-1980s, spore biologists formed a large, wellorganized international community, and conferences specifically focusing on spore biology were held in the USA and Europe. Scientists such as Halvorson (both father and son, see Halvorson, 1997) and Grahame Gould dedicated their life to spore-research, and became icons in the field. Proceedings from the spore-conferences (entitled Spores vol. 1-7 and Spore Research 1970, 1973, 1976), together with the huge amounts of spore-related literature from these years contain an enormous wealth of information on all aspects of spore-formers and their spores. Much of the information gathered in this era is still highly valuable to spore research today. With the rise of genetically oriented studies in the 1970s and 1980s, Bacillus subtilis 168 became the model spore-former, because of its suitability for genetic studies (reviewed in Nicholson, 2004). B. subtilis was the first spore-former from which the genome sequence was determined completely (Kunst et al., 1997), is by far the best understood Gram-positive bacterium, and serves as a model for cellular differentiation studies even today (Eichenberger et al., 2004;

7

Chapter 1

Errington, 2003; Nicholson, 2004). Recently, bio-terrorist attacks in the US, in which Bacillus anthracis spores were deployed (see Enserink, 2002; Higgins et al., 2003; Jernigan et al., 2002), sparked a new boost of budget for spore research, focusing on B. anthracis, a bacterium closely related to Bacillus cereus (Rasko et al., 2005; see paragraph 2.1-2.3). 1.3 Spores in space and spore longevity An interesting aspect of bacterial spores is their ability to survive space travel. Since the early 19th century, theories have been formulated that life has not exclusively developed on earth, but exists and travels throughout space. Well-known scientists such as Wagner, Kelvin, F. Cohn, and H. v. Helmholtz have been intrigued by this idea. The most extensive treatment of the subject was provided by Arrhenius, who in 1903 introduced the name panspermia. The theory of panspermia postulated that microscopic forms of life, such as spores, are dispersed in space by the radiation pressure from the sun, thereby seeding life from one planet to the other. This theory was heavily criticized, for example because it could not be experimentally tested and because spores were considered unable to survive long-time exposure to the hostile environment of space, especially the vacuum and radiation (Horneck et al., 2001; Mileikowsky et al., 2000; Nicholson et al., 2000; Nicholson, 2004; Sussman and Halvorson, 1969). However, in recent years spore survival in the space environment has been extensively tested (reviewed in Nicholson, 2003; 2004). What we know at present is: (i) highly resistant spores are present in rocks at the surface of our planet, (ii) these rocks are ejected into space upon impact of asteroids, (iii) spores associated with these rocks are able to survive the ejection into space, (iv) these spores are able to persist in space for at least several years, and (v) these spores are able to survive the process of descent and impact on another planet. So the theory of panspermia was modified by introduction of rock ejecta that serve as vehicles for spore space travel. The major complicating factor with respect to the current theory of panspermia is the fact that the time-frame involved is not resolved (see Nicholson, 2003; 2004). It may be that the transfer between the planets takes millions of years (see Nicholson, 2004); however, spores have been reported to survive for such periods of time, as pointed out below. Because of their metabolic dormancy, bacterial spores do not require any nutrients or energy sources to persist (Keynan, 1972; Lewis, 1969). This dormancy, combined with their resistance, enables them to last for extreme periods of time. Numerous reports have documented the isolation of viable spores from environmental samples such as dried soil in herbarium collections, paleosols, ancient lake sediments, permafrost soils, ice cores and old cans of meat. These samples ranged in age from several decades to thousands of years (Gest and Mandelstam, 1987; Nicholson, 2004; Parkes, 2000; Potts, 1994; Sussman and Halvorson, 1969b). To convincingly claim isolation of a real ancient bacterium, caution must be taken to avoid contamination of the ancient sample with “modern” bacteria. Early studies frequently lacked proper controls, and claims on the isolation of ancient spores were easily rejected (Nicholson, 2004; Parkes, 2000; Vreeland and Rosenzweig, 2002). However, in 1995, a report appeared on the isolation of a Bacillus sphaericus from a 25-40 Million year old bee fossilized in amber (Cano and Borucki, 1995). Careful sample selection, stringent sterilization techniques and numerous controls were used to support the claim of a truly ancient isolate (Cano, 1995; Parkes, 2000). In 2000, another report appeared on the isolation of a halo-tolerant Salibacillus spp. from a brine inclusion inside a 250 million-year-old primary salt crystal (Vreeland et al., 2000). Again, the study was performed with rigorous sample selection and strict controls. The validity of the claims of millions-of-years old isolates has been questioned from several points of view (reviewed in Kminek et al., 2003; Nicholson, 2004; Vreeland and Rosenzweig, 2002), however, these claims could not be definitively disaffirmed (see Maughan et al., 2002; Nicholson, 2003; 2004; Parkes, 2000; Vreeland and Rosenzweig, 2002).

8

Introduction and outline of this thesis

Fig 3. Schematic representation of the internal structure of a bacterial spore. Reproduced from Foster and Johnstone (1990), with permission from publisher and authors.

1.4 Spore formation and structure The remarkable properties of bacterial spores are brought about by their unique structure (Fig 3; for early reviews see Tipper and Gauthier, 1972; Warth, 1978). Spores consist of a core, surrounded by the inner membrane, the cortex, the outer membrane (not depicted), the coat and in some species the exosporium (Fig. 3). Each of these components is highlighted in this paragraph, with its respective role in dormancy, resistance and germination. Furthermore, this paragraph will summarize the most important events of spore formation, and focus briefly on the process of spore germination, in which spores return to a metabolically active state. The core The innermost part of the spore, the core, contains the spore cytoplasm with the regular cellular components, such as cytoplasmatic proteins, ribosomes and DNA. The physical state of the core cytoplasm, however, is far from regular in comparison to vegetative cell cytoplasm, having a water content of only 30-50%, instead of the 70-88% in vegetative cytoplasm (Potts, 1994; Setlow, 1994; 2000). This dehydrated state plays an important role in spore longevity, dormancy and

9

Chapter 1

resistance (Beaman et al., 1982; 1984; Gould, 1986; Nakashio and Gerhardt, 1985; Russell, 2003; Setlow, 1994; 2000). The pH in the spore core is 6.3-6.5, which is substantially lower than the pH of the cytoplasm of vegetative cells (Setlow and Setlow, 1980). The core cytoplasm lacks most of the common high-energy compounds found in the cytoplasm of vegetative cells (reviewed in Setlow, 1994; 2000), and the core harbors large quantities of a particular kind of proteins, termed small acid soluble proteins or SASP (Setlow, 1988). These proteins form a complex with the spore DNA, thereby forcing the DNA into a special compressed format (Douki et al., 2004; FrenkielKrispin et al., 2004), which protects the DNA against many types of damage (reviewed in Setlow, 1988; 1995). Cytoplasmatic proteins in the core are immobile (Cowan et al., 2003), and the core cytoplasm contains very large amounts of divalent cations, mainly calcium, which are complexed with the spore-specific compound Pyridine-2,6-dicarboxylic acid (dipicolinic acid or DPA; Powell, 1953). DPA is associated with core dehydration (Paidhungat et al., 2000), and plays a role in wet heat resistance (Halvorson and Howitt, 1961; Paidhungat et al., 2000) and UV resistance in environmentally relevant conditions (Slieman and Nicholson, 2001). The amount of DPA in the core is extremely high, accounting for about 5-15% of the total spore weight (Halvorson and Howitt, 1961; Murrell and Warth, 1965). DPA and calcium are excreted from the core during germination, and play important roles in several steps of the germination process (Paidhungat et al., 2001; de Vries, 2004). The inner membrane The spore core is surrounded by the inner membrane. The inner membrane becomes the cytoplasmatic membrane of the nascent vegetative cell upon germination, and swells 2-fold without de novo lipid synthesis. Therefore, the inner membrane is in a special, compressed state. Indeed, the lipids in the inner membrane of dormant spores are largely immobile, while in germinated spores and vegetative cells, the membrane is fluid and membrane lipids are highly mobile (Cowan et al., 2004). The inner membrane was proposed to be the main permeability barrier of spores (Setlow, 1994; 2000), and is the site where the spore germination receptors are located (see germination below; Hudson et al., 2001; Paidhungat et al., 2001). Furthermore, the inner membrane is a key target for several sporicidal chemicals (Cortezzo et al., 2004; Genest et al., 2002; Setlow et al., 2003; Young and Setlow, 2004; 2004b). Thus, the inner membrane is a structure of prime importance for spore resistance and germination. The cortex and germ cell wall The cortex is a thick cell wall composed of specifically modified peptidoglycan, built around the inner membrane (Warth and Strominger, 1972). The cortex is of crucial importance for the maintenance of spore core dehydration and thus resistance and dormancy (reviewed in Popham, 2002). It has been postulated that the cortex is involved in the acquisition of core dehydration, by exerting a large osmotic and/or mechanical pressure on the core during the final stages of sporulation (Alderton and Snell, 1963; Gould and Dring, 1975; reviewed in Gould, 1977; Lewis et al., 1960), but more recent results indicated that it merely serves as a static structure that maintains dehydration (Popham et al., 1996; Popham, 2002). Cortex peptidoglycan is electro-negatively charged (Gould and Dring, 1975) and loosely cross-linked (Popham and Setlow, 1993; Warth and Strominger, 1972). The degree of cross-linking was found to vary within the cortex regions, and this may play a role in the pressure that the cortex exerts on the core (Meador-Parton and Popham, 2000; Popham, 2002). The specific structure of the cortex is conserved among species, and may play a role in spore heat resistance, although a clear correlation between cortex structure and heat resistance has not been found (Atrih and Foster, 2001; discussed in Popham, 2002). During germination, the cortex peptidoglycan is rapidly degraded by lytic enzymes already present in the dormant spore (see germination below).

10

Introduction and outline of this thesis

The inner part of the cortex lacks the specific modifications that are characteristic of cortex peptidoglycan and is called the germ cell wall or primordial cell wall. The germ cell wall is not degraded upon germination and forms the initial cell wall of the freshly germinated spore (see Foster, 1994; Popham et al., 1996; Popham, 2002). The outer membrane Around the cortex lies the relatively poorly studied outer membrane (Crafts-Lighty and Ellar, 1980). The outer membrane (not to be confused with the outer membrane in gram-negative bacteria) has opposite polarity to the inner membrane (Wilkinson et al., 1975), and can easily be seen by electron microscopy at the early stages of spore formation (see Ellar and Lundgren, 1966; Young and Fitz-James, 1959). The integrity of the outer membrane has been doubted, and it was suggested to have a function only during spore formation (Setlow, 2000; Tipper and Gauthier, 1972). In the 1960s Philipp Gerhardt and Samuel Black published a series of papers on the permeability of bacterial spores, in which they concluded that the outer membrane was quite permeable even to large molecules (Black et al., 1960; Black and Gerhardt, 1961; 1962; 1962b; Gerhardt and Black, 1961; 1961b; Gerhardt et al., 1972). However, in later papers the authors have changed their views, and identified the outer membrane as the main permeability barrier in bacterial spores (see Gerhardt et al., 1982; Koshikawa et al., 1984; Nakashio and Gerhardt, 1985). However, this change went largely unnoticed and at present the old permeability papers are still cited (see Cowan et al., 2004; Setlow, 1994). It has been reported that the outer membrane is intact in dormant B. megaterium spores (Crafts-Lighty and Ellar, 1980), and in our experiments described in chapter 5 we found that the outer membrane in B. subtilis and B. cereus is a functional barrier to the diffusion of large ions. This suggests that the outer membrane may be of more importance than hitherto assumed, and deserves further attention in future studies. The coat The coat (for an in-depth review, Driks, 1999) is built around the outer membrane, and is a dynamic, intricate protein structure generally consisting of three distinct layers (Henriques et al., 2004). The coat shields the cortex peptidoglycan from enzymatic attack (Driks, 1999; 2002; Setlow, 2000). Furthermore, the coat is involved in resistance to environmental UV radiation (Riesenman and Nicholson, 2000) and to a variety of chemicals including oxidative agents (Genest et al., 2002; Kim et al., 2004; Loshon et al., 2001; Riesenman and Nicholson, 2000; Setlow, 2000 and references therein; Tennen et al., 2000; Young and Setlow, 2004; 2004b;). However, the coat does not seem to be of great importance for resistance to wet heat (Koshikawa et al., 1984; reviewed in Driks, 1999). The coat contains certain enzymes, such as laccases (Hullo et al., 2001; Martins et al., 2002), which may be active even when the spore core is devoid of metabolic activity. These enzymes may have a significant function in spore ecology by modifying the spore’s micro-environment (Francis and Tebo, 2002; Nicholson, 2004). The inner layer of the coat harbors a lytic enzyme, which helps degrading the cortex during germination (see Paidhungat et al., 2001; Bagyan and Setlow, 2002; Ragkousi et al., 2003). Several other coat proteins are also involved in spore germination, by facilitating the passage of specific germinant molecules through the coat (Behravan et al., 2000). Despite all this knowledge, there is still much to learn about the roles of the individual coat proteins. Recently, with the application of techniques new to spore-research, such as atomic force microscopy (Dufrene, 2002) and automated scanning microscopy (Westphal et al., 2003), it has become clear that the coat is a very dynamic structure that responds to changes in humidity (Driks, 2003; Chada et al., 2003; Plomp et al., 2005; Westphal et al., 2003).

11

Chapter 1

The Exosporium In many species the spore coat is surrounded by a loose, membrane-like structure called the exosporium (Fig 1, 3). The exosporium is important for spore hydrophobicity and adherence properties (Faille et al., 2002; Koshikawa et al., 1989). Since long, spores of B. cereus and B. anthracis are known to possess an exosporium (Matz et al., 1970), however, the B. subtilis 168 model spore-former does not (compare Fig 1 A-B). Furthermore, it is difficult to obtain sufficient quantities of exosporium for study, and therefore the exosporium has received little attention over the years (A. Moir, pers. comm.). Nevertheless, with renewed efforts in recent years the exosporium from B. cereus and B. anthracis has been analyzed in considerable detail. Next to important antigens that could serve for detection and identification (Fox et al., 2003; Steichen et al., 2003), the exosporium was found to contain a number of enzymes, some of which are possibly involved in germination (Charlton et al., 1999; Redmond et al., 2004; Todd et al., 2003). Another interesting finding is the presence of a manganese oxidizing enzyme in the exosporium from a marine Bacillus spp., which in the natural environment encases the spore in a metal shell, thereby increasing its resistance (Francis et al., 2002; Nicholson, 2004). Recently, it was found that the ExsA protein is indispensable for anchoring the exosporium to the coat, while the ExsA equivalent in B. subtilis 168 is essential for proper coat assembly (Bailey-Smith et al., 2005). Several other proteins of the B. cereus exosporium have homology to B. subtilis coat proteins, and therefore it has been proposed that the exosporium is a specialized and further decorated coat layer (Bailey-Smith et al., 2005). Spore formation The process of spore formation, also called sporulation, has been studied in detail using B. subtilis as a model, and reviewed extensively recently. Therefore, only a brief summary of events is given here; for more detail, the reader may refer to Barák, 2004; Eichenberger et al., 2004; Errington, 2003; Jedrzejas and Huang, 2003; Phillips and Strauch, 2002; Piggot and Hilbert, 2004; and Piggot and Losick, 2002. Sporulation is initiated by phosphorylation of the master transcription regulator, Spo0A. Together with σH, phosphorylated Spo0A triggers the asymmetric sporulation division, in a process called septation. Septation produces two distinct cells (Fig. 4) with very different fates, the smaller pre-spore (also known as fore-spore), which develops into the spore, and the larger mother cell, which is necessary for spore formation but ultimately lyses (programmed cell death). Soon after septation is completed, distinct programs of gene expression are initiated in the two cell types. These are directed by sporulation-specific RNA polymerase (RNAP) sigma-factors, σF in the prespore and σE in the mother cell. Sigma-factors are protein subunits transiently associated with RNAP. The associated sigma-factor determines the binding specificity of RNAP to certain promoter regions, enhancing transcription of specific sub-sets of genes involved in specific processes (Gruber and Cross, 2003; Haldenwang, 1995), in this case the sporulation process. The next hallmark of sporulation is the engulfment of the pre-spore by the mother cell. After completion of engulfment, there is a substantial change in transcription, with σG becoming active in the pre-spore and σK in the mother cell (Fig. 4). These global changes in gene regulation are coupled to morphogenesis and to each other by inter-compartmental signaling, eventually leading to the development of the resistance properties that characterize bacterial spores. When the spore is fully mature, the mother-cell lyses in a process of programmed cell death and the mature spore is released into the environment (Fig. 2).

12

Introduction and outline of this thesis

Fig. 4. Morphogenesis and gene regulation during spore formation. (a) Activation of Spo0A and σH in the pre-divisional cell leads to asymmetric division (b) and early compartmentalized gene expression with σF becoming active in the pre-spore and σE in the mother cell. (c) A series of proteins produced in the mother cell degrade the asymmetric septum and trigger migration of the membrane around the pre-spore, a process called engulfment, represented here by the curved arrows. (d) When the membranes fuse at the pole of the cell, the pre-spore is released as a protoplast in the mother cell, and a second round of compartmentalized gene expression occurs, with σG becoming active in the pre-spore and σK in the mother cell. These late factors activate transcription of genes that build the structural components of the spore that provide its resistance qualities. Adapted from Piggot and Hilbert (2004), with permission from publisher and authors.

Spore germination Spore germination involves a series of rapid degradative reactions, leading to dismantlement of the unique spore structure and loss of spore dormancy and resistance (Fig. 5). The subsequent steps that lead to cell-enlargement and cell-division are termed outgrowth, which is considered a separate process, distinct from germination (see Campbell and Leon, 1958). Germination can be enhanced by several treatments, including heat-treatments, time, and certain chemicals (Keynan and Evenchick, 1969). This enhancement is called activation (Foster and Johnstone, 1990), and the underlying mechanisms have not been resolved yet. The process of germination has been touched upon in the description of the various spore components above, and in several recent reviews (Moir et al., 2002; Setlow, 2003; de Vries, 2004). A brief overview of key events is given below. The dormant spore is equipped with sensors to choose the right moment of germination. These sensors are specific proteins called nutrient receptors or germinant receptors, and are located at the inner membrane (Hudson et al., 2001; Paidhungat et al., 2001). The germinant receptors recognize specific molecules (germinants) as a signal of nutrient-rich conditions, i. e. conditions suitable for growth. Effective germinants are amino acids (especially L-alanine), sugars and ribosides (Hornstra et al., 2005; L. Hornstra, pers. comm.). Some germinant receptors have been shown to specifically recognize one single germinant (gerA), while other germinant receptors are involved in the response to more than one germinant (gerB, gerK). Alternatively, some germinants are recognized by more than one receptor (e.g. gerI and gerQ for inosine; Moir et al., 2002 and references therein). Recent evidence indicates that there is interaction between sub-domains from

13

Chapter 1

different receptors (Igarashi and Setlow, 2005), strengthening the earlier proposed hypothesis that several different receptors may interact with each other and form complexes (McCann et al., 1996). Upon binding of the germinant to its receptor, an as yet unidentified signal is transmitted, and a number of very fast, irreversible changes occurs, ultimately leading to a fully germinated spore as summarized in Fig. 5.

Fig 5. Schematic representation of spore germination. Adapted from Setlow (2003), with permission from publisher and author.

1.5. Concluding remarks Bacterial spores are the ultimate survival vehicles, and have astonished scientists for over a century. Spore-formation is a very successful survival and dispersal strategy: spores are present virtually everywhere, and may persist for long times as they are very hard to destroy. With this in mind, it is not difficult to imagine that spores cause major problems in settings where sterility and hygiene are of importance, such as the medicare- and food-industry. Thus, bacterial spores are of prime interest from both fundamental and applied perspectives. Moreover, several spore-formers have pathogenic properties, extending the occurrence of their spores from a hygiene issue to a health issue. In the following section, such a pathogenic spore-former is introduced in a food context: Bacillus cereus.

14

Introduction and outline of the thesis

II.

Bacillus cereus

2.1 Introduction: what is Bacillus cereus? Bacillus cereus is a spore-forming, facultative anaerobic bacterium. Its cells are rod-shaped, hence the name “Bacillus” (“rod”). The name “cereus” (“wax”) was given because its colonies have a waxy appearance on agar plates (Frankland and Frankland, 1887). Together with 5 other Bacillus species, B. cereus belongs to the Bacillus cereus group, a group of bacteria so closely related that distinction between them is often problematic (see paragraph 2.3). The other members are B. weihenstephanensis, B. mycoides, B. pseudomycoides, B. thuringiensis, and B. anthracis (Jensen et al., 2003). While B. mycoides owes its name to its fungal-like growth on agar plates (Flügge, 1886; see Di Franco et al., 2002), and B. pseudomycoides and B. weihenstephanensis were recently described as new sub-groups (Lechner et al., 1998; Nakamura, 1998), the best known and studied members of the B. cereus group are B. cereus, B. thuringiensis and B. anthracis. B. thuringiensis synthesizes crystalline inclusions and δ-endotoxins that are lethal to insects, and is applied as the leading biorational pesticide (for reviews, see Aronson et al., 1986; Schnepf et al., 1998; Whalon and Wingerd, 2003). B. anthracis is a notoriously potent pathogen, being the etiological agent of anthrax (for an extensive review, see Mock and Fouet, 2001). B. cereus can also cause disease, has been associated with systemic and local infections, and is one of the most important microorganisms found in severe ocular infections (Callegan et al., 2003; Chan et al., 2003; Kotiranta et al., 2000). However, B. cereus is best known for its ability to cause food-poisoning (see paragraph 2.4), and is an important spoilage organism in pasteurized dairy products (paragraph 2.5). 2.2 B. cereus ecology The ecological niches and life-cycles of B. anthracis and B. thuringiensis have been established to a considerable extent (Jensen et al., 2003; Rasko et al., 2005). The life cycle of B. anthracis involves infection of a vertebrate host (usually a mammalian herbivore) through its spores. The spores are taken up by macrophages of the host immune system, and germinate inside the lymph nodes. The host is killed by the toxins (see Mourez et al., 2002) produced by the numerous B. anthracis cells in its system (>107 /ml of serum), which form new spores in the decaying carcass. The new spores are disseminated by scavengers and insects, and infect new hosts, closing the circle (Fig. 6). For B. thuringiensis the host organism is an insect rather than a mammal (Fig. 6). Furthermore, the interaction of B. thuringiensis with its insect host is thought to be not necessarily lethal to the host (Jensen et al., 2003). The whereabouts of B. cereus are more obscure. Its spores are omnipresent, and can be isolated from a wide variety of environmental samples (Granum, 2001; Kotiranta et al., 2000 and refs therein; Kramer and Gilbert, 1989). For this reason, B. cereus was since long considered to be a soil inhabitant. However, recent genome analysis indicated that B. cereus specializes on protein metabolism, which points towards a symbiotic or parasitic niche rather than a saprophytic life-style as a benign soil bacterium (Ivanova et al., 2003). The B. cereus genome includes numerous genes for invasion, establishment and propagation within a host, including several genes particularly useful for degradation of fungal cell walls or the insect gut wall (Huang et al., 2005; Ivanova et al., 2003). Indeed, it was proposed that B. cereus, like B. thuringiensis, is a natural inhabitant of the insect gut (Margulis et al., 1998; reviewed in Jensen et al., 2003; Parkhill and Berry, 2003; Sebaihia et al., 2003).

15

Chapter 1

Fig. 6. An illustration of the known pathogenic life cycles of B. anthracis and B. thuringiensis. Although a human pathogen, B. cereus has not been shown to enter a pathogenic life cycle similar to those of B. anthracis and B. thuringiensis. Reproduced from Jensen et al. (2003), with permission from publisher and authors.

2.3 Genetic versus phenotypic traits Originally, the distinction between members of the B. cereus group was solely based on their different phenotypic traits, but recently, genetic analysis tools have been included for species identification. This has led to confusion, because genetically, B. cereus, B. thuringiensis and B. anthracis are so similar that they can be considered as variants of one single species (Helgason et al., 2000). Furthermore, the distinction between B. cereus and B. weihenstephanensis is problematic (Stenfors and Granum, 2001), and analysis of mobile genetic elements indicated that also B. mycoides is genetically highly similar to other members of the B. cereus group (Léonard et al., 1997). Two independent analyses of seven different household genes from large numbers of representative strains indicated a clonality of the B. cereus group, and it was proposed that all members are descendants of a common ancestor that supposedly lived as a saprophyte or insect gut commensal (Helgason et al., 2004; Priest et al., 2004). The shared similarity was confirmed recently with the elucidation of the complete genomes of B. anthracis and two B. cereus strains (Ivanova et al., 2003; Rasko et al., 2004; Read et al., 2003). Apparently, a large “core-set” of genes is shared between the different members of the B. cereus group (Carlson and Kolsto, 1994; Parkhill and Berry, 2003; Rasko et al., 2005). Apart from this core-set, the genetic diversity in the B. cereus group is extensive (Helgason et al., 1998; 2004). Especially genes with phage-related functions and genes located on plasmids contribute to this diversity, and actually the genes largely responsible for the major phenotypic traits that distinguish B. thuringiensis and B. anthracis from B. cereus are located on such plasmids (Helgason et al., 2000; Parkhill and Berry, 2003; Radnedge et al., 2003; Rasko et al., 2004; 2005; Read et al., 2002). A large number of plasmids is known to occur within the B. cereus group, and these plasmids are frequently exchanged between the different members (Jensen et al.. 2003; Read et al., 2003). In addition, several plasmid-encoded genes, including genes with virulence-related functions, have been shown to switch location between plasmids and the chromosome (Carlson and Kolsto, 1994; Rasko et al., 2004; 2005). Thus, the genetic material causing the specific phenotypes that distinguish the members of the B. cereus group from one another is exchangeable between these members, and therefore the phenotypes are exchangeable as well (Helgason et al., 2000; Rasko et al., 2005). Illustrations of such exchanges are B. thuringiensis strains showing high virulence in humans after wound infection (Damgaard et al., 1997; Hernandez et al., 1998), B. thuringiensis implicated in gastro-enteritis (typically caused by B. cereus; Jackson et al., 1995) and more recently B. cereus capable of causing anthrax-like symptoms due to the presence of a plasmid

16

Introduction and outline of the thesis

that is highly similar to one of the characteristic plasmids of B. anthracis (Hoffmaster et al., 2004). In other pathogenic bacteria, a similar situation is encountered, but not acknowledged with separate species names. For example, the difference between Escherichia coli strains K12 and O157:H7 is extensive, the shared 4.1 Mb genomic core-set being interrupted by strain-specific genetic islands of in total 1.4 Mb for O157:H7 and 0.5 Mb for K12 (reviewed in Fux et al., 2005). Just as plasmids in the B. cereus group, these islands often contain genes with virulence and pathogenicity related functions, and are subject to frequent horizontal transfer. Such findings were also reported for Pseudomonas aeruginosa and Staphylococcus aureus (Fux et al., 2005). Thus, it may be concluded that the different members of the B. cereus group should be regarded as strains from one single species, i.e. B. cereus. However, the implications of renaming B. thuringiensis strains as B. cereus would be severe for the biocontrol industry, and renaming B. anthracis to B. cereus obviously would cause much confusion in the food, medical, and military research fields. For these practical reasons it is advisable to maintain the classical nomenclature, as was recently pointed out by Priest et al. (2004). 2.4 B. cereus and food poisoning Food-borne disease is a significant health threat. The microbial safety of food has improved substantially over the last few decades, but, nevertheless, in a recent survey conducted by the Dutch Institute for National Health and the Environment (RIVM), gastro-enteritis caused by microorganisms in food was identified as a significant threat to national health. The resulting annual health loss for the Dutch population was 3,000-10,000 DALY (Disability Adjusted Life Years, a common measure for health-loss described by Murray and Lopez (1996)), a value comparable to the loss caused by HIV/AIDS in the Netherlands (van Kreijl en Knaap, 2004). In the US, each year 76 million food-borne infections occur, resulting in 325,000 hospitalizations and approximately 5,000 deaths. Productivity losses may amount USD 7-37 billion annually in the US alone (Steele, 2001; Scott Smith and Pillai, 2004 and references therein). B. cereus causes two types of food poisoning syndromes: the emetic type and the diarrheal type (Reviewed by Granum, 1994; 2001; Granum and Lund, 1997; Schoeni and Wong, 2005). The emetic type is associated with farinaceous foods, particularly fried or cooked rice and pasta. In most outbreaks, these foods are stored after preparation, in conditions (room temperature) that allow rapid growth and toxin production. The toxin causing the emetic symptoms is cereulide, a heat stable, circular peptide antibiotic (Agata et al., 1995), which is produced by non-ribosomal peptide synthetase (Horwood et al., 2004) and toxic to mitochondria by acting as a potassium ion channel. These potassium ionophore properties are thought to be responsible for the disease symptoms caused by cereulide (Mikkola et al., 1999). B. cereus cells growing during storage form cereulide in the food, which is not destroyed upon heating and induces emetic symptoms shortly (1-5h) after ingestion. The symptoms include nausea, vomiting, and malaise, in some cases followed by diarrhea (Kramer and Gilbert, 1989). Generally, the symptoms are relatively mild and disappear within 24 hours, however, one case was reported in which cereulide caused fulminant liver failure and death (Mahler et al., 1997). The diarrheal type is caused by enterotoxins, and is mostly associated with proteinaceous foods such as meat. B. cereus produces several different enterotoxins, and also other toxins and virulence factors, such as phospholipases and haemolysins (Granum and Lund, 1997; Schoeni and Wong, 2005). The recent elucidation of two B. cereus genomes revealed the presence of even more toxin genes (Rasko et al., 2004) and the presence of many toxin genes in a reputedly nonpathogenic strain (Ivanova et al., 2003). The enterotoxins are not heat-stable, and thought to be formed by B. cereus cells growing in the digestive tract after ingestion of food containing B. cereus spores (Granum, 2001; Kramer and Gilbert, 1989). Symptoms start within 8-16 hours, and include abdominal pain, profuse watery diarrhea, rectal tenesmus, and occasionally nausea and vomiting

17

Chapter 1

(Kramer and Gilbert, 1989). Generally, the symptoms resolve within 12-24 hours, however in some rare cases symptoms perpetuate and can eventually lead to death. For example recently, a case was reported in which a new powerful enterotoxin, CytK, was produced by a B. cereus strain isolated from a severe outbreak that killed three people in France (Lund et al., 2000). The accurate number of food poisonings caused by B. cereus is difficult to estimate, for a number of reasons. First, B. cereus poisoning is usually not diagnosed because the symptoms are relatively mild (Kramer and Gilbert, 1989). Secondly, the reporting procedures are inconsistent and vary between countries (Ehling-Schulz et al., 2004; Schoeni and Wong, 2005). Thirdly, the symptoms closely match those caused by other food-poisoning organisms such as S. aureus and C. perfringens (Anonymous, 2005). In fact, until recently, plates containing high numbers of B. cereus colonies were regarded to be contaminated by diagnostic laboratories, and not included in the statistics. Thus, the number B. cereus-caused food-poisonings is probably highly underestimated (Granum, 1994) and large variations occur in the number of reported cases between countries (Ehling-Schultz, 2004; Granum, 2001; Kotiranta et al., 2000; Kramer and Gilbert, 1989; Shinagawa, 1990). In several cases, B. cereus has emerged as a dominant cause of food-poisoning. For instance, careful investigation of food-borne outbreaks in the German Federal Armed Forces showed that B. cereus was by far the most frequently isolated pathogen in the retained food samples (EhlingSchultz et al., 2004), being responsible for nearly half (42%) of the outbreaks reported between 1985 and 2000 (Kleer et al., 2001). Indeed, risk assessment studies have repeatedly indicated that B. cereus is a hazard of major importance in various foodstuffs, especially cooked chilled foods (Carlin et al., 2000; Nauta et al., 2003). 2.5 B. cereus and the dairy industry In the dairy industry, B. cereus is an important cause of microbe-related problems (Andersson et al., 1995). It is practically impossible to completely avoid the presence of B. cereus in raw milk samples, because B. cereus spores are ubiquitously present in the farm environment: in soil, on cattle-feed and in cattle-faeces. From these sources, the spores easily contaminate the udders and the raw milk (Christiansson et al., 1999; te Giffel et al., 2002). Furthermore, B. cereus spores are very hydrophobic (Koshikawa et al., 1989; Wiencek et al., 1990;), and attach to the surfaces of processing equipment of the dairy industry (Andersson et al., 1995; Faille et al., 2002). Attachment of the spores increases their heat resistance (Simmonds et al., 2003), while heat treatments designed to clean the equipment may increase spore hydrophobicity and thus increase attachment even further (Wiencek et al., 1990). Spores attached to processing equipment may germinate, multiply and re-sporulate (Andersson et al., 1995), resulting in a continuous source of contamination (e.g., Eneroth et al., 2001; te Giffel and Beumer, 1998). While pasteurization is insufficient to kill the spores, competition from vegetative cells of other bacteria, which are dominant in raw milk (Holm et al., 2004; Lafarge et al., 2004), is eliminated (Andersson et al., 1995). With its large array of genes for protein metabolism (Ivanova et al., 2003), B. cereus is excellently equipped for growth in milk. In addition, B. cereus spores germinate much more efficiently in milk than spores from other Bacilli (Wilkinson and Davies, 1974). Thus, although B. cereus spores are generally less abundant in milk than spores from other Bacilli (such as B. licheniformis, B. sphaericus and B. sutbilis, see Larsen and Jorgensen, 1999 and references therein), B. cereus spores can germinate and outgrow rapidly after pasteurization, thereby easily outcompeting the other Bacilli (Larsen and Jorgensen, 1999). In fact, B. cereus spores germinate more efficiently in pasteurized milk than in raw milk (te Giffel et al., 1995; Wilkinson and Davies, 1974). Hence, germination prior to pasteurization is reduced, but enhanced after pasteurization, thus increasing not only the chances for survival of the pasteurization process, but also for outgrowth and spoilage during storage. Importantly, several B. cereus strains are psychrotolerant (not to

18

Introduction and outline of the thesis

confuse with psychrophilic [Morita, 1975; Gounot, 1986]). Such psychrotolerant mesophiles are frequently referred to as psychrotrophs, especially in the food and dairy microbiology (Morita, 1975; Gounot, 1986). These strains are able to grow at temperatures as low as 4-7°C (Larsen and Jorgensen, 1999; Lechner et al., 1998; Stenfors and Granum, 2001), which is the temperature of storage for pasteurized milk (Andersson et al., 1995; Larsen and Jorgensen, 1999). This leads to enrichment of pasteurized milk with psychrotolerant strains during storage (te Giffel and Beumer, 1998). In this way, the milk process is selecting for B. cereus, and the number of B. cereus per ml is the determining factor for the shelf-life of pasteurized milk and several other heat-treated milk products (Andersson et al., 1995; te Giffel et al., 2002). While B. cereus is the main spoilage organism for pasteurized milk, a relatively low number of poisoning events has been attributed to milk containing B. cereus. It was found that the B. cereus strains generally found in milk do not produce cereulide, and only rarely enterotoxins, although enterotoxin genes are often detected in strains isolated from milk (Torkar and Mozina, 2000). Some B. cereus found in milk actually produced enterotoxins in the milk, however, these strains grow only very slowly at 37 ºC, reducing the risk of diarrhea (te Giffel and Beumer, 1998). Indeed, Langeveld et al. (1996) showed that the probability of disease caused by consumption of pasteurized milk containing high numbers of B. cereus is very low. Thus, with respect to B. cereus, the main issue in dairy products has been spoilage rather than poisoning.

19

Chapter 1

III. Outline of this thesis Bacterial spores are a major nuisance in the food industry, being omnipresent, thus occurring frequently in food ingredients and raw materials. Their high resistance enables survival of food processing treatments designed to inactivate bacteria. In the final food products, the spores can germinate, outgrow and multiply, leading to food-spoilage and -poisoning. To reduce spore-related problems, the industry employs costly processing conditions that require much time and energy, and decrease the organoleptic properties of the final products. Despite decades of dedicated research, we still have no proper basic understanding of the exact mechanisms that enable spores to be so resistant (Newsome, 2003). A more thorough understanding of these mechanisms might enable the industry to design processes that more efficiently eliminate spores. This would lead to an extended shelf-life of products, a reduction of spoilage and poisoning events, cheaper processes and better product quality. B. cereus is a spore-former, an important food-borne pathogen, and the most important spoilage organism of pasteurized dairy products. For these reasons, key properties of B. cereus are investigated within the Microbial Functionality and Safety research program of the Wageningen Centre for Food Sciences (WCFS), including stress response mechanisms and biofilm formation (de Vries et al., 2004). The research described in this thesis covers the aspects of spore formation, spore structure, and spore germination. Chapter 2 describes the construction of a firm basis for spore-research on B. cereus: defined growth- and sporulation conditions are developed and spore-analysis protocols are established. The possibilities of this methodology are exploited in Chapter 3, which contributes to clarification of the influence of carbon and nitrogen sources on the processes of growth and sporulation as well as on spore properties. Sporulation is the ultimate stress response, and it has become clear that next to sporulation sigma factors, stress sigma factor σB may play a role in sporulation, and affects spore properties, as described in Chapter 4. In Chapter 5 a novel method for the study of the structure of bacterial spores is presented, yielding novel insights for the discussion on the causes of spore resistance. Chapter 6 describes an innovative method to study the behavior of individual spores during the germination process, while Chapter 7 focuses on the properties of environmental and industrial B. cereus isolates. Finally, in Chapter 8, the findings of this PhD study are summarized and placed in perspective with future directions and a discussion on their practical application. References 1. 2. 3. 4. 5.

20

Agata, N., M. Ohta, M. Mori, and M. Isobe. 1995. A novel dodecadepsipeptide, cereulide, is an emetic toxin of Bacillus cereus. FEMS Microbiol Lett 129:17-20. Alderton, G., and N. Snell. 1963. Base exchange and heat resistance in bacterial spores. Biochem Biophys Res Commun 10:139-143. Andersson, A., U. Ronner, and P. E. Granum. 1995. What problems does the food industry have with the spore-forming pathogens Bacillus cereus and Clostridium perfringens? Int J Food Microbiol 28:145-155. Anonymous 2005, posting date. Bacillus cereus and other Bacillus sp. US Food & Drug Administration; Centre for Food Safety and Applied Nutrition. [Online: http://vm.cfsan.fda.gov/~mow/chap12.html] Aronson, A. I., W. Beckman, and P. Dunn. 1986. Bacillus thuringiensis and related insect pathogens. Microbiol Rev 50:1-24.

Introduction and outline of the thesis

6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

22. 23.

Atrih, A., and S. J. Foster. 2001. Analysis of the role of bacterial endospore cortex structure in resistance properties and demonstration of its conservation amongst species. J Appl Microbiol 91:364-372. Bagyan, I., and P. Setlow. 2002. Localization of the cortex lytic enzyme CwlJ in spores of Bacillus subtilis. J Bacteriol 184:1219-1224. Bailey-Smith, K., S. J. Todd, T. W. Southworth, J. Proctor, and A. Moir. 2005. The ExsA protein of Bacillus cereus is required for assembly of coat and exosporium onto the spore surface. J Bacteriol 187:3800-3806. Barák, I. 2004. Sporulation in Bacillus subtilis and other bacteria, p. 53-64. In E. Ricca, A. O. Henriques, and S. M. Cutting (ed.), Bacterial spore formers: probiotics and emerging applications. Horizon Bioscience, Wymondham, Norfolk UK. Beaman, T. C., J. T. Greenamyre, T. R. Corner, H. S. Pankratz, and P. Gerhardt. 1982. Bacterial spore heat resistance correlated with water content, wet density, and protoplast/sporoplast volume ratio. J Bacteriol 150:870-877. Beaman, T. C., T. Koshikawa, H. S. Pankratz, and P. Gerhardt. 1984. Dehydration partitioned within core protoplast accounts for heat resistance of bacterial spores. FEMS Microbiol Lett 24:47-51. Behravan, J., H. Chirakkal, A. Masson, and A. Moir. 2000. Mutations in the gerP locus of Bacillus subtilis and Bacillus cereus affect access of germinants to their targets in spores. J Bacteriol 182:1987-1994. Black, S. H., and P. Gerhardt. 1961. Permeability of bacterial spores. I. Characterization of glucose uptake. J Bacteriol 82:743-749. Black, S. H., and P. Gerhardt. 1962. Permeability of bacterial spores. III. Permeation relative to germination. J Bacteriol 83:301-308. Black, S. H., and P. Gerhardt. 1962b. Permeability of bacterial spores. IV. Water content, uptake, and distribution. J Bacteriol 83:960-967. Black, S. H., R. E. MacDonald, T. Hashimoto, and P. Gerhardt. 1960. Permeability of dormant bacterial spores. Nature 185:782-783. Callegan, M. C., S. T. Kane, D. C. Cochran, M. S. Gilmore, M. Gominet, and D. Lereclus. 2003. Relationship of plcR-regulated factors to Bacillus endophthalmitis virulence. Infect Immun 71:3116-3124. Campbell, L. L. J. 1958. Bacterial spore germination. Definitions and methods of study, p. 33-38. In H. O. Halvorson (ed.), Spores, vol. I. American Institute of Biological Sciences, Washington. Cano, R. J. 1995. Age of bacteria in amber (Response). Science 270:2016-2017. Cano, R. J., and M. K. Borucki. 1995. Revival and identification of bacterial spores in 2540-million-year-old Dominican amber. Science 268:1060-1064. Carlin, F., H. Girardin, M. W. Peck, S. C. Stringer, G. C. Barker, A. Martinez, A. Fernandez, P. Fernandez, W. M. Waites, S. Movahedi, F. van Leusden, M. Nauta, R. Moezelaar, M. D. Torre, and S. Litman. 2000. Research on factors allowing a risk assessment of spore-forming pathogenic bacteria in cooked chilled foods containing vegetables: a FAIR collaborative project. Int J Food Microbiol 60:117-135. Carlson, C. R., and A. B. Kolsto. 1994. A small (2.4 Mb) Bacillus cereus chromosome corresponds to a conserved region of a larger (5.3 Mb) Bacillus cereus chromosome. Mol Microbiol 13:161-169. Chada, V. G., E. A. Sanstad, R. Wang, and A. Driks. 2003. Morphogenesis of bacillus spore surfaces. J Bacteriol 185:6255-6261.

21

Chapter 1

24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42.

22

Chan, W. M., D. T. Liu, C. K. Chan, K. K. Chong, and D. S. Lam. 2003. Infective endophthalmitis caused by Bacillus cereus after cataract extraction surgery. Clin Infect Dis 37:e31-34. Charlton, S., A. J. Moir, L. Baillie, and A. Moir. 1999. Characterization of the exosporium of Bacillus cereus. J Appl Microbiol 87:241-245. Christiansson, A., J. Bertilsson, and B. Svensson. 1999. Bacillus cereus spores in raw milk: factors affecting the contamination of milk during the grazing period. J Dairy Sci 82:305-314. Cortezzo, D. E., K. Koziol-Dube, B. Setlow, and P. Setlow. 2004. Treatment with oxidizing agents damages the inner membrane of spores of Bacillus subtilis and sensitizes spores to subsequent stress. J Appl Microbiol 97:838-852. Cowan, A. E., D. E. Koppel, B. Setlow, and P. Setlow. 2003. A soluble protein is immobile in dormant spores of Bacillus subtilis but is mobile in germinated spores: implications for spore dormancy. Proc Natl Acad Sci U S A 100:4209-4214. Cowan, A. E., E. M. Olivastro, D. E. Koppel, C. A. Loshon, B. Setlow, and P. Setlow. 2004. Lipids in the inner membrane of dormant spores of Bacillus species are largely immobile. Proc Natl Acad Sci U S A 101:7733-7738. Crafts-Lighty, A., and D. J. Ellar. 1980. The structure and function of the spore outer membrane in dormant and germinating spores of Bacillus megaterium. J Appl Bacteriol 48:135-145. Damgaard, P. H., P. E. Granum, J. Bresciani, M. V. Torregrossa, J. Eilenberg, and L. Valentino. 1997. Characterization of Bacillus thuringiensis isolated from infections in burn wounds. FEMS Immunol Med Microbiol 18:47-53. de Vries, Y. P. 2004. The role of calcium in bacterial spore germination. Microbes Environ 19:199-202. de Vries, Y. P., M. van der Voort, J. Wijman, W. van Schaik, L. M. Hornstra, W. M. De Vos, and T. Abee. 2004. Progress in food-related research focussing on Bacillus cereus. Microbes Environ 19:265-269. Di Franco, C., E. Beccari, T. Santini, G. Pisaneschi, and G. Tecce. 2002. Colony shape as a genetic trait in the pattern-forming Bacillus mycoides. BMC Microbiol 2:33. Douki, T., B. Setlow, and P. Setlow. 2005. Effects of the binding of alpha/beta-type small, acid-soluble spore proteins on the photochemistry of DNA in spores of Bacillus subtilis and in vitro. Photochem Photobiol 81:163-169. Driks, A. 1999. Bacillus subtilis spore coat. Microbiol Mol Biol Rev 63:1-20. Driks, A. 2003. The dynamic spore. Proc Natl Acad Sci U S A 100:3007-3009. Driks, A. 2002. Maximum shields: the assembly and function of the bacterial spore coat. Trends Microbiol 10:251-254. Dufrene, Y. F. 2002. Atomic force microscopy, a powerful tool in microbiology. J Bacteriol 184:5205-5213. Ehling-Schulz, M., M. Fricker, and S. Scherer. 2004. Identification of emetic toxin producing Bacillus cereus strains by a novel molecular assay. FEMS Microbiol Lett 232:189-195. Ehrenberg, C. G. 1838. Die Infusionstierchen als vollkommene Organismen. Verlag Leopold Voss, Leipzig. Eichenberger, P., M. Fujita, S. T. Jensen, E. M. Conlon, D. Z. Rudner, S. T. Wang, C. Ferguson, K. Haga, T. Sato, J. S. Liu, and R. Losick. 2004. The program of gene transcription for a single differentiating cell type during sporulation in Bacillus subtilis. PLoS Biol 2:e328.

Introduction and outline of the thesis

43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54.

55. 56. 57. 58. 59. 60. 61. 62.

Ellar, D. J., and D. G. Lundgren. 1966. Fine structure of sporulation in Bacillus cereus grown in a chemically defined medium. J Bacteriol 92:1748-1764. Eneroth, A., B. Svensson, G. Molin, and A. Christiansson. 2001. Contamination of pasteurized milk by Bacillus cereus in the filling machine. J Dairy Res 68:189-196. Enserink, M. 2002. Microbial genomics. TIGR begins assault on the anthrax genome. Science 295:1442-1443. Errington, J. 2003. Regulation of endospore formation in Bacillus subtilis. Nat Rev Microbiol 1:117-126. Faille, C., C. Jullien, F. Fontaine, M. N. Bellon-Fontaine, C. Slomianny, and T. Benezech. 2002. Adhesion of Bacillus spores and Escherichia coli cells to inert surfaces: role of surface hydrophobicity. Can J Microbiol 48:728-738. Foster, S. J. 1994. The role and regulation of cell wall structural dynamics during differentiation of endospore-forming bacteria. Soc Appl Bacteriol Symp Ser 23:25S-39S. Foster, S. J., and K. Johnstone. 1990. Pulling the trigger: the mechanism of bacterial spore germination. Mol Microbiol 4:137-141. Fox, A., G. C. Stewart, L. N. Waller, K. F. Fox, W. M. Harley, and R. L. Price. 2003. Carbohydrates and glycoproteins of Bacillus anthracis and related bacilli: targets for biodetection. J Microbiol Methods 54:143-152. Francis, C. A., K. L. Casciotti, and B. M. Tebo. 2002. Localization of Mn(II)-oxidizing activity and the putative multicopper oxidase, MnxG, to the exosporium of the marine Bacillus sp. strain SG-1. Arch Microbiol 178:450-456. Francis, C. A., and B. M. Tebo. 2002. Enzymatic manganese(II) oxidation by metabolically dormant spores of diverse Bacillus species. Appl Environ Microbiol 68:874880. Frankland, G. C., and P. F. Frankland. 1887. Studies on some new micro-organsisms from air. Philosophical transactions of the Royal Society of London. Series B, biological sciences 173:257-287. Frenkiel-Krispin, D., R. Sack, J. Englander, E. Shimoni, M. Eisenstein, E. Bullitt, R. Horowitz-Scherer, C. S. Hayes, P. Setlow, A. Minsky, and S. G. Wolf. 2004. Structure of the DNA-SspC complex: implications for DNA packaging, protection, and repair in bacterial spores. J Bacteriol 186:3525-3530. Fux, C. A., M. Shirtliff, P. Stoodley, and J. W. Costerton. 2005. Can laboratory reference strains mirror 'real-world' pathogenesis? Trends Microbiol 13:58-63. Genest, P. C., B. Setlow, E. Melly, and P. Setlow. 2002. Killing of spores of Bacillus subtilis by peroxynitrite appears to be caused by membrane damage. Microbiology 148:307314. Gerhardt, P., T. C. Beaman, T. R. Corner, J. T. Greenamyre, and L. S. Tisa. 1982. Photometric immersion refractometry of bacterial spores. J Bacteriol 150:643-648. Gerhardt, P., and S. H. Black. 1961b. Permeability of bacterial spores, p. 218-229. In H. O. Halvorson (ed.), Spores, vol. II. Burgess Publishing Company, Minneapolis. Gerhardt, P., and S. H. Black. 1961. Permeability of bacterial spores. II. Molecular variables affecting solute permeation. J Bacteriol 82:750-760. Gerhardt, P., R. Scherrer, and S. H. Black. 1972. Melecular sieving by dormant spore structures, p. 68-77. In H. O. Halvorson, R. Hanson, and L. L. Campbell (ed.), Spores, vol. 5. ASM, Fontana, Wisconsin. Gest, H., and J. Mandelstam. 1987. Longevity of microorganisms in natural environments. Microbiol Sci 4:69-71. Gould, G. W. 1977. Recent advances in the understanding of resistance and dormancy in bacterial spores. J Appl Bacteriol 42:297-309.

23

Chapter 1

63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75.

76. 77.

78.

79.

24

Gould, G. W. 1986. Water and the survival of bacterial spores, p. 143-156. In A. C. Leopold (ed.), Membranes, Metabolism, and Dry Organisms. Cornell University Press, Ithaca, USA. Gould, G. W., and G. J. Dring. 1975. Heat resistance of bacterial endospores and concept of an expanded osmoregulatory cortex. Nature 258:402-405. Granum, P. E. 2001. Bacillus cereus, p. 373-381. In M. P. e. a. Doyle (ed.), Food Microbiology: Fundamentals and Frontiers. ASM Press, Washington DC. Granum, P. E. 1994. Bacillus cereus and its toxins. Soc Appl Bacteriol Symp Ser 23:61S66S. Granum, P. E., and T. Lund. 1997. Bacillus cereus and its food poisoning toxins. FEMS Microbiol Lett 157:223-228. Gruber, T. M., and C. A. Gross. 2003. Multiple sigma subunits and the partitioning of bacterial transcription space. Annu Rev Microbiol 57:441-466. Haldenwang, W. G. 1995. The sigma factors of Bacillus subtilis. Microbiol. Rev. 59:1-30. Halvorson, H., and C. Howitt. 1961. The role of DPA in bacterial spores, p. 149-165. In H. O. Halvorson (ed.), Spores, vol. II. Burgess Publishing Company, Minneapolis. Halvorson, H. O. 1997. Two generations of spore research: from father to son. Microbiologia 13:131-148. Helgason, E., D. A. Caugant, M. M. Lecadet, Y. Chen, J. Mahillon, A. Lovgren, I. Hegna, K. Kvaloy, and A. B. Kolsto. 1998. Genetic diversity of Bacillus cereus/B. thuringiensis isolates from natural sources. Curr Microbiol 37:80-87. Helgason, E., O. A. Okstad, D. A. Caugant, H. A. Johansen, A. Fouet, M. Mock, I. Hegna, and Kolsto. 2000. Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis-one species on the basis of genetic evidence. Appl Environ Microbiol 66:2627-2630. Helgason, E., N. J. Tourasse, R. Meisal, D. A. Caugant, and A. B. Kolsto. 2004. Multilocus sequence typing scheme for bacteria of the Bacillus cereus group. Appl Environ Microbiol 70:191-201. Henriques, A. O., T. V. Costa, L. O. Martins, and R. Zilhão. 2004. The functional architecture and assembly of the spore coat, p. 65-86. In E. Ricca, A. O. Henriques, and S. M. Cutting (ed.), Bacterial spore formers: probiotics and emerging applications. Horizon Bioscience, Wymondham, Norfolk UK. Hernandez, E., F. Ramisse, J. P. Ducoureau, T. Cruel, and J. D. Cavallo. 1998. Bacillus thuringiensis subsp. konkukian (serotype H34) superinfection: case report and experimental evidence of pathogenicity in immunosuppressed mice. J Clin Microbiol 36:2138-2139. Higgins, J. A., M. Cooper, L. Schroeder-Tucker, S. Black, D. Miller, J. S. Karns, E. Manthey, R. Breeze, and M. L. Perdue. 2003. A field investigation of Bacillus anthracis contamination of U.S. Department of Agriculture and other Washington, D.C., buildings during the anthrax attack of october 2001. Appl Env Microbiol 69:593-599. Hoffmaster, A. R., J. Ravel, D. A. Rasko, G. D. Chapman, M. D. Chute, C. K. Marston, B. K. De, C. T. Sacchi, C. Fitzgerald, L. W. Mayer, M. C. Maiden, F. G. Priest, M. Barker, L. Jiang, R. Z. Cer, J. Rilstone, S. N. Peterson, R. S. Weyant, D. R. Galloway, T. D. Read, T. Popovic, and C. M. Fraser. 2004. Identification of anthrax toxin genes in a Bacillus cereus associated with an illness resembling inhalation anthrax. Proc Natl Acad Sci U S A 101:8449-8454. Horneck, G., P. Rettberg, G. Reitz, J. Wehner, U. Eschweiler, K. Strauch, C. Panitz, V. Starke, and C. Baumstark-Khan. 2001. Protection of bacterial spores in space, a contribution to the discussion on Panspermia. Orig Life Evol Biosph 31:527-547.

Introduction and outline of the thesis

80. 81. 82. 83. 84. 85. 86.

87. 88. 89. 90.

91. 92. 93. 94.

Hornstra, L. M., Y. P. de Vries, W. M. de Vos, T. Abee, and M. H. Wells-Bennik. 2005. gerR, a novel ger operon involved in L-alanine- and inosine-initiated germination of Bacillus cereus ATCC 14579. Appl Environ Microbiol 71:774-781. Horwood, P. F., G. W. Burgess, and H. J. Oakey. 2004. Evidence for non-ribosomal peptide synthetase production of cereulide (the emetic toxin) in Bacillus cereus. FEMS Microbiol Lett 236:319-324. Huang, C. J., T. K. Wang, S. C. Chung, and C. Y. Chen. 2005. Identification of an antifungal chitinase from a potential biocontrol agent, Bacillus cereus 28-9. J Biochem Mol Biol 38:82-88. Hudson, K. D., B. M. Corfe, E. H. Kemp, I. M. Feavers, P. J. Coote, and A. Moir. 2001. Localization of GerAA and GerAC germination proteins in the Bacillus subtilis spore. J Bacteriol 183:4317-4322. Hullo, M. F., I. Moszer, A. Danchin, and I. Martin-Verstraete. 2001. CotA of Bacillus subtilis is a copper-dependent laccase. J Bacteriol 183:5426-5430. Igarashi, T., and P. Setlow. 2005. Interaction between individual protein components of the GerA and GerB nutrient receptors that trigger germination of Bacillus subtilis spores. J Bacteriol 187:2513-2518. Ivanova, N., A. Sorokin, I. Anderson, N. Galleron, B. Candelon, V. Kapatral, A. Bhattacharyya, G. Reznik, N. Mikhailova, A. Lapidus, L. Chu, M. Mazur, E. Goltsman, N. Larsen, M. D'Souza, T. Walunas, Y. Grechkin, G. Pusch, R. Haselkorn, M. Fonstein, S. D. Ehrlich, R. Overbeek, and N. Kyrpides. 2003. Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis. Nature 423:87-91. Jackson, S. G., R. B. Goodbrand, R. Ahmed, and S. Kasatiya. 1995. Bacillus cereus and Bacillus thuringiensis isolated in a gastroenteritis outbreak investigation. Lett Appl Microbiol 21:103-105. Jedrzejas, M. J., and W. J. Huang. 2003. Bacillus species proteins involved in spore formation and degradation: from identification in the genome, to sequence analysis, and determination of function and structure. Crit Rev Biochem Mol Biol 38:173-198. Jensen, G. B., B. M. Hansen, J. Eilenberg, and J. Mahillon. 2003. The hidden lifestyles of Bacillus cereus and relatives. Environ Microbiol 5:631-640. Jernigan, D. B., P. L. Raghunathan, B. P. Bell, R. Brechner, E. A. Bresnitz, J. C. Butler, M. Cetron, M. Cohen, T. Doyle, M. Fischer, C. Greene, K. S. Griffith, J. Guarner, J. L. Hadler, J. A. Hayslett, R. Meyer, L. R. Petersen, M. Phillips, R. Pinner, T. Popovic, C. P. Quinn, J. Reefhuis, D. Reissman, N. Rosenstein, A. Schuchat, W. J. Shieh, L. Siegal, D. L. Swerdlow, F. C. Tenover, M. Traeger, J. W. Ward, I. Weisfuse, S. Wiersma, K. Yeskey, S. Zaki, D. A. Ashford, B. A. Perkins, S. Ostroff, J. Hughes, D. Fleming, J. P. Koplan, and J. L. Gerberding. 2002. Investigation of bioterrorism-related anthrax, United States, 2001: epidemiologic findings. Emerg Infect Dis 8:1019-1028. Keynan, A. 1972. Cryptobiosis: a review of the mechanisms of the ametabolic state in bacterial spores, p. 355-363. In H. O. Halvorson, R. Hanson, and L. L. Campbell (ed.), Spores, vol. 5. ASM, Fontana, Wisconsin. Keynan, A., and Z. Evenchik. 1969. Activation, p. 359-396. In G. W. Gould and A. Hurst (ed.), The Bacterial Spore, vol. 1. Academic Press, London and New York. Keynan, A., and N. Sandler. 1983. Spore research in a historical perspective, p. 2-49. In A. Hurst and G. W. Gould (ed.), The Bacterial Spore, vol. 2. Academic Press, London and New York. Kim, H. S., D. Sherman, F. Johnson, and A. I. Aronson. 2004. Characterization of a major Bacillus anthracis spore coat protein and its role in spore inactivation. J Bacteriol 186:2413-2417.

25

Chapter 1

95. 96. 97. 98. 99. 100. 101.

102. 103. 104.

105. 106. 107. 108. 109. 110. 111.

26

Kleer, J., A. Bartholoma, R. Levetzow, T. Reiche, H. J. Sinell, and P. Teufel. 2001. Bakterielle Lebensmittel-Infektionen und -Intoxikationen in Einrichtungen zur Gemeinschaftsverpflegung 1985 bis 2000. Arch Lebensm Hyg 52:73-112. Kminek, G., J. L. Bada, K. Pogliano, and J. F. Ward. 2003. Radiation-dependent limit for the viability of bacterial spores in halite fluid inclusions and on Mars. Radiat Res 159:722-729. Koshikawa, T., T. C. Beaman, H. S. Pankratz, S. Nakashio, T. R. Corner, and P. Gerhardt. 1984. Resistance, germination, and permeability correlates of Bacillus megaterium spores successively divested of integument layers. J Bacteriol 159:624-632. Koshikawa, T., M. Yamazaki, M. Yoshimi, S. Ogawa, A. Yamada, K. Watabe, and M. Torii. 1989. Surface hydrophobicity of spores of Bacillus spp. J Gen Microbiol 135 ( Pt 10):2717-2722. Kotiranta, A., K. Lounatmaa, and M. Haapasalo. 2000. Epidemiology and pathogenesis of Bacillus cereus infections. Microbes Infect 2:189-198. Kramer, J. M., and R. J. Gilbert. 1989. Bacillus cereus and other Bacillus species, p. 2170. In M. P. Doyle (ed.), Food-borne Bacterial Pathogens. Marcel Dekker, Inc., New York, N.Y. Kunst, F., N. Ogasawara, I. Moszer, A. M. Albertini, G. Alloni, V. Azevedo, M. G. Bertero, P. Bessieres, A. Bolotin, S. Borchert, R. Borriss, L. Boursier, A. Brans, M. Braun, S. C. Brignell, S. Bron, S. Brouillet, C. V. Bruschi, B. Caldwell, V. Capuano, N. M. Carter, S. K. Choi, J. J. Codani, I. F. Connerton, A. Danchin, and et al. 1997. The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390:249-256. Langeveld, L. P. M., W. A. v. Spronsen, E. C. H. v. Beresteijn, and S. H. W. Notermans. 1996. Consumption by healthy adults of pasteurized milk with a high concentration of Bacillus cereus: a double-blind study. J Food Prot 59:723-726. Larsen, H. D., and K. Jorgensen. 1999. Growth of Bacillus cereus in pasteurized milk products. Int J Food Microbiol 46:173-176. Lechner, S., R. Mayr, K. P. Francis, B. M. Pruss, T. Kaplan, E. Wiessner-Gunkel, G. S. Stewart, and S. Scherer. 1998. Bacillus weihenstephanensis sp. nov. is a new psychrotolerant species of the Bacillus cereus group. International Journal of Systematic Bacteriology 48 Pt 4:1373-1382. Leonard, C., Y. Chen, and J. Mahillon. 1997. Diversity and differential distribution of IS231, IS232 and IS240 among Bacillus cereus, Bacillus thuringiensis and Bacillus mycoides. Microbiology 143:2537-2547. Lewis, J. C. 1969. Dormancy, p. 301-358. In G. W. Gould and A. Hurst (ed.), The Bacterial Spore, vol. 1. Academic Press, London and New York. Lewis, J. C., N. S. Snell, and H. K. Burr. 1960. Water permeability of bacterial spores and the concept of a contractile cortex. Science 132:544-545. Loshon, C. A., E. Melly, B. Setlow, and P. Setlow. 2001. Analysis of the killing of spores of Bacillus subtilis by a new disinfectant, Sterilox. J Appl Microbiol 91:1051-1058. Lund, T., M. L. De Buyser, and P. E. Granum. 2000. A new cytotoxin from Bacillus cereus that may cause necrotic enteritis. Mol Microbiol 38:254-261. Mahler, H., A. Pasi, J. M. Kramer, P. Schulte, A. C. Scoging, W. Bar, and S. Krahenbuhl. 1997. Fulminant liver failure in association with the emetic toxin of Bacillus cereus. N Engl J Med 336:1142-1148. Margulis, L., J. Z. Jorgensen, S. Dolan, R. Kolchinsky, F. A. Rainey, and S. C. Lo. 1998. The Arthromitus stage of Bacillus cereus: intestinal symbionts of animals. Proc Natl Acad Sci U S A 95:1236-1241.

Introduction and outline of the thesis

112.

113. 114. 115. 116. 117. 118.

119. 120. 121. 122.

123. 124. 125. 126. 127. 128. 129. 130.

Martins, L. O., C. M. Soares, M. M. Pereira, M. Teixeira, T. Costa, G. H. Jones, and A. O. Henriques. 2002. Molecular and biochemical characterization of a highly stable bacterial laccase that occurs as a structural component of the Bacillus subtilis endospore coat. J Biol Chem 277:18849-18859. Matz, L. L., T. C. Beaman, and P. Gerhardt. 1970. Chemical composition of exosporium from spores of Bacillus cereus. J Bacteriol 101:196-201. Maughan, H., C. W. Birky, Jr., W. L. Nicholson, W. D. Rosenzweig, and R. H. Vreeland. 2002. The paradox of the "ancient" bacterium which contains "modern" proteincoding genes. Mol Biol Evol 19:1637-1639. McCann, K. P., C. Robinson, R. L. Sammons, D. A. Smith, and B. M. Corfe. 1996. Alanine germination receptors of Bacillus subtilis. Lett Appl Microbiol 23:290-294. Meador-Parton, J., and D. L. Popham. 2000. Structural analysis of Bacillus subtilis spore peptidoglycan during sporulation. J Bacteriol 182:4491-4499. Mikkola, R., N. E. Saris, P. A. Grigoriev, M. A. Andersson, and M. S. SalkinojaSalonen. 1999. Ionophoretic properties and mitochondrial effects of cereulide: the emetic toxin of B. cereus. Eur J Biochem 263:112-117. Mileikowsky, C., F. A. Cucinotta, J. W. Wilson, B. Gladman, G. Horneck, L. Lindegren, H. J. Melosh, H. Rickman, M. Valtonen, and J. Q. Zheng. 2000. Natural transfer of viable microbes in space. Part 1: From Mars to Earth and Earth to Mars. Icarus 145:391-427. Mock, M., and A. Fouet. 2001. Anthrax. Ann Rev Microbiol 55:647-671. Moir, A., B. M. Corfe, and J. Behravan. 2002. Spore germination. Cell Mol Life Sci 59:403-409. Mourez, M., D. B. Lacy, K. Cunningham, R. Legmann, B. R. Sellman, J. Mogridge, and R. J. Collier. 2002. 2001: a year of major advances in anthrax toxin research. Trends in Microbiology 10:287-293. Murray, J. C. L., and A. D. Lopez. 1996. The global burden of disease: a comparative assessment of mortality and disability from disease, injuries, and risk factors in 1990 and projected to 2020. Harvard School of Public Health, on behalf of the WHO and World Bank, Cambridge, Mass. Murrell, W. G., and A. D. Warth. 1965. Composition and heat resistance of bacterial spores, p. 1-25. In L. L. Campbell and H. O. Halvorson (ed.), Spores, vol. III. ASM Press, Ann Arbor. Nakamura, L. K. 1998. Bacillus pseudomycoides sp. nov. Int J Syst Bacteriol 48 Pt 3:1031-1035. Nakashio, S., and P. Gerhardt. 1985. Protoplast dehydration correlated with heat resistance of bacterial spores. J Bacteriol 162:571-578. Nauta, M. J., S. Litman, G. C. Barker, and F. Carlin. 2003. A retail and consumer phase model for exposure assessment of Bacillus cereus. Int J Food Microbiol 83:205-218. Newsome, R. 2003. Dormant microbes: research needs. Food Tech. 57:38-42. Nicholson, W. L. 2004. Ubiquity, longevity, and ecological roles of Bacillus spores, p. 115. In E. Ricca, A. O. Henriques, and S. M. Cutting (ed.), Bacterial spore formers: probiotics and emerging applications. Horizon Bioscience, Wymondham, Norfolk UK. Nicholson, W. L. 2003. Using thermal inactivation kinetics to calculate the probability of extreme spore longevity: implications for paleomicrobiology and lithopanspermia. Orig Life Evol Biosph 33:621-631. Nicholson, W. L., N. Munakata, G. Horneck, H. J. Melosh, and P. Setlow. 2000. Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiol Mol Biol Rev 64:548-572.

27

Chapter 1

131. 132. 133. 134. 135. 136. 137. 138. 139. 140. 141. 142. 143. 144. 145. 146. 147. 148.

149.

28

Paidhungat, M., K. Ragkousi, and P. Setlow. 2001. Genetic requirements for induction of germination of spores of Bacillus subtilis by Ca(2+)-dipicolinate. J Bacteriol 183:48864893. Paidhungat, M., B. Setlow, A. Driks, and P. Setlow. 2000. Characterization of spores of Bacillus subtilis which lack dipicolinic acid. J Bacteriol 182:5505-5512. Parkes, R. J. 2000. A case of bacterial immortality? Nature 407:844-845. Parkhill, J., and C. Berry. 2003. Genomics: Relative pathogenic values. Nature 423:23-25. Phillips, Z. E., and M. A. Strauch. 2002. Bacillus subtilis sporulation and stationary phase gene expression. Cell Mol Life Sci 59:392-402. Piggot, P. J., and D. W. Hilbert. 2004. Sporulation of Bacillus subtilis. Curr Opin Microbiol 7:579-586. Piggot, P. J., and R. Losick. 2002. Sporulation genes and intercompartmental regulation, p. 483-519. In A. L. Sonenshein, J. A. Hoch, and R. Losick (ed.), Bacillus subtilis and its closest relatives: from genes to cells. ASM Press, Washington D. C. Plomp, M., T. J. Leighton, K. E. Wheeler, and A. J. Malkin. 2005. The high-resolution architecture and structural dynamics of Bacillus spores. Biophys J 88:603-608. Popham, D. L. 2002. Specialized peptidoglycan of the bacterial endospore: the inner wall of the lockbox. Cell Mol Life Sci 59:426-433. Popham, D. L., J. Helin, C. E. Costello, and P. Setlow. 1996. Muramic lactam in peptidoglycan of Bacillus subtilis spores is required for spore outgrowth but not for spore dehydration or heat resistance. Proc Natl Acad Sci U S A 93:15405-15410. Popham, D. L., and P. Setlow. 1993. The cortical peptidoglycan from spores of Bacillus megaterium and Bacillus subtilis is not highly cross-linked. J Bacteriol 175:2767-2769. Potts, M. 1994. Desiccation tolerance of prokaryotes. Microbiol Rev 58:755-805. Powell, J. F. 1953. Isolation of dipicolinic acid (pyridine-2:6-dicarboxylic acid) from spores of Bacillus megatherium. Biochem J 54:210-211. Priest, F. G., M. Barker, L. W. Baillie, E. C. Holmes, and M. C. Maiden. 2004. Population structure and evolution of the Bacillus cereus group. J Bacteriol 186:7959-7970. Radnedge, L., P. G. Agron, K. K. Hill, P. J. Jackson, L. O. Ticknor, P. Keim, and G. L. Andersen. 2003. Genome differences that distinguish Bacillus anthracis from Bacillus cereus and Bacillus thuringiensis. Appl Environ Microbiol 69:2755-2764. Ragkousi, K., P. Eichenberger, C. van Ooij, and P. Setlow. 2003. Identification of a new gene essential for germination of Bacillus subtilis spores with Ca2+-dipicolinate. J Bacteriol 185:2315-2329. Rasko, D. A., M. R. Altherr, C. S. Han, and J. Ravel. 2005. Genomics of the Bacillus cereus group of organisms. FEMS Microbiol Rev 29:303-329. Rasko, D. A., J. Ravel, O. A. Okstad, E. Helgason, R. Z. Cer, L. Jiang, K. A. Shores, D. E. Fouts, N. J. Tourasse, S. V. Angiuoli, J. Kolonay, W. C. Nelson, A. B. Kolsto, C. M. Fraser, and T. D. Read. 2004. The genome sequence of Bacillus cereus ATCC 10987 reveals metabolic adaptations and a large plasmid related to Bacillus anthracis pXO1. Nucleic Acids Res 32:977-988. Read, T. D., S. N. Peterson, N. Tourasse, L. W. Baillie, I. T. Paulsen, K. E. Nelson, H. Tettelin, D. E. Fouts, J. A. Eisen, S. R. Gill, E. K. Holtzapple, O. A. Okstad, E. Helgason, J. Rilstone, M. Wu, J. F. Kolonay, M. J. Beanan, R. J. Dodson, L. M. Brinkac, M. Gwinn, R. T. DeBoy, R. Madpu, S. C. Daugherty, A. S. Durkin, D. H. Haft, W. C. Nelson, J. D. Peterson, M. Pop, H. M. Khouri, D. Radune, J. L. Benton, Y. Mahamoud, L. Jiang, I. R. Hance, J. F. Weidman, K. J. Berry, R. D. Plaut, A. M. Wolf, K. L. Watkins, W. C. Nierman, A. Hazen, R. Cline, C. Redmond, J. E. Thwaite, O. White, S. L. Salzberg, B. Thomason, A. M. Friedlander, T. M. Koehler, P. C. Hanna,

Introduction and outline of the thesis

150.

151. 152. 153. 154. 155. 156. 157. 158. 159. 160. 161. 162. 163. 164. 165. 166. 167. 168. 169.

A. B. Kolsto, and C. M. Fraser. 2003. The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteria. Nature 423:81-86. Read, T. D., S. L. Salzberg, M. Pop, M. Shumway, L. Umayam, L. Jiang, E. Holtzapple, J. D. Busch, K. L. Smith, J. M. Schupp, D. Solomon, P. Keim, and C. M. Fraser. 2002. Comparative genome sequencing for discovery of novel polymorphisms in Bacillus anthracis. Science 296:2028-2033. Redmond, C., L. W. Baillie, S. Hibbs, A. J. Moir, and A. Moir. 2004. Identification of proteins in the exosporium of Bacillus anthracis. Microbiology 150:355-363. Riesenman, P. J., and W. L. Nicholson. 2000. Role of the spore coat layers in Bacillus subtilis spore resistance to hydrogen peroxide, artificial UV-C, UV-B, and solar UV radiation. Appl Env Microbiol 66:620-626. Russell, A. D. 2003. Lethal effects of heat on bacterial physiology and structure. Sci Progr 86:115-137. Schnepf, E., N. Crickmore, J. Van Rie, D. Lereclus, J. Baum, J. Feitelson, D. R. Zeigler, and D. H. Dean. 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62:775-806. Schoeni, J. L., and A. C. Wong. 2005. Bacillus cereus food poisoning and its toxins. J Food Prot 68:636-648. Scott Smith, J., and S. Pillai. 2004. Irradiation and food safety. Food Tech 58:48-55. Sebaihia, M., S. Bentley, L. Crossman, N. Thomson, and J. Parkhill. 2003. A bad combination. Trends Microbiol 11:297-299. Setlow, B., A. E. Cowan, and P. Setlow. 2003. Germination of spores of Bacillus subtilis with dodecylamine. J Appl Microbiol 95:637-648. Setlow, B., and P. Setlow. 1980. Measurements of the pH within dormant and germinated bacterial spores. Proc Natl Acad Sci U S A 77:2474-2476. Setlow, P. 1995. Mechanisms for the prevention of damage to DNA in spores of Bacillus species. Annu Rev Microbiol 49:29-54. Setlow, P. 1994. Mechanisms which contribute to the long-term survival of spores of Bacillus species. Soc Appl Bacteriol Symp Ser 23:49S-60S. Setlow, P. 2000. Resistance of bacterial spores, p. 217-230. In G. Storz and R. HenggeAronis (ed.), Bacterial stress responses. ASM press, Washington, D.C. Setlow, P. 1988. Small, acid-soluble spore proteins of Bacillus species: structure, synthesis, genetics, function, and degradation. Annu Rev Microbiol 42:319-338. Setlow, P. 2003. Spore germination. Curr Opin Microbiol 6:1-7. Shinagawa, K. 1990. Analytical methods for Bacillus cereus and other Bacillus species. Int J Food Microbiol 10:125-141. Simmonds, P., B. L. Mossel, T. Intaraphan, and H. C. Deeth. 2003. Heat resistance of Bacillus spores when adhered to stainless steel and its relationship to spore hydrophobicity. J Food Prot 66:2070-2075. Slieman, T. A., and W. L. Nicholson. 2001. Role of dipicolinic acid in survival of Bacillus subtilis spores exposed to artificial and solar UV radiation. Appl Environ Microbiol 67:1274-1279. Steele, J. H. 2001. Food irradiation: a public health challenge for the 21st century. Clin Infect Dis 33:376-377. Steichen, C., P. Chen, J. F. Kearney, and C. L. Turnbough, Jr. 2003. Identification of the immunodominant protein and other proteins of the Bacillus anthracis exosporium. J Bacteriol 185:1903-1910.

29

Chapter 1

170. 171. 172. 173. 174. 175. 176. 177. 178. 179. 180. 181. 182. 183. 184. 185. 186. 187. 188. 189. 190.

30

Stenfors, L. P., and P. E. Granum. 2001. Psychrotolerant species from the Bacillus cereus group are not necessarily Bacillus weihenstephanensis. FEMS Microbiology Letters 197:223-228. Sussman, A. S., and H. O. Halvorson. 1969b. Longevity and survivability of spores, p. 45100, Spores, their dormancy and germination. Harper and Row, New York. Sussman, A. S., and H. O. Halvorson. 1969. Spores in space, p. 101-115, Spores, their dormancy and germination. Harper and Row, New York. te Giffel, M. C., and R. R. Beumer. 1998. [Isolation, identification and characterization of Bacillus cereus in the dairy industry]. Tijdschr Diergeneesk 123:628-632. te Giffel, M. C., R. R. Beumer, J. Hoekstra, and F. M. Rombouts. 1995. Germination of bacterial spores during sample preparation. Food Microbiol 12:327-332. te Giffel, M. C., A. Wagendorp, A. Herrewegh, and F. Driehuis. 2002. Bacterial spores in silage and raw milk. Antonie van Leeuwenhoek 81:625-630. Tennen, R., B. Setlow, K. L. Davis, C. A. Loshon, and P. Setlow. 2000. Mechanisms of killing of spores of Bacillus subtilis by iodine, glutaraldehyde and nitrous acid. J Appl Microbiol 89:330-338. Tipper, D. J., and J. J. Gauthier. 1972. Structure of the bacterial endospore, p. 3-13. In H. O. Halvorson, R. Hanson, and L. L. Campbell (ed.), Spores, vol. 5. ASM, Fontana, Wisconsin. Todd, S. J., A. J. Moir, M. J. Johnson, and A. Moir. 2003. Genes of Bacillus cereus and Bacillus anthracis encoding proteins of the exosporium. J Bacteriol 185:3373-3378. Torkar, K. G., and S. S. Mozina. 2000. Differentiation of Bacillus cereus isolates from milk and milk products with biochemical, immunological, AP-PCR and PCR-RFLP methods. Food technol biotechnol 38:135-142. van Kreijl, C. F., and A. G. A. C. Knaap. 2004. Ons eten gemeten, gezonde voeding en veilig voedsel in Nederland 270555007; ISBN 90-313-4411-7. RIVM (Dutch National Institute for Public Health and the Environment). Vreeland, R. H., and W. D. Rosenzweig. 2002. The question of uniqueness of ancient bacteria. J Ind Microbiol Biotechnol 28:32-41. Vreeland, R. H., W. D. Rosenzweig, and D. W. Powers. 2000. Isolation of a 250 millionyear-old halotolerant bacterium from a primary salt crystal. Nature 407:897-900. Warth, A. D. 1978. Molecular structure of the bacterial spore. Adv Microb Physiol 17:1-45. Warth, A. D., and J. L. Strominger. 1972. Structure of the peptidoglycan from spores of Bacillus subtilis. Biochemistry 11:1389-1396. Westphal, A. J., P. B. Price, T. J. Leighton, and K. E. Wheeler. 2003. Kinetics of size changes of individual Bacillus thuringiensis spores in response to changes in relative humidity. Proc Natl Acad Sci U S A 100:3461-3466. Whalon, M. E., and B. A. Wingerd. 2003. Bt: mode of action and use. Arch Insect Biochem Physiol 54:200-211. Wiencek, K. M., N. A. Klapes, and P. M. Foegeding. 1990. Hydrophobicity of Bacillus and Clostridium spores. Appl Environ Microbiol 56:2600-2605. Wilkinson, B. J., J. A. Deans, and D. J. Ellar. 1975. Biochemical evidence for the reversed polarity of the outer membrane of the bacterial forespore. Biochem J 152:561-569. Wilkinson, G., and F. L. Davies. 1974. Some aspects of the germination of Bacillus cereus in milk, p. 153-159. In A. N. Barker, G. W. Gould, and J. Wolf (ed.), Spore Research 1973. Academic Press, London. Young, I. E., and P. C. Fitz-James. 1959. Chemical and morphological studies of bacterial spore formation. II. Spore and parasporal protein formation in Bacillus cereus var. alesti. J Biophys Biochem Cytol 6:483-498.

Introduction and outline of the thesis

191. 192.

Young, S. B., and P. Setlow. 2004. Mechanisms of Bacillus subtilis spore resistance to and killing by aqueous ozone. J Appl Microbiol 96:1133-1142. Young, S. B., and P. Setlow. 2004b. Mechanisms of killing of Bacillus subtilis spores by Decon and Oxone, two general decontaminants for biological agents. J Appl Microbiol 96:289-301.

31

2. Growth and Sporulation of Bacillus cereus ATCC14579 under Defined Conditions: Temporal Expression of Genes for Key Sigma Factors Ynte P. de Vries, Luc M. Hornstra, Willem M. de Vos, and Tjakko Abee Excerpt published in Applied and Environmental Microbiology, 2004, 70:2514-2519

ABSTRACT An airlift fermenter system allowing precise regulation of pH and aeration combined with a chemically defined medium was used to study growth and sporulation of B. cereus ATCC14579. Sporulation was complete and synchronous. Expression of sigA, sigB, sigF and sigG was monitored with real-time reverse transcription-PCR, and the pattern qualitatively resembled that of B. subtilis. This method allows reproducible production of stable spores, while the synchronous growth and defined conditions are excellently suitable for further geneexpression studies on cellular differentiation of B. cereus.

33

Chapter 2

Introduction Bacillus cereus is a gram-positive, facultative anaerobic rod-shaped bacterium able to form spores. It is an ubiquitous bacterium found in soil and in many raw and processed foods such as rice, milk and dairy products, spices and vegetables (Roberts et al., 1982; Carlin et al., 2000; Christiansson et al., 1999; Sarrias et al., 2002; Guinebretiere et al., 2003). Many strains of B. cereus are able to produce toxins and cause distinct types of food-poisoning (Lund et al., 2000; Granum, 1994). Concerns over B. cereus contamination have increased over the past few years, because of the rapidly expanding market of chilled foods that may be pasteurized but still contain viable spores (Guinebretiere et al., 2003; Carlin et al., 2000). Spores from B. cereus can germinate and outgrow during storage, even at low temperatures (Carlin et al., 2000; Choma et al., 2000; Guinebretiere et al., 2003). To battle this increasing problem, major efforts focus on determining the causes of the spore’s resistance and the mechanisms of germination. There is ample literature on spores and sporulation of B. cereus, but for most sporulation studies that focus on genetics, derivatives of Bacillus subtilis 168 are used, due to their easy handling and genetic accessibility (Driks, 2002). B. subtilis has a much smaller genome (Kunst et al., 1997) than B. cereus, and contains no plasmids, while B. cereus strains are commonly known to harbor one or more plasmids (Helgason et al., 2000). Genome analysis indicates that B. cereus and B. subtilis have a completely different ecological background: whereas B. subtilis can be seen as a benign soil dweller specialized on degrading plant-derived polysaccharides, B. cereus seems to be adapted to thrive as a pathogen or parasite, preferring a more carnivorous diet of proteins and amino acids (Ivanova et al., 2003; Parkhill and Berry, 2003). Differences include important sporulation genes, for example in gerA type operons, the products of which are essential to nutrient and nonnutrient mediated germination of spores (Paidhungat et al., 2000; Wuytack et al., 2000). The B. subtilis genome contains 5 gerA type operons of which only 3 are expressed during sporulation (Paidhungat et al., 2000), while data mining of the B. cereus genome has revealed the presence of 7 gerA-like operons, which are all expressed during sporulation (L. M. Hornstra, Y. P. de Vries, M. H. Bennik, W. M. de Vos, and T. Abee, presented at Functional Genomics of Gram-Positive Microorganisms, 12th International Conference on Bacilli, Baveno, Italy, 22 to 27 June 2003). Furthermore, B. cereus spores are surrounded by an exosporium, whereas B. subtilis spores are not (Koshikawa et al., 1989; Todd et al., 2003). Thus, although many of the genes that play a role in sporulation are believed to be conserved among Bacilli (Eichenberger et al., 2003; Stragier, 2002), there may be important differences between B. cereus and B. subtilis sporulation genes and their expression. It has been well established that bacterial spore properties are affected by the conditions during sporulation (e.g. Atrih and Foster, 2001; Evans et al., 1997; Gonzalez et al., 1999; Raso et al., 1998), but in most studies spores are routinely produced from fortified agar or rich liquid media, which results in heterogeneous sporulation conditions for the individual cells. Homogeneous sporulation conditions and precise regulation of growth and sporulation parameters are of great importance for obtaining reproducible and homogeneous spore batches. Furthermore, the study of gene-expression during cell-differentiation requires homogeneous and synchronized cultures. In this study we describe defined conditions for growth and sporulation of B. cereus ATCC14579, which has been characterized at the genome level (Ivanova, 2003), using a chemically defined medium in combination with an airlift fermenter system. We monitored the expression of four genes coding for sigma factors involved in housekeeping, stress-response, and sporulation (σA, σB, σF, and σG) during growth and sporulation. Important properties of the resulting spores are established and discussed, and the temporal expression of the sigma factor genes is compared to the sporulation model developed for B. subtilis.

34

Growth and sporulation of Bacillus cereus under defined conditions

Materials and Methods Strains, medium and fermentation B. cereus strain ATCC14579 was obtained from the American Type Culture Collection and grown on a chemically defined medium modified from the work of Nakata (1964), which contained the following components (final concentrations): D-glucose, 10mM; L-glutamic acid, 20 mM; Lleucine, 6 mM; L-valine, 2.6 mM; L-threonine, 1.4 mM; L-methionine, 0.47 mM; L-histidine, 0.32 mM; D/L-lactic acid, 5 mM; acetic acid, 1 mM; FeCl3, 50 µM; CuCl2, 2.5 µM; ZnCl2, 12.5 µM; MnSO4, 66 µM; MgCl2, 1 mM; (NH4)2SO4, 5 mM; Na2MoO4, 2.5 µM; CoCl2, 2.5 µM; Ca(NO3)2, 1 mM. The medium was buffered at pH 7.2 with 100 mM potassium phosphate buffer. The fermenter system consisted of an autoclavable 2 liter glass bioreactor from Applikon (Schiedam, the Netherlands), equipped with an ADI 1020 biocontroller unit, an ADI 1012 motor controller and a ADI 1018 thermocirculator. Sterile air was fed through the culture at a constant rate while the oxygen concentration was kept at 50% saturation level by automatic adjustment of the stirring speed. For an indication of the oxygen consumption in the vessel, the relative oxygen consumption (RO) was calculated by dividing the stirring speed in rpm by the oxygen concentration measured in the vessel. Fermentations were carried out at a constant temperature of 30 °C. The fermenter was inoculated with 3-4.108 B. cereus spores that had been pasteurized at 75 °C for 15 minutes and subsequently germinated with a mixture of L-alanine and inosine (25 and 1 mM, resp.) in 10 mM potassium phosphate buffer at pH 8.0 for 5 minutes at 30 °C. Analytical procedures Concentrations of D- and L-lactic acid and glucose were determined in culture supernatant by the UV method with enzymatic bioanalysis kits from Boehringer Mannheim (Darmstadt, Germany). Phase-contrast microscopy was performed with an axioskop microscope from Zeiss, equipped with a coolpix 995 digital camera from Nikon. Amino acids were determined in culture supernatant with the PicoTaq HPLC method (Bidingmeyer et al., 1984). The amino acids and NH4+ from culture supernatant were derivatized according to manufacturer’s instructions with the AccQFluorTM kit from Waters (Etten-Leur, the Netherlands). DPA was measured with a fluorescent assay modified from Hindle and Hall (1999). Spores or cells were suspended in Tris-NaCl-Tw (Tris-HCl (10 mM, pH 7.5) buffer with 10 mM NaCl and 0.1% Tween80), destroyed by autoclaving (121 °C, 15 min), a treatment known to release all DPA (Janssen et al., 1958), and pelletted at maximum speed in an eppendorf centrifuge for 2 minutes. The supernatant was assayed for DPA as follows: 80 µl of supernatant was used in a final volume of 200 µl Tris-NaCl-Tw with freshly added TbCl3 at a final concentration of 100 µM, and the fluorescence was measured with the Safire microplate reader from Tecan (Salzburg, Austria) in combination with XFLUOR4 software version 4.40. Standard curves of 0-150 µM of DPA showed straight lines (R2 > 0.99) with this method, which had a detection limit of approx. 0.5 µM. The settings of the machine were as follows. Excitation wavelength: 272 nm; Emission wavelength: 547 nm; Bandwith excitation and emission wavelengths: 7.5 nm; Lag time 10 µs; Integration time 2000 µs. Spore handling and properties Spores were harvested through airlifting as the spores stabilized the foam formed in the top of the fermenter vessel. The foam containing the spores passed the fermenter air-outlet, and was led through 2 bottles in which it slowly collapsed. What remained was a highly concentrated suspension containing 90-99% phasebright spores, as monitored by phase-contrast microscopy. This suspension was centrifuged at 10,000 g for 30 min. The spores formed a solid pellet with a light creme color, on top of which in some cases a thin, loosely packed, dark brownish upper layer was present. This upper layer consisted of cell-debris, and was discarded while the remainder of the

35

Chapter 2

pellet was resuspended in sterile water. The spores were kept at room temperature and subjected to daily washes in water until there were no cell debris, vegetative cells or phase dark spores left (typically three to seven washings). Subsequently, the spores were suspended in 10 mM K-PO4 buffer at pH 7 and stored in the dark at 4 °C. The heat resistance assay was modified from the work of Kooiman (1974). Spores (106 – 107 per ml) were injected at regular time intervals in screwcap tubes containing 10 mM K-PO4 (pH 7) at 100 °C in an oilbath. After a set time, all tubes were cooled simultaneously on ice. Serial dilutions were made in 10 mM K-PO4 (pH 7) and plated onto diluted nutrient broth (2.6 g/l; Difco) solidified with 1.5 % agar. Colonies were counted after 3 days of incubation at 30 °C. Spore surface hydrophobicity was measured according to the method described by Rosenberg et al. (1980). Spores were suspended in water, and after measurement of the absorbance at 660 nm (A before; values at 0.4-0.5), 0.1 ml of n-hexadecane (Sigma Aldrich) was added to 2 ml of spore suspension in a glass tube. This mixture was vortexed for 1 min, after which the phases were allowed to separate for 15 min. Then, the absorbance of the aqueous phase was determined again (A after), and the % transfer to the n-hexadecane was deduced by calculating 100-[(A after/A before)*100]. The wet density of the whole spore was measured with a Percoll gradient according to the method described by Tisa et al. (1982). Spores were concentrated in 0.15 M NaCl, added to 90% (vol/vol) Percoll (Amersham Pharmacia Biotech) solution with 0.15 M NaCl, and subsequently centrifugated for 20 min at 30,000 g. Whole spore wet density was derived by comparison of the positions of the spores with those of density marker beads (Amersham Pharmacia Biotech) in the self-established gradient. The spore core density was determined with Nycodenz (Nygaard, Oslo, Norway) density gradients (Rickwood et al., 1982) according to Lindsay et al. (1985) as follows; Spores were permeabilised with a decoating solution containing 0.1 M DTT, 0.1 M NaCl and 0.5 % SDS according to Vary (1973). Permeabilised spores were equilibrated in 30 % Nycodenz (1.159 g/ml). Semi-continuous gradients were made by letting discontinuous gradients diffuse overnight at 5 °C (Rickwood et al., 1982) or for a few hours at room temperature. After centrifugation in a swingout rotor for 45 minutes at 3700 rpm in a Centaur 2 centrifuge, the spores formed a band at a certain position. The refractive index, which was linearly correlated with the density, from samples taken just above and below the band was measured with a precision refractometer, and the average yielded the spore core density. Core water content was calculated according to Lindsay et al. (1985). RNA isolation and real-time RT-PCR For RNA extraction we used the RNeasy kit from Qiagen. Cells from 2 ml of culture were resuspended in 0.6 ml RLT buffer provided in the RNeasy kit and lysed by bead-beating (3 minutes at 30 Hz) with 0.6 grams of zirkonia/silica beads (0.1 mm diameter) from Biospec (Bartlesville, Okla.). The lysate was centrifuged for 2 minutes at the highest speed in an Eppendorf centrifuge, and the supernatant was used in the protocol provided with the RNeasy kit. The purified RNA was treated with RNA-se free DNA-se (Amersham Pharmacia Biotech). In silico analyses were performed with the ERGO genome and discovery system (Overbeek, 2003) from Integrated Genomics (Chicago, Ill.). Appropriate primers (all 5’ - 3’) were designed for sigA, sigB, sigF and sigG (Table 1). One hundred twenty nanograms of total RNA of each sample was used for cDNAsynthesis with Superscript II (Invitrogen) according to manufacturer instructions, in a final volume of 20 µl. For real-time PCR we used the qPCR core kit for Sybr Green I - No ROX, from Eurogentec. Amplification reactions contained 2 µl of cDNA template and 10 pmol of each primer (R + F) in a final volume of 50 µl. In deviation from the protocol delivered with the qPCR core kit, 0.5µl 1,000-fold-diluted Sybr green I solution from Molecular Probes was used instead of the Sybr

36

Growth and sporulation of Bacillus cereus under defined conditions

green from the qPCR core kit. The amplification profile was 30s at 94 °C, 30s at 58 °C and 60s at 72 °C for σA, σF, and σG. For σB the temperature of step 2 was lowered from 58 °C to 55 °C. PCR products were detected directly by monitoring the increase in fluorescence caused by Sybr Green I intercalation using the iCycler system (Biorad Laboratories). A threshold was set, which intersected the amplification curves in the linear region of the semi-log plot. The original amounts of cDNA in unknown samples are compared to each-other by interpolation from a standard curve of CT (threshold crossing) values generated from different concentrations (10, 1, 0.1 and 0.01 ng/µl) of genomic DNA, which was isolated from exponentially growing B. cereus cells according to Pospiech and Neumann (1995). The sigma factor expression level was calculated according to the formula Expression level = a*eb*Ct, in which a and b are constants dependent on the primer-set and amplification parameters, experimentally derived from the standard curve obtained from amplification reactions with the different genomic DNA concentrations as a template. Ct is the threshold crossing value found in the amplification reaction with the specific primer-set, reaction parameters and cDNA from the specific time-point as a template. All reactions were carried out in duplicate, and the Ct values of the duplicates differed in all cases less than 5 %. All of the amplified fragments used to generate datapoints had melt-curves identical to the positive controls and formed single bands of the expected size on agarose gel, confirming that the rise in fluorescence measured by the iCycler system was caused by amplification of only the specific gene fragments we targeted. Table 1. Sigma factor genes from B. subtilis and B. cereus with ERGO database open reading frame (ORF) numbers, their similarity based on amino acid sequence, and the primers used in this study. Sigma ORF number Similarity Identity % Primers (5’ - 3’) factor B. subtilis B. cereus % F: CTATGTAGGCCGTTGGTATGCCT 2514 856 93 88.5 σA R: AGGCGATGTTGCTTCTTGGTCTT F: GAAATCGCAAATCATTTAGG 473 5124 73 52.5 σB R: CTTTTAATACGAGAAACGTG F: GGATGTATTGGGCTCTTGAAATCGG 2341 2311 91 79.8 σF R: CGGCTCGCTTCTTGTGCTAGAACAAC F: GCAAAGTGGAGAGATAAGCGCAAGAG 1534 532 94 89.2 σG R: TACGCGAATCGGATTATTATCACGC

Results and Discussion Growth and substrate utilization A major advantage of a chemically defined growth medium over rich media is the possibility to monitor exactly which substrates are used, to what degree and at what growth-stage (Hageman et al., 1984). To place the temporal expression of genes in a context with substrate availability and growth-stage, we started out with determining the growth characteristics and the substrate utilization of B. cereus on the defined medium. The growth and pH development (Fig. 1A) were roughly similar to those previously described (Kennedy et al., 1971; Nakata, 1963; Nakata and Halvorson, 1960). However, we could clearly distinguish five different growth phases, starting with a lag phase (phase 1). In the exponential phase (phase 2, Fig. 1) cells occurred singly or in short chains and were highly motile. During this phase, the pH of the medium dropped from 7.2 to 7.0, but started to rise again as soon as the glucose was depleted (phase 3, Fig. 1B). This indicates that the pH drop resulted from fermentation of glucose, although oxygen was present at a 50% saturation level and was consumed actively by the growing bacteria (Fig. 1A). For respiration of glycolysis products, an active TCA cycle is required, and glucose represses TCA cycle activity (Hederstedt, 1993). Therefore, fermentation products will accumulate as long as glucose is present. Homolactic acid fermention did not occur, because the lactic acid concentration did not increase.

37

Chapter 2

However, B. cereus is able to ferment glucose to a variety of other acids, such as pyruvic acid and acetic acid (Nakata and Halvorson, 1960; Wang and Wang, 2002). Furthermore, lactic acid was not metabolized by the bacteria as long as glucose was present (Fig. 1B phase 1 and 2), presumably due to catabolite repression and repression of the TCA cycle by glucose. Amino acid metabolism was not repressed by glucose. Amino acids were metabolized rapidly in phases 2 and 3, in a specific order: Threonine first, then valine, histidine, leucine, methionine, and glutamate as the last (Fig. 2B).

Fig. 1. Growth and sporulation of B. cereus in a defined medium. A. Absorbance at 600 nm (closed diamonds); pH (closed squares); and relative oxygen consumption (open circles; calculated as described in the methods section). B. Carbon sources in culture supernatant and spore formation. Glucose (closed circles); D-lactic acid (closed triangles down); L-lactic acid (open circles); The % of cells with a phasebright spore (gray bars); DPA concentration (closed squares). C. Relative expression levels of genes encoding key sigma factors (calculated as described in the methods section). SigA (closed circles); SigB (open circles); SigF(closed triangles down); SigG (open triangles down). The vertical broken lines divide the graphs into 5 developmental phases: lag-phase (1), exponential (2), early stationary (3), late stationary, early sporulation (4), late sporulation (5).

38

Growth and sporulation of Bacillus cereus under defined conditions

The amino acids added in the lowest concentrations were not necessarily depleted first (compare Fig. 2B and C). Metabolism of amino acids resulted in an increase of NH4+ in the medium. The amount of NH4+ released was half of the total amount of nitrogen in the amino acids consumed (Fig. 2A), indicating that only half of the nitrogen was accumulated into biomass.

Fig. 2. Amino acids and ammonia in culture supernatant. A. Absorbance at 600 nm (closed circles); Total nitrogen in amino acids (closed squares); Glutamate (closed diamonds); Ammonia (closed triangles up); Nitrogen in amino acids other than glutamate (closed triangles down). B. Relative amounts of amino acids in culture supernatant; Glutamate (open circles); Threonine (closed circles); Leucine (open triangles up); Methionine (closed triangles up); Valine (open squares); Histidine (closed squares). C. Absolute amounts of amino acids in culture supernatant. Symbols: see B.

39

Chapter 2

Upon glucose exhaustion the cells entered stationary phase (phase 3), lost their motility, and became arranged into aggregates (Fig. 3A), while lactic acid was metabolized and the oxygen consumption reached its maximum (Fig. 1A and B). Cell aggregation in B. cereus is a specific event (Wise and Frazer, 1972) and may play a role in the transfer of genetic material, which occurs frequently within the B. cereus group (Helgason et al., 2000). B. cereus metabolized both D- and Lisomers of lactic acid with a slight preference for the L-isomer (Fig. 1B). Lactic acid metabolism and amino acid metabolism resulted in a dramatic increase of the pH. The burst in oxygen demand is indicative of the intense metabolic activity that is needed for spore formation (Halvorson, 1957). At the end of the late stationary, early sporulation phase (phase 4) the first phase-gray spores could be seen in the aggregated cells. The number of phase-bright spores increased suddenly and dramatically in the final sporulation phase, phase 5 (Fig. 3B), and at the same time rapid production of dipicolinic acid took place (Fig. 1B). The bulk of the glutamate was used during these two final phases. This depletion of glutamate was not accompanied by NH4+ release (Fig. 2A), indicating that glutamate served as a nitrogen source during sporulation. During the final phase, the absorbance increased further, and the spore-specific compound dipicolinic acid (DPA) was rapidly formed in the culture. DPA formation coincided with phase-brightening of the fore-spores, and in the end over 99 % of the cells had a phasebright spore (Fig. 1B). Finally, the aggregates of cells fell apart after which the cells lysed and the spores were released into the medium. Spore purification was facilitated by the high degree of sporulation, because almost no vegetative cells were left in the spore suspensions. Furthermore, the cells formed spores at the same time, in phase 5, confirming synchronicity in the culture. After release from the sporangia, the spores aggregated in large amounts of foam that formed in the fermenter and were airlifted out of the fermenter vessel. We used this property to harvest the spores without the need to centrifuge all the culture liquid. The foam was collected, and microscopic observation confirmed that it consisted almost exclusively of phase-bright spores (Fig. 3C). The total number of spores produced in the culture was approximately 1,2.1012 from which 1 – 2.1011 spores (10-20%) were routinely obtained in highly pure suspensions (>99.9 % phase-bright spores, no debris, vegetative or sporulating cells as determined by phase-contrast microscopy).

Fig. 3. Microscopic images of B. cereus cells. Bars indicate 5 µm. Vegetative cells aggregating in stationary phase (A), aggregated cells forming spores (B), spores present in foam (C).

Spore properties We tested the mature spores for a number of parameters that are important in relation to food processing, such as wet heat resistance and hydrophobicity. Spore heat-inactivation curves had a shoulder and a tail, which could be caused by clumping of the spores because of their hydrophobic surface. However, addition of 0.1 % TWEEN 80, a powerful nonionic surfactant that can prevent protein aggregation at elevated temperatures (Arakawa and Kita, 2000), did not change the shape of the curves. We derived the decimal reduction time from the slope of the middle section of the inactivation curves, which yielded a reproducible D-value of 1 minute at 100 °C, in line with previous reports (Algie, 1983).

40

Growth and sporulation of Bacillus cereus under defined conditions

Spore surface hydrophobicity is caused by the presence of an exosporium (Koshikawa et al., 1989) and plays a key role in adherence of the spores to surfaces (Charlton et al., 1999; Wiencek et al., 1990 and references therein), for example of food or food processing equipment (Faille et al., 2002). The obtained B. cereus spores were highly hydrophobic since over 95% of the spores transferred from the water to the n-hexadecane phase in the BATH assay (Rosenberg et al., 1980). In contrast, of B. cereus vegetative cells and spores from B. subtilis, which lack an exosporium, over 98% remained in the water phase (Fig. 4). This is consistent with previous reports (Koshikawa et al., 1989; Wiencek et al., 1990). We measured the whole spore density to investigate possible heterogeneity in the spore batches using self-establishing Percoll gradients. The spores were of uniform density. Dormant spores formed one single band at 1.13 g/ml, comparable to what was previously reported (Tisa et al., 1982). Autoclaved and germinated spores settled between 1.062 and 1.075 g/ml, and cell-debris typically at 1.049 g/ml. Note that these values reflect the density of the whole spore; for determination of the core density we used semi-continuous Nycodenz gradients. In these gradients the spores again settled in a single band, at a density of 1.3240 ± 0.0012 g/ml. From the core density, the core water content can be derived, which is believed to play an important role in spore heat resistance (Melly et al., 2002; Popham et al., 1995; Setlow, 2000). The B. cereus spores obtained in this study had a core-water content of 53.5 %, in concordance with previous reports (Lindsay et al., 1985). Furthermore, the spores were stable in time (more than 3 years), and germinated readily upon addition of alanine and/or inosine. In conclusion, this method of spore production is excellent for production of large, pure and homogeneous spore-batches that are stable over time and suitable for detailed studies. Fig. 4. Partitioning in a hexadecane-water system as a measure of hydrophobicity. A. B. cereus spores; B. B. cereus vegetative cells; C. B. subtilis spores.

A

B

C

0%

20%

40%

60%

n-hexadecane

80%

100%

water

Gene expression Sporulation in B. subtilis is orchestrated through the compartmentalized action of four sigma factors σF, σE, σG, and σK (Piggot and Losick, 2002). Sigma factor σA is the primary sigma factor in B. subtilis, regulating macromolecular synthesis (MMS) and playing a key role in the housekeeping functions of the cell. The alternative sigma factor σB plays a role in general stress response including entry into stationary phase (Helmann and Moran, 2002). The B. cereus genome is predicted to contain a single homologue to B. subtilis sigA, sigB, sigF and sigG (Table 1). We used

41

Chapter 2

quantitative PCR to measure sigF, sigG, sigA and sigB expression during the various growth phases in B. cereus. With quantitative PCR the amount of product after each amplification cycle is measured. The obtained data were normalized with a dilution series of genomic DNA. In this way, efficiencies of the specific primer annealing and amplification reactions are taken into account, which allows quantitative comparison of the sigA, sigB, sigF and sigG transcripts. During phase 1 and the first half of phase 2 the cell density was too low for efficient harvesting. Halfway through phase 2, when the absorbance reached 0.2, the first cell samples were taken and transcripts of sigA, sigB and sigF were detected (Fig. 1C). Phase 2 was characterized by a high expression of sigA, low expression of sigB and sigF, and absence of sigG transcripts. The sigA transcript was most abundant, while the sigB and sigF expression was 100- and 20-fold lower, respectively. The high expression of sigA, which continues through phase 3 and 4 (see below), confirms its anticipated pivotal role in all cellular processes. The detection of small amounts of sigB and sigF transcript during exponential growth may be explained by either some heterogeneity or low expression by the majority of the cells. However, at the end of phase 2, upon entry into early stationary phase (phase 3), transcription of sigB and sigF increased markedly (see below), to 8- and 30-fold compared to initial values, respectively, while sigA expression remained essentially constant. This indicates that the vast majority of the cells enter stationary phase at the same time, confirming synchronicity of the culture. Phase 3, the early stationary phase, was characterized by peaks of sigB and sigF expression, while sigA transcription remained high and the first sigG transcripts were detected at a low abundance. We detected a peak in sigB expression early in phase 3, at the time of glucose exhaustion; subsequently lactate was metabolized (Fig. 1B). In B. subtilis, σB is expressed upon entry in stationary phase (Helmann and Moran, 2002) and when the cell encounters stress, directly affecting cell- and carbon metabolism (Price, 2002). Our findings fit this idea, although of the 3 lactate dehydrogenase genes in the B. cereus genome none contain a σB consensus sequence, and none are upregulated when σB is activated (Willem van Schaik, pers. comm.). This suggests that, in B. cereus, if σB influences carbon metabolism, it does so indirectly. SigF transcription was upregulated parallel to sigB transcription, but to a higher level (30-fold, Fig. 1C) and showing a broader peak that lasted through phases 3 and 4. This is well before the first irreversible step of sporulation, as observed in B. subtilis (Driks, 1999). Halfway phase 3, sigG transcripts could be detected for the first time (Fig. 1C). Initial sigG transcription intensity was, like sigB and sigF transcription, much lower than that of sigA, and remained low during phase 3. Sigma factor σG plays a role in the final stages of sporulation. It is expressed in the forespore and regulates synthesis of SASP and Ger proteins (Helmann and Moran, 2002). Phase 4, the late stationary and early sporulation phase, was characterized by a significant increase (140-fold) of sigG expression. In B. subtilis σG is, just like σF, held inactive until the appropriate moment, while sigG transcription is, in addition to stimulation by σF, stimulated by active σG (Piggot and Losick, 2002). Indeed, upstream of the B. cereus sigG we found a clear σG consensus sequence (data not shown) that fits with those previously described (Barlass et al., 2002). This, in combination with the sharp peak of sigG expression, shows that in B. cereus auto stimulation of sigG probably takes place. Interestingly, sigF transcription remained high in phase 4 after its peak in phase 3. This suggests that while σG was already active, sigF was still transcribed, or, more likely, that the sigF transcript was quite stable in time. In phase 5 the bulk of the spores acquired a phase-bright appearance, soon after the peak in sigG expression. This indicates forespore core dehydration in response to σG activity, like in B. subtilis (Driks, 1999). The oxygen consumption (Fig. 1A) decreased dramatically, confirming that metabolic activity significantly decreased, while as anticipated, the transcripts of sigA, sigB, sigF and sigG decreased to low levels.

42

Growth and sporulation of Bacillus cereus under defined conditions

Conclusions In conclusion, we have developed an easy and efficient way of producing synchronized and homogeneous B. cereus spore batches. The chemically defined medium in combination with the fermenter system allows precise monitoring and manipulation of key growth- and sporulation parameters, and results in synchronous growth and sporulation, which facilitates gene-expression studies. The kinetics of expression of sigA, sigB, sigF and sigG follow the model developed for B. subtilis, underscoring the conservation of sporulation mechanisms among bacilli. Future studies will focus on gene-expression differences during growth and sporulation with different sets of parameters, for example, pH and carbon source. Acknowledgements We thank Dr. Willem van Schaik for providing the primers for sigB. References 1. 2. 3. 4. 5. 6.

7. 8. 9. 10. 11. 12.

Algie, J. E. 1983. The heat resistance of bacterial spores and its relationship to the contraction of the forespore protoplasm during sporulation. Curr Microbiol 9:173-176. Arakawa, T., and Y. Kita. 2000. Protection of bovine serum albumin from aggregation by Tween 80. J Pharm Sci 89:646-651. Atrih, A., and S. J. Foster. 2001. Analysis of the role of bacterial endospore cortex structure in resistance properties and demonstration of its conservation amongst species. J Appl Microbiol 91:364-372. Barlass, P. J., C. W. Houston, M. O. Clements, and A. Moir. 2002. Germination of Bacillus cereus spores in response to L-alanine and to inosine: the roles of gerL and gerQ operons. Microbiology 148:2089-2095. Bidingmeyer, B. A., S. A. Cohen, and T. L. Tarvin. 1984. Rapid analysis of amino acids using pre-column derivatization. J Chromat 336:93-104. Carlin, F., H. Girardin, M. W. Peck, S. C. Stringer, G. C. Barker, A. Martinez, A. Fernandez, P. Fernandez, W. M. Waites, S. Movahedi, F. van Leusden, M. Nauta, R. Moezelaar, M. D. Torre, and S. Litman. 2000. Research on factors allowing a risk assessment of spore-forming pathogenic bacteria in cooked chilled foods containing vegetables: a FAIR collaborative project. Int J Food Microbiol 60:117-135. Charlton, S., A. J. Moir, L. Baillie, and A. Moir. 1999. Characterization of the exosporium of Bacillus cereus. J Appl Microbiol 87:241-245. Choma, C., M. H. Guinebretiere, F. Carlin, P. Schmitt, P. Velge, P. E. Granum, and C. Nguyen-The. 2000. Prevalence, characterization and growth of Bacillus cereus in commercial cooked chilled foods containing vegetables. J Appl Microbiol 88:617-625. Christiansson, A., J. Bertilsson, and B. Svensson. 1999. Bacillus cereus spores in raw milk: factors affecting the contamination of milk during the grazing period. J Dairy Sci 82:305-314. Driks, A. 1999. Bacillus subtilis spore coat. Microbiol Mol Biol Rev 63:1-20. Driks, A. 2002. Maximum shields: the assembly and function of the bacterial spore coat. Trends Microbiol 10:251-254. Eichenberger, P., S. T. Jensen, E. M. Conlon, C. van Ooij, J. Silvaggi, J. E. GonzalezPastor, M. Fujita, S. Ben-Yehuda, P. Stragier, J. S. Liu, and R. Losick. 2003. The sigmaE regulon and the identification of additional sporulation genes in Bacillus subtilis. J Mol Biol 327:945-972.

43

Chapter 2

13. 14. 15. 16. 17.

18.

19. 20. 21. 22. 23. 24. 25.

26. 27. 28. 29.

44

Evans, R. I., N. J. Russell, G. W. Gould, and P. J. McClure. 1997. The germinability of spores of a psychrotolerant, non-proteolytic strain of Clostridium botulinum is influenced by their formation and storage temperature. J Appl Microbiol 83:273-280. Faille, C., C. Jullien, F. Fontaine, M. N. Bellon-Fontaine, C. Slomianny, and T. Benezech. 2002. Adhesion of Bacillus spores and Escherichia coli cells to inert surfaces: role of surface hydrophobicity. Can J Microbiol 48:728-738. Gonzalez, I., M. Lopez, S. Martinez, A. Bernardo, and J. Gonzalez. 1999. Thermal inactivation of Bacillus cereus spores formed at different temperatures. Int J Food Microbiol 51:81-84. Granum, P. E. 1994. Bacillus cereus and its toxins. Soc. Appl. Bacteriol. Symp. Ser. 23:61S-66S. Guinebretiere, M. H., H. Girardin, C. Dargaignaratz, F. Carlin, and C. Nguyen-The. 2003. Contamination flows of Bacillus cereus and spore-forming aerobic bacteria in a cooked, pasteurized and chilled zucchini puree processing line. Int J Food Microbiol 82:223-232. Hageman, J. H., G. W. Shankweiler, P. R. Wall, K. Franich, G. W. McCowan, S. M. Cauble, J. Grajeda, and C. Quinones. 1984. Single, chemically defined sporulation medium for Bacillus subtilis: growth, sporulation, and extracellular protease production. J Bacteriol 160:438-41. Halvorson, H. O. 1957. Rapid and simultaneous sporulation. J Appl Bacteriol 20:305-314. Hederstedt, L. 1993. p. 181-197. In A. L. Sonenshein, J. A. Hoch, and R. Losick (ed.), Bacillus subtilis and Other Gram-Positive Bacteria: Biochemistry, Physiology and Molecular Genetics. ASM, Washington, DC. Helgason, E., O. A. Okstad, D. A. Caugant, H. A. Johansen, A. Fouet, M. Mock, I. Hegna, and Kolsto. 2000. Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis-one species on the basis of genetic evidence. Appl Environ Microbiol 66:2627-2630. Helmann, J. D., and C. P. Moran, Jr. 2002. RNA polymerase and sigmafactors, p. 289313. In A. L. Sonenshein, J. A. Hoch, and R. Losick (ed.), Bacillus subtilis and its closest relatives: from genes to cells. ASM Press, Washington D. C. Hindle, A. A., and E. A. H. Hall. 1999. Dipicolinic acid (DPA) assay revisited and appraised for spore detection. Analyst 124:1599-1604. Hornstra, L. M., Y. P. d. Vries, M. H. Bennik, W. M. d. Vos, and T. Abee. 2003. Presented at the Functional genomics of gram-positive microorganisms, Baveno, Italy. Ivanova, N., A. Sorokin, I. Anderson, N. Galleron, B. Candelon, V. Kapatral, A. Bhattacharyya, G. Reznik, N. Mikhailova, A. Lapidus, L. Chu, M. Mazur, E. Goltsman, N. Larsen, M. D'Souza, T. Walunas, Y. Grechkin, G. Pusch, R. Haselkorn, M. Fonstein, S. D. Ehrlich, R. Overbeek, and N. Kyrpides. 2003. Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis. Nature 423:87-91. Janssen, F. W., A. J. Lund, and L. E. Anderson. 1958. Colorimetric assay for dipicolinic acid in bacterial spores. Science 127:26-27. Kennedy, R. S., F. J. Malveaux, and J. J. Cooney. 1971. Effects of glutamic acid on sporulation of Bacillus cereus and on spore properties. Can J Microbiol 17:511-519. Kooiman, W. J. 1974. The screw-cap tube technique: a new and accurate technique for the determination of the wet heat resistance of bacterial spores, p. 87-92. In A. N. Barker, G. W. Gould, and J. Wolf (ed.), Spore Research 1973. Academic Press, London. Koshikawa, T., M. Yamazaki, M. Yoshimi, S. Ogawa, A. Yamada, K. Watabe, and M. Torii. 1989. Surface hydrophobicity of spores of Bacillus spp. J Gen Microbiol 135 ( Pt 10):2717-2722.

Growth and sporulation of Bacillus cereus under defined conditions

30.

31. 32. 33. 34. 35. 36. 37.

38. 39. 40. 41. 42. 43. 44. 45. 46. 47.

Kunst, F., N. Ogasawara, I. Moszer, A. M. Albertini, G. Alloni, V. Azevedo, M. G. Bertero, P. Bessieres, A. Bolotin, S. Borchert, R. Borriss, L. Boursier, A. Brans, M. Braun, S. C. Brignell, S. Bron, S. Brouillet, C. V. Bruschi, B. Caldwell, V. Capuano, N. M. Carter, S. K. Choi, J. J. Codani, I. F. Connerton, A. Danchin, and et al. 1997. The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390:249-256. Lindsay, J. A., T. C. Beaman, and P. Gerhardt. 1985. Protoplast water content of bacterial spores determined by buoyant density sedimentation. J Bacteriol 163:735-737. Lund, T., M. L. De Buyser, and P. E. Granum. 2000. A new cytotoxin from Bacillus cereus that may cause necrotic enteritis. Mol Microbiol 38:254-261. Melly, E., P. C. Genest, M. E. Gilmore, S. Little, D. L. Popham, A. Driks, and P. Setlow. 2002. Analysis of the properties of spores of Bacillus subtilis prepared at different temperatures. J Appl Microbiol 92:1105-1115. Nakata, H. M. 1963. Effect of pH on intermediates produced during growth and sporulation of Bacillus cereus. J Bacteriol 86:577-581. Nakata, H. M. 1964. Organic nutrients required for growth and sporulation of Bacillus cereus. J Bacteriol 88:1522-1524. Nakata, H. M., and H. O. Halvorson. 1960. Biochemical changes occurring during growth and sporulation of Bacillus cereus. J Bacteriol 80:801-810. Overbeek, R., N. Larsen, T. Walunas, M. D'Souza, G. Pusch, E. Selkov, Jr., K. Liolios, V. Joukov, D. Kaznadzey, I. Anderson, A. Bhattacharyya, H. Burd, W. Gardner, P. Hanke, V. Kapatral, N. Mikhailova, O. Vasieva, A. Osterman, V. Vonstein, M. Fonstein, N. Ivanova, and N. Kyrpides. 2003. The ERGO genome analysis and discovery system. Nucleic Acids Res 31:164-171. Paidhungat, M., and P. Setlow. 2000. Role of ger proteins in nutrient and nonnutrient triggering of spore germination in Bacillus subtilis. J. Bacteriol. 182:2513-2519. Parkhill, J., and C. Berry. 2003. Genomics: Relative pathogenic values. Nature 423:23-25. Piggot, P. J., and R. Losick. 2002. Sporulation Genes and Intercompartmental Regulation, p. 483-519. In A. L. Sonenshein, J. A. Hoch, and R. Losick (ed.), Bacillus subtilis and its closest relatives: from genes to cells. ASM Press, Washington D. C. Popham, D. L., S. Sengupta, and P. Setlow. 1995. Heat, hydrogen peroxide, and UV resistance of Bacillus subtilis spores with increased core water content and with or without major DNA-binding proteins. Appl Environ Microbiol 61:3633-3638. Pospiech, A., and B. Neumann. 1995. A versatile quick-prep of genomic DNA from grampositive bacteria. Trends Genet. 11:217-218. Price, C. W. 2002. General stress response, p. 369-385. In A. L. Sonenshein, J. A. Hoch, and R. Losick (ed.), Bacillus subtilis and its closest relatives: from genes to cells. ASM Press, Washington D. C. Raso, J., M. M. Gongora-Nieto, G. V. Barbosa-Canovas, and B. G. Swanson. 1998. Influence of several environmental factors on the initiation of germination and inactivation of Bacillus cereus by high hydrostatic pressure. Int J Food Microbiol 44:125-132. Rickwood, D., T. Ford, and J. Graham. 1982. Nycodenz: a new nonionic iodinated gradient medium. Anal Biochem 123:23-31. Roberts, T. A., and J. A. Thomas. 1982. Germination and outgrowth of single spores of Clostridium botulinum and putrefactive anaerobes. J Appl Bacteriol 53:317-321. Rosenberg, M., D. Gutnick, and E. Rosenberg. 1980. Adherence of bacteria to hydrocarbons: a simple method for measuring cell-surface hydrophobicity. FEMS Microbiol Lett 9:29-33.

45

Chapter 2

48. 49. 50. 51. 52. 53. 54. 55. 56. 57.

46

Sarrías, J. A., M. Valero, and M. C. Salmerón. 2002. Enumeration, isolation and characterization of Bacillus cereus strains from Spanish raw rice. Food Microbiol 19:589595. Setlow, P. 2000. Resistance of bacterial spores, p. 217-230. In G. Storz and R. HenggeAronis (ed.), Bacterial stress responses. ASM press, Washington, D.C. Stragier, P. 2002. A gene odyssey: exploring the genomes of endospore-forming bacteria, p. 519-527. In A. L. Sonenshein, J. A. Hoch, and R. Losick (ed.), Bacillus subtilis and its closest relatives: from genes to cells. ASM Press, Washington D. C. Tisa, L. S., T. Koshikawa, and P. Gerhardt. 1982. Wet and dry bacterial spore densities determined by buoyant sedimentation. Appl Environ Microbiol 43:1307-1310. Todd, S. J., A. J. Moir, M. J. Johnson, and A. Moir. 2003. Genes of Bacillus cereus and Bacillus anthracis encoding proteins of the exosporium. J Bacteriol 185:3373-3378. Vary, J. C. 1973. Germination of Bacillus megaterium spores after various extraction procedures. J Bacteriol 116:797-802. Wang, J., and H. Wang. 2002. Fermentation products and carbon balance of spoilage Bacillus cereus. J Food Drug Anal 10:64-68. Wiencek, K. M., N. A. Klapes, and P. M. Foegeding. 1990. Hydrophobicity of Bacillus and Clostridium spores. Appl Environ Microbiol 56:2600-2605. Wise, J. A., and D. K. Fraser. 1972. Developmental stages during growth and sporulation of Bacillus cereus, p. 203-212. In H. O. Halvorson, R. Hanson, and L. L. Campbell (ed.), Spores, vol. 5. ASM, Fontana, Wisconsin. Wuytack, E. Y., J. Soons, F. Poschet, and C. W. Michiels. 2000. Comparative study of pressure- and nutrient-induced germination of Bacillus subtilis spores. Appl Environ Microbiol 66:257-261.

3. Influence of Glutamate on Growth, Sporulation and Spore Properties of Bacillus cereus ATCC14579 in a Defined Medium Ynte P. de Vries, Ratna D. Atmadja, Luc M. Hornstra, Willem M. de Vos, and Tjakko Abee. Excerpt published in Applied and Environmental Microbiology, 2005, 71:3248-3254

ABSTRACT A chemically defined medium in combination with an airlift fermenter system was used to study growth and sporulation of Bacillus cereus ATCC14579. The medium contained six amino acids and lactate as the main carbon sources. The amino acids were depleted during exponential growth, while lactate was metabolized mainly during stationary phase. Two concentrations of glutamate were used: high (20 mM, YLHG) and low (2.5 mM, YLLG). Under both conditions, sporulation was complete and synchronous. Sporulation started and was completed while significant amounts of carbon and nitrogen sources were still present in the medium, indicating that starvation was not the trigger for sporulation. Analysis of amino acids and NH4+ in the culture supernatant showed that most of the nitrogen assimilated by the bacteria was taken up during sporulation. The consumption of glutamate depended on the initial concentration; in YLLG, all of the glutamate was used early during exponential growth, while in YLHG, almost all of the glutamate was used during sporulation. In YLLG, but not in YLHG, NH4+ was taken up by the cells during sporulation. The total amount of nitrogen used by the bacteria in YLLG was lower than that used by the bacteria in YLHG, although a significant amount of NH4+ was present in the medium throughout sporulation. Despite these differences, growth and temporal expression of key sigma factors involved in sporulation were parallel, indicating that the genetic time frames of sporulation were similar under both conditions. Nevertheless, in YLHG dipicolinic acid production started later and the spores were released from the mother cells much later than in YLLG. Notably, spores had a higher heat resistance when obtained after growth in YLHG than when obtained after growth in YLLG, and the spores germinated more rapidly and completely in response to inosine, L-alanine, and a combination of these two germinants.

47

Chapter 3

Introduction It has been well established that bacterial spore properties are affected by the conditions during sporulation. In most studies, spores are routinely produced from fortified agar or rich liquid media, which results in heterogeneous sporulation conditions for the individual cells. This prevents careful analysis of the metabolism during growth and sporulation. Recent studies describing the effect of sporulation conditions on spore properties involved modulation of sporulation temperature (Atrih and Foster 2001; Evans et al., 1997; Gonzalez et al., 1999; Melly et al., 2002; Raso et al., 1998; 1998b) or compared spores produced from different media (Cazemier et al., 2001; Mazas et al., 1995). Studies employing defined conditions and media to link specific substrate use with sporogenesis are relatively rare; moreover, the effect of carbon sources on sporulation has not been studied systematically in recent decades. The most common carbon source used in sporulation media is glucose, but in natural conditions B. cereus may encounter rather different substrates, such as lactate. We have previously shown that B. cereus is able to metabolize lactate (de Vries et al., 2004), which is formed in the natural environment by fermentation of a variety of naturally occurring polymers, such as lactose in dairy or plant sugars in silage. Indeed, silage is a known source of B. cereus contamination of milk (te Giffel et al., 2002). Furthermore, in contrast to glucose, lactate does not cause catabolite repression or repression of the TCA cycle. An active TCA cycle is required for activation of the Spo0A phosphorelay, which leads to sporulation (Ireton et al., 1995), and a defect in the TCA cycle blocks sporulation in B. subtilis (Jin et al., 1997). Therefore, we investigated the growth and sporulation of B. cereus ATCC14579, which has been characterized at the genome level (Ivanova et al., 2003), on a chemically defined medium with lactate instead of glucose as the main carbon source. Secondly, because glutamate has been reported to have a large impact on sporulation as well as spore properties in bacilli (Buono et al., 1966; Charba and Nakata, 1977; Kennedy et al., 1971), we used two different concentrations of glutamate: low (2.5 mM; YLLG) and high (20 mM; YLHG). Materials and Methods Strains, medium and fermentation B. cereus strain ATCC14579 was obtained from the American Type Culture Collection and used throughout this study. It was cultivated in the chemically defined medium described in Chapter 2, with modifications as indicated in Table 1. Fermentation conditions and inoculation were as described in Chapter 2. The spores were harvested and washed as described in Chapter 2, and stored in 10 mM K-PO4, pH 7, with 0.1 % Tween80 to prevent clumping and attachment of spores to plastic laboratory equipment. The addition of Tween80 had no effect on spore heat resistance. Analytical procedures Concentrations of D- and L-lactic acid and glucose were determined in culture supernatant by the UV method with enzymatic bioanalysis kits from Boehringer Mannheim (Darmstadt, Germany). Phasecontrast microscopy was performed with an axioskop microscope from Zeiss. Amino acids were determined in culture supernatant, cells and spores with the PicoTaq HPLC method as described in Chapter 2. For cell and spore analysis, samples were autoclaved and acidhydrolyzed prior to derivatization. The cultures were assayed for DPA as in Chapter 2. Spore properties For the heat resistance assay, 100µl spores suspended at an A600 of 0.7-1.0 in Tris-NaCl-Tw buffer (Tris-HCl (10 mM, pH 7.5) buffer with 10 mM NaCl and 0.1% Tween80), were sealed in the tip of a Pasteur pipette, placed in an oil bath calibrated at the designated temperature and cooled

48

Influence of glutamate on growth, sporulation and spore properties of Bacillus cereus

after a set time in ice-cold water. Because of the small diameter of the Pasteur pipette tips and the small volume of spore suspension, we assume that the heating and cooling of the spore-suspensions was instantaneous. Serial dilutions were made in Tris-NaCl-Tw buffer and plated onto diluted nutrient broth (2.6 g/l; Difco) solidified with 1.5% agar. Colonies were counted after overnight incubation at 30°C. Whole spore and spore core density were determined as in Chapter 2. Spore germination was measured by the drop in A600 of spore suspensions at 30 °C, using a Safire microplate reader from Tecan (Salzburg, Austria) in combination with XFLUOR4 software version 4.40. Spores were suspended at an A600 of 0.7-1.0 in Tris-NaCl-Tw buffer and after addition of the germinants, the A600 was followed automatically with intermittent shaking to prevent settling of the spores. Table 1. Medium composition of YLHG and YLLG

Components D/L – lactic acid (mM) L-glutamic acid (mM) L-leucine (mM) L-valine (mM) L-threonine (mM) L-methionine (mM) L-histidine (mM) Acetic acid (mM) FeCl3 (µM) CuCl2 (µM) ZnCl2 (µM) MnSO4 (µM) MgCl2 (mM) (NH4)2SO4 (mM) Na2MoO4 (µM) CoCl2 (µM) Ca(NO3)2 (mM)

Concentration YLHG 25 20 6 2.6 1.4 0.47 0.32 1 50 2.5 12.5 66 1 5 2.5 2.5 1

YLLG 25 2.5 6 2.6 1.4 0.47 0.32 1 50 2.5 12.5 66 1 5 2.5 2.5 1

RT-PCR

Real-time RT-PCR was used to monitor the expression of the genes for key sigma factors (sigA, sigB, sigF and sigG), and performed as described in Chapter 2. The level of expression was calibrated using the positive control, sigA expression, as a reference. All the samples in this communication were handled in exactly the same way, to enable a good comparison between the gene expression in the two conditions tested. QPCR reactions were carried out in duplicate, and the duplicates differed less than 5 % in all cases. Results and Discussion Carbon and nitrogen usage during growth and sporulation B. cereus was able to grow and sporulate in the chemically defined medium devoid of glucose. With lactate as the main carbon source, the exponential growth phases of B. cereus with low and high glutamate, YLLG and YLHG, respectively, were identical (Fig. 1A). The A600 and pH curves ran parallel up to the point where the glutamate is used up in YLLG (Fig. 1B), while during growth on YLHG glutamate was barely consumed at this time. From the moment of glutamate

49

Chapter 3

depletion in YLLG, the absorbance and pH differed between cultures grown in YLLG or YLHG, although lactic acid was metabolized in a parallel manner for both media (Fig. 1B). During growth on YLHG, the extra rise in pH coincided with glutamate consumption, indicating it was a result of glutamate metabolism. The higher A600 that was reached during growth in YLHG suggests that many more cells are present in this culture as compared to the culture grown in YLLG. However, neither plate counts nor direct microscopic observation confirmed that this was the case, although the interpretation of plate counts was difficult because the cells formed large clusters at the end of exponential growth, as we observed before (Chapter 2, Fig. 3A,B). It is possible that the A600 is influenced by the pH, because we noticed that, in the defined medium, a high pH (above 8.5) results in some precipitation, presumably of phosphate with calcium or magnesium. Furthermore, a dark brown color developed in the supernatant during sporulation in YLHG, but not in YLLG, further raising the A600.

Fig. 1. Changes observed during growth and sporulation of B. cereus on YLHG (closed symbols) vs. YLLG (open symbols). A. A600 (circles) and pH (squares). B. L-lactate (triangles up), D-lactate (triangles down), DPA (diamonds) and glutamate (circles).

This makes it plausible that although the final A600 reached in the culture grown on YLHG was higher, the actual cell density was quite similar to that in the culture grown on YLLG. Indeed, the total biomasses, as measured by nitrogen content, were similar for both cultures at the end of sporulation, both cultures produced similar amounts of dipicolinic acid (DPA), sporulation frequency was >99 % in both cases, and the cleaned spores had a similar DPA content (Table 2). This confirmed that equal amounts of cells were present in the cultures on YLLG and YLHG. We initially expected the maximum growth rate of B. cereus on the medium with lactate to be lower than on the same medium with the much more favorable substrate glucose, but the rates

50

Influence of glutamate on growth, sporulation and spore properties of Bacillus cereus

were identical (about 0.45 h-1). However, the lag-phase was much longer without glucose. This indicates that the exponential growth rate is primarily determined by the amino acids in the medium. Indeed, the bacteria metabolized the amino acids present in the medium during exponential growth. The metabolism of 10 mM of amino acids during exponential growth was accompanied by a 10 mM rise of NH4+ in the supernatant of both cultures, indicating that B. cereus used the amino acids mainly as carbon source and fuel, but not for assimilation into proteins. When grown with glucose, only half of the nitrogen in the amino acids metabolized during exponential growth was converted to NH4+ (see Chapter 2, Fig. 2A). Most of the lactic acid was metabolized after the cells had entered stationary phase (Fig. 2; Fig. 1B). These findings confirm the anticipated preference of B. cereus for amino acids, as derived from genome analysis studies (Ivanova et al., 2000). In the presence of either glucose or lactate, the amino acids were depleted in the same order: Threonine first, then valine, histidine, leucine and methionine as the last (see Chapter 2, Fig. 2B,C). The moment of glutamate consumption depended on the initial concentration: B. cereus metabolized glutamate earlier in YLLG than in YLHG, and during cultivation in YLLG glutamate was depleted early, after threonine. In YLHG the concentration of glutamate started to decrease 3 h later, and glutamate was still present in the medium when all the other amino acids were depleted. Fig. 2. Nitrogen sources during growth and sporulation of B. cereus in YLHG (A); YLLG (B). Symbols: A600 (circles); Total nitrogen in supernatant (squares); Nitrogen in glutamate (diamonds); Nitrogen in amino acids other than glutamate (triangles down); NH4 (triangles up).

Because the metabolism of amino acids during exponential growth resulted in a nearly equimolar increase of NH4+ in the supernatant, the total amount of nitrogen assimilated by B. cereus was very small during this growth phase. Indeed, our measurements clearly indicated that most of the nitrogen assimilated by B. cereus was taken up during sporulation (Fig. 2), underscoring the high nitrogen requirement of sporulating cells. The total amount of nitrogen initially present was 40 mM

51

Chapter 3

in YLHG and 22.5 mM in YLLG, a significant part of which (10 mM) consisted of NH4+, which was routinely added to the media as (NH4)2SO4 (Fig. 2; Table 1). When cultivated in YLHG, B. cereus used the bulk of the glutamate in the stationary and sporulation phase, after the other amino acids had disappeared. During this phase, the NH4+ concentration in the supernatant remained 20 mM, indicating that the nitrogen in the glutamate was completely assimilated (Fig. 2A). When cultivated in YLLG however, B. cereus assimilated NH4+ during the stationary and sporulation phase (Fig. 2B). At the end of sporulation, a total of 20 mM of nitrogen had disappeared from the culture supernatant in YLHG, of which we could trace back 16 mM as cell-bound nitrogen (Table 2). The remaining 4 mM might be present in the brown pigment that was formed in the culture supernatant during sporulation, as a brown pigment produced by B. subtilis during sporulation was shown to contain a significant proportion of nitrogen (Barnett and Hageman, 1983). The culture grown in YLLG had assimilated 15 mM of nitrogen in total at the end of sporulation, all of which we could trace back as cell-bound nitrogen (Table 2). Fig. 3. Sigma-factor expression level (calculated as described in the text) of B. cereus grown and sporulated in YLHG (closed symbols) vs. YLLG (open symbols). (A) Expression of sigA (circles) and sigB (squares); DPA concentration (diamonds). (B) Expression of sporulation sigma-factors sigF (triangles) and sigG (squares); DPA concentration (diamonds).

In both media tested, 99 % of the cells produced a phase-bright spore and the concentration of dipicolinic acid (DPA) reached about 115 µM, after which the oxygen consumption of the cultures decreased dramatically (data not shown). DPA production and phasebrightening of the forespores are indicative of the final phases of sporulation (Errington, 1993). Notably, DPA production, which parallels the production of phase-bright spores (see Chapter 2, Fig. 1B), started much earlier in YLLG as compared to YLHG, and release of the spores from the mother-cells was delayed concomitantly in YLHG. In our experiments, significant amounts of carbon (D-lactate, glutamate) and nitrogen (NH4+, glutamate) were present when the cells started to sporulate. Even after sporulation was completed, a significant amount of these C and N sources remained.

52

Influence of glutamate on growth, sporulation and spore properties of Bacillus cereus

Nutrient limitation therefore seems an unlikely trigger for sporulation under the conditions we employed. From B. subtilis it is known that in addition to starvation, quorum sensing can be involved in the initiation of sporulation (Sonenshein, 2000). In silico analysis of the B. cereus ATCC14579 genome with the ERGO genome and discovery system (Overbeek et al., 2003) revealed the presence of the gram-positive quorum sensing and competence II pathway which is also present in B. subtilis. Thus, although additional experiments in which cell density is carefully manipulated are needed to provide definite evidence, it is tempting to speculate that in our experiments cell density rather than nutrient limitation may have been the factor that triggered the cells to sporulate. Because extra glutamate did not result in extra biomass, the cell-density in YLHG and YLLG was similar at all times. Indeed, sporulation as judged by the rise in sigF and sigG transcription started at the same moment under both conditions (see below; Fig. 3). Gene expression The differences we found in the nitrogen metabolism and timing of DPA formation prompted us to check for differences in the genetic program of sporulation on YLLG and YLHG. Therefore, we measured the expression of genes for four key sigma factors involved in stress response, growth and sporulation: sigB, sigA, and sigF and sigG, respectively (Fig. 3). The product of sigA, sigma factor σA, is the primary sigma factor. It regulates macromolecular synthesis, plays a key role in the housekeeping functions of the cell (Helmann and Moran, 2002), and is expressed by B. cereus at a high level throughout growth and sporulation (see Chapter 2, Fig. 1C); therefore, we used sigA expression as a reference and positive control. During cultivation on YLLG and YLHG, sigB transcription was higher than the levels of transcription during growth with glucose, and showed no clear peak upon entry into the stationary phase (Fig. 3A). SigB transcription is increased upon entry in stationary phase (Chapter 2, Fig 1C; Helmann and Moran, 2002) and when the cell encounters stress (van Schaik et al., 2004), directly affecting cell- and carbon metabolism (Price, 2002). Our present findings indicate that B. cereus experiences more stress when grown in a medium with lactate instead of glucose, and that the sigB peak observed before (see Chapter 2, Fig. 1C) was associated with the depletion of glucose rather than with changes in growth-rate upon entry into the stationary phase. During the cultivations in YLLG and YLHG, the expression patterns of sigF and sigG (Fig. 3B) were comparable to those on glucose in the sense that we observed a high peak of sigF transcription followed by a sigG peak. However, with lactate as a carbon source sigF expression showed a sharp peak, while during sporulation with glucose, we observed a broad peak (Chapter 2, Fig. 1C). The product of sigF, sporulation sigma factor σF, is produced in the predivisional cell (Piggot and Losick, 2002), well before the first irreversible step of sporulation (Driks, 1999). After septation, when the cells have become committed to sporulate, σF is released from its anti-sigma factor to become active in the forespore only (Piggot and Losick, 2002). The gene encoding σG, sigG, is under the control of σF and expressed in the forespore compartment just after the appearance of the sporulation septum (Driks, 1999). Thus, increase of sigG transcription indicates σF activity. σG plays a role in the final stages of sporulation, regulating the synthesis of SASP and Ger proteins (Helmann and Moran, 2002; Hornstra et al., 2005; Barlass et al., 2002). The maximum levels of sigF and sigG expression were slightly higher during sporulation on YLHG as compared to YLLG. Interestingly, the times at which the maximum sigF and sigG expressions occurred were almost the same under both conditions, with only a slight delay of 30 min for the YLHG cultivation. This suggested that there was no considerable difference in the programming of sporulation when judged by the temporal expression of the key sigma factors measured. Nevertheless, in YLLG B. cereus started to produce DPA and phase-bright spores more than 3 h earlier than in YLHG, indicating that the length of the sporulation process was increased by a

53

Chapter 3

high concentration of glutamate. In contrast, Kennedy et al. (1971) reported that lower glutamate concentrations led to a longer sporulation time. These authors used different media and a different strain, which may have contributed to this difference. DPA synthesis is regulated by σK (Errington, 1993), a sporulation specific sigma factor expressed by the mother-cell compartment in response to σG activity in the forespore during the final stages of sporulation (Piggot and Losick, 2002). It is possible that although sigG expressions are parallel in YLLG and YLHG, the actual σG activity is delayed in YLHG, or that σG activity is also parallel but σK activity in the mother cell is delayed. Either way, our findings suggest that there is a certain degree of plasticity in the tightly regulated sporulation program. Spore properties Important spore properties are germinability and heat resistance. Therefore, we determined the resistance of the spores from YLHG and YLLG to 95°C, and their germination response to two well-known germinants for B. cereus: L-alanine and inosine. Spores from YLHG were more resistant to 95°C than those from YLLG (Table 2). Because heat resistance has been associated with the core water content (Popham et al., 1995; Beaman et al., 1984) we measured the core density from which the water content can be calculated (Popham et al., 1995). The whole spore density and the DPA content of spores from YLLG were rather similar to those of spores from YLHG, but the core density was somewhat higher (Table 2). This was in contrast to our expectations, because core density is generally believed to be positively correlated with heat resistance. Indeed, as was stated by Atrih and Foster (2001), complex co-operative phenomena are involved in the assembly of spores, and a multi-component system is the basis for spore heat resistance. Table 2. Properties of spores from YLLG vs. YLHG. D95, decimal reduction time at 95ºC; OD600, optical density at 600 nm.

Spores from

YLLG YLHG

Cell-bound N in the culture at 35 hours ± SD (mM)

DPA content ± SD (µM/OD600 unit)

15.10 ± 3.20 16.03 ± 1.05

112 ± 1.35 115 ± 2.56

Density (g/ml) Whole spore 1.13 1.13

Spore core ± SD 1.345 ± 0.008 1.309 ± 0.012

D95 (± SD) (min)

1.52 ± 0.07 2.64 ± 0.16

We measured the germinability of the spores without a prior heat-activation step, to prevent any possible interference by compounds released from the spores themselves (Preston and Douthit, 1984), and because spores from YLLG tended to germinate spontaneously after a heat shock of 15 minutes at 75 °C, which reflects their greater heat sensitivity. Clear differences in germination speed and extent were observed between spores from YLHG vs. YLLG, as can be seen in Fig. 4. The response to L-alanine was severely limited in spores from YLLG, while these spores responded faster to inosine, but much slower still than spores from YLHG. The most powerful germinant known for B. cereus is a combination of L-alanine and inosine (Clements et al., 1998). Although the difference was less pronounced, even with this powerful germinant, spores from YLHG germinated quicker and more completely than spores from YLLG. Thus, with the different germinants tested, spores from YLHG germinated faster and to a larger extent than spores from YLLG. It is known that spores from different media may have a different response to L-alanine because of differences in the alanine racemase content (A. Moir, pers. comm.), an enzyme that is present in the exosporium of B. cereus (Todd et al., 2003) and converts the germinant L-alanine into the germination inhibitor D-alanine. In addition to containing alanine racemase, the exosporium of B. cereus contains nucleoside hydrolase, an enzyme that could inhibit inosine induced germination (Todd et al., 2003). The presence of higher levels of nucleoside hydrolase and alanine racemase in

54

Influence of glutamate on growth, sporulation and spore properties of Bacillus cereus

spores from YLLG could therefore explain the differences we observed in inosine-induced germination. We tried germination in the presence of D-cycloserine, a known alanine racemase inhibitor (Bron et al., 2004), but this resulted in an overall lower response in all cases. Experiments with other alanine racemase inhibitors (e.g. O-carbamyl-D-serine; Jones and Gould, 1968) and nucleoside hydrolase inhibitors could bring more clarity to those issues. Our data clearly show that the concentration of glutamate affected not only the sporulation process but also the spore properties. Indeed, glutamate has been reported to have a large impact on sporulation as well as spore properties of bacilli. In Bacillus thuringiensis, a close relative of B. cereus (Helgason et al., 2000), glutamate appears to regulate TCA cycle activity as well as influence spore heat resistance and DPA content (Nickerson et al., 1974). B. cereus has been reported to metabolize glutamate primarily as an energy source, during the time of sporogenesis (Charba and Nakata, 1977). Several reports have attempted to determine the fate of the carbon skeleton of glutamate, and it was concluded that glutamate used during sporulation is respired almost completely to CO2, largely via the TCA cycle (Charba and Nakata, 1977; Nickerson et al., 1974). Our results confirm that glutamate is used largely during sporulation when present in a high concentration, but an interesting outcome is that glutamate metabolism during sporulation does not result in an increase of NH4+ in the medium. This implies that the glutamate served as a nitrogen donor during the sporulation process, not only as a source of energy and reducing power, as was stated by Charba and Nakata (1977). Indeed, in YLLG, NH4+ was taken up by the cells during sporulation, but D-lactate, which can serve as a source of reducing potential and energy, partly remained in the supernatant. Fig. 4. Germination measured by decrease in A600 of B. cereus spores from YLHG (closed symbols) vs. YLLG (open symbols). A600 at time 0 was 0.8-1.0 in all cases; curves are averages of 4 independent experiments. Germinants used: L-alanine, 10 mM (circles); inosine, 5 mM (triangles); inosine in combination with L-alanine, 0.5 mM each (squares).

Conclusions In conclusion, we have found that B. cereus is able to form spores on a chemically defined medium without glucose but with lactate as a main carbon source. Sporulation was not induced by nutrient limitation. Glutamate delayed the final stages of sporulation, but not the moment of sporulation initiation. Clearly, the concentration of glutamate influenced key spore properties such as heat resistance and germination. Our defined approach to sporulation and spore properties is highly suitable for future gene expression analysis employing DNA micro-arrays. This research provides a firm basis for experiments coupling defined sporulation conditions with full transcriptome analysis and spore properties, which will allow for careful dissection of the molecular basis of such important spore properties as heat resistance and germination.

55

Chapter 3

Acknowledgements We thank Dr. Willem van Schaik for providing the primers for sigB. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

56

Atrih, A., and S. J. Foster. 2001. Analysis of the role of bacterial endospore cortex structure in resistance properties and demonstration of its conservation amongst species. J Appl Microbiol 91:364-372. Barlass, P. J., C. W. Houston, M. O. Clements, and A. Moir. 2002. Germination of Bacillus cereus spores in response to L-alanine and to inosine: the roles of gerL and gerQ operons. Microbiology 148:2089-2095. Barnett, T. A., and J. H. Hageman. 1983. Characterization of a brown pigment from Bacillus subtilis cultures. Can J Microbiol 29:309-315. Beaman, T. C., T. Koshikawa, H. S. Pankratz, and P. Gerhardt. 1984. Dehydration partitioned within core protoplast accounts for heat resistance of bacterial spores. FEMS Microbiol Lett 24:47-51. Bidingmeyer, B. A., S. A. Cohen, and T. L. Tarvin. 1984. Rapid analysis of amino acids using pre-column derivatization. J Chromatogr 336:93-104. Bron, P. A., S. M. Hoffer, I. I. van Swam, W. M. De Vos, and M. Kleerebezem. 2004. Selection and characterization of conditionally active promoters in Lactobacillus plantarum, using alanine racemase as a promoter probe. Appl Environ Microbiol 70:310-317. Buono, F., R. Testa, and D. G. Lundgren. 1966. Physiology of growth and sporulation in Bacillus cereus. I. Effect of glutamic and other amino acids. J Bacteriol 91:2291-2299. Cazemier, A. E., S. F. Wagenaars, and P. F. ter Steeg. 2001. Effect of sporulation and recovery medium on the heat resistance and amount of injury of spores from spoilage bacilli. J Appl Microbiol 90:761-770. Charba, J. F., and H. M. Nakata. 1977. Role of glutamate in the sporogenesis of Bacillus cereus. J Bacteriol 130:242-248. Choma, C., H. Clavel, H. Dominguez, N. Razafindramboa, H. Soumille, C. Nguyen-the, and P. Schmitt. 2000. Effect of temperature on growth characteristics of Bacillus cereus TZ415. Int J Food Microbiol 55:73-77. Clements, M. O., and A. Moir. 1998. Role of the gerI operon of Bacillus cereus 569 in the response of spores to germinants. J Bacteriol 180:6729-6735. de Vries, Y. P., L. M. Hornstra, W. M. de Vos, and T. Abee. 2004. Growth and sporulation of Bacillus cereus ATCC 14579 under defined conditions: temporal expression of genes for key sigma factors. Appl Environ Microbiol 70:2514-2519. Driks, A. 1999. Bacillus subtilis spore coat. Microbiology and Molecular Biology Reviews 63:1-20. Errington, J. 1993. Bacillus subtilis sporulation: regulation of gene expression and control of morphogenesis. Microbiol Rev 57:1-33. Evans, R. I., N. J. Russell, G. W. Gould, and P. J. McClure. 1997. The germinability of spores of a psychrotolerant, non-proteolytic strain of Clostridium botulinum is influenced by their formation and storage temperature. J Appl Microbiol 83:273-280. Gonzalez, I., M. Lopez, S. Matrinez, A. Bernardo, and J. Gonzalez. 1999. Thermal inactivation of Bacillus cereus spores formed at different temperatures. Int J Food Microbiol 51:81-84.

Influence of glutamate on growth, sporulation and spore properties of Bacillus cereus

17. 18. 19. 20. 21. 22.

23. 24. 25. 26. 27. 28. 29. 30. 31.

32. 33.

Helgason, E., O. A. Okstad, D. A. Caugant, H. A. Johansen, A. Fouet, M. Mock, I. Hegna, and Kolsto. 2000. Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis-one species on the basis of genetic evidence. Appl Environ Microbiol 66:2627-2630. Helmann, J. D., and C. P. Moran, Jr. 2002. RNA polymerase and sigmafactors, p. 289313. In A. L. Sonenshein, J. A. Hoch, and R. Losick (ed.), Bacillus subtilis and its closest relatives: from genes to cells. ASM Press, Washington D. C. Hindle, A. A., and E. A. H. Hall. 1999. Dipicolinic acid (DPA) assay revisited and appraised for spore detection. Analyst 124:1599-1604. Hornstra, L. M., Y. P. de Vries, W. M. de Vos, T. Abee, and M. H. Wells-Bennik. 2005. gerR, a novel ger operon involved in L-alanine- and inosine-initiated germination of Bacillus cereus ATCC 14579. Appl Environ Microbiol 71:774-781. Ireton, K., S. Jin, A. D. Grossman, and A. L. Sonenshein. 1995. Krebs cycle function is required for activation of the Spo0A transcription factor in Bacillus subtilis. Proc Natl Acad Sci U S A 92:2845-2849. Ivanova, N., A. Sorokin, I. Anderson, N. Galleron, B. Candelon, V. Kapatral, A. Bhattacharyya, G. Reznik, N. Mikhailova, A. Lapidus, L. Chu, M. Mazur, E. Goltsman, N. Larsen, M. D'Souza, T. Walunas, Y. Grechkin, G. Pusch, R. Haselkorn, M. Fonstein, S. D. Ehrlich, R. Overbeek, and N. Kyrpides. 2003. Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis. Nature 423:87-91. Janssen, F. W., A. J. Lund, and L. E. Anderson. 1958. Colorimetric assay for dipicolinic acid in bacterial spores. Science 127:26-27. Jin, S., P. A. Levin, K. Matsuno, A. D. Grossman, and A. L. Sonenshein. 1997. Deletion of the Bacillus subtilis isocitrate dehydrogenase gene causes a block at stage I of sporulation. J Bacteriol 179:4725-4732. Jones, A., and G. W. Gould. 1968. Stimulation of germination of bacterial spores by analogues of D- alanine. J Gen Microbiol 53:383-394. Kennedy, R. S., F. J. Malveaux, and J. J. Cooney. 1971. Effects of glutamic acid on sporulation of Bacillus cereus and on spore properties. Can J Microbiol 17:511-519. Lindsay, J. A., T. C. Beaman, and P. Gerhardt. 1985. Protoplast water content of bacterial spores determined by buoyant density sedimentation. J Bacteriol 163:735-737. Mazas, M., I. Gonzalez, M. Lopez, and R. Martin. 1995. Effects of sporulation media and strain on thermal resistance of Bacillus cereus spores. Int J Food Sci Technol 30:71-78. Melly, E., P. C. Genest, M. E. Gilmore, S. Little, D. L. Popham, A. Driks, and P. Setlow. 2002. Analysis of the properties of spores of Bacillus subtilis prepared at different temperatures. J Appl Microbiol 92:1105-1115. Nickerson, K. W., J. De Pinto, and L. A. Bulla, Jr. 1974. Sporulation of Bacillus thuringiensis without concurrent derepression of the tricarboxylic acid cycle. J Bacteriol 117:321-323. Overbeek, R., N. Larsen, T. Walunas, M. D'Souza, G. Pusch, E. Selkov, Jr., K. Liolios, V. Joukov, D. Kaznadzey, I. Anderson, A. Bhattacharyya, H. Burd, W. Gardner, P. Hanke, V. Kapatral, N. Mikhailova, O. Vasieva, A. Osterman, V. Vonstein, M. Fonstein, N. Ivanova, and N. Kyrpides. 2003. The ERGO genome analysis and discovery system. Nucleic Acids Res 31:164-171. Piggot, P. J., and R. Losick. 2002. Sporulation genes and intercompartmental regulation, p. 483-519. In A. L. Sonenshein, J. A. Hoch, and R. Losick (ed.), Bacillus subtilis and its closest relatives: from genes to cells. ASM Press, Washington D. C. Popham, D. L., S. Sengupta, and P. Setlow. 1995. Heat, hydrogen peroxide, and UV resistance of Bacillus subtilis spores with increased core water content and with or without major DNA-binding proteins. Appl Environ Microbiol 61:3633-3638.

57

Chapter 3

34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44.

58

Preston, R. A., and H. A. Douthit. 1984. Stimulation of germination of unactivated Bacillus cereus spores by ammonia. J Gen Microbiol 130 ( Pt 5):1041-1050. Price, C. W. 2002. General stress response, p. 369-385. In A. L. Sonenshein, J. A. Hoch, and R. Losick (ed.), Bacillus subtilis and its closest relatives: from genes to cells. ASM Press, Washington D. C. Raso, J., G. Barbosa-Canovas, and B. G. Swanson. 1998. Sporulation temperature affects initiation of germination and inactivation by high hydrostatic pressure of Bacillus cereus. J Appl Microbiol 85:17-24. Raso, J., M. M. Gongora-Nieto, G. V. Barbosa-Canovas, and B. G. Swanson. 1998b. Influence of several environmental factors on the initiation of germination and inactivation of Bacillus cereus by high hydrostatic pressure. Int J Food Microbiol 44:125-132. Rickwood, D., T. Ford, and J. Graham. 1982. Nycodenz: a new nonionic iodinated gradient medium. Anal Biochem 123:23-31. Sonenshein, A. L. 2000. Control of sporulation initiation in Bacillus subtilis. Curr Opin Microbiol 3:561-566. te Giffel, M. C., A. Wagendorp, A. Herrewegh, and F. Driehuis. 2002. Bacterial spores in silage and raw milk. Antonie van Leeuwenhoek 81:625-630. Tisa, L. S., T. Koshikawa, and P. Gerhardt. 1982. Wet and dry bacterial spore densities determined by buoyant sedimentation. Appl Environ Microbiol 43:1307-1310. Todd, S. J., A. J. Moir, M. J. Johnson, and A. Moir. 2003. Genes of Bacillus cereus and Bacillus anthracis encoding proteins of the exosporium. J Bacteriol 185:3373-3378. van Schaik, W., M. H. Tempelaars, J. A. Wouters, W. M. de Vos, and T. Abee. 2004. The alternative sigma factor sigmaB of Bacillus cereus: response to stress and role in heat adaptation. J Bacteriol 186:316-325. Vary, J. C. 1973. Germination of Bacillus megaterium spores after various extraction procedures. J Bacteriol 116:797-802.

4. Deletion of sigB in Bacillus cereus affects Spore Properties Ynte P. de Vries, Luc M. Hornstra, Ratna D. Atmadja, Willem van Schaik, Willem M. de Vos and Tjakko Abee. Excerpt published in FEMS Microbiology Letters, 2005, 252:169-173

ABSTRACT In Bacillus cereus and other gram-positive bacteria the alternative sigma factor σB is an important regulator of the stress response. Deletion of the sigB gene generally leads to a stress-sensitive phenotype of vegetative cells. In this study, we describe the effect of the deletion of the sigB gene in B. cereus on spore properties. In particular, spores of the sigB deletion mutant showed a defect in germination upon exposure to the germinants alanine and inosine.

59

Chapter 4

Introduction B. cereus spores can withstand many commonly used practices for food preservation, including pasteurization. Upon germination of the spores, the response of the vegetative cells to the stresses imposed on them in the final product, will determine their capacity to grow out, causing spoilage and/or food-borne infections. One of the major regulators of the stress response is the alternative sigma-factor σB, which plays an important role in the survival of B. cereus under adverse conditions (van Schaik et al., 2004). Previously, we observed that at the start of stationary phase, the genes encoding σB and sporulation sigma factor σF, sigB and sigF respectively, were upregulated both at the same time (Chapter 2). Here we assess the effect of the deletion of sigB on the process of sporulation and spore properties of B. cereus. Materials and methods Strains, media and analytical procedures The sigB null mutant of B. cereus ATCC14579 was constructed in a previous study (van Schaik et al., 2004), and its growth and sporulation characteristics were compared to the parental strain in defined conditions as described in Chapter 2. Analytical procedures were as described in Chapters 2 and 3. To determine spore wet heat resistance, survivors were counted after exposure to 95 °C for 5 minutes using the same procedures as in Chapter 3. RT-qPCR was performed as described in Chapters 2 and 3, and the sigA expression level was used as a reference and internal standard for comparison of the datasets from the two different fermenters. Cryo-SEM Cryo-planing Scanning Electron Microscopy (Nijsse and van Aelst, 1999) was performed as follows. Droplets of concentrated spore-slurry were put on aluminum rivets and immediately rapidly frozen in liquid propane. The cryo-fixed samples were placed in a sample holder in a cryo ultra microtome (Reichert Ultracut E/FC4D) and cut at specimen temperature of -100°C. The samples were first planed with a glass knife, after which the surface was planed with a diamond knife (Histo no trough, 8 mm 45°, Drukker International, The Netherlands). After planing the samples were placed in a dedicated cryo-preparation chamber (CT 1500 HF, Oxford instruments, UK), freeze dried for 3 minutes at -90°C at 1,33 x 10-3 Pa and subsequently sputtered with a layer of 10 nm Pt. The samples were cryo-transferred into a field emission scanning microscope (JEOL 6300F, Japan) on the sample stage at -190°C. All images were recorded digitally (Orion, 6 E.L.I. sprl, Belgium) at a scan rate of 100 seconds (full frame) and at the size of 2528 x 2030, 8 bit. The images were optimized and resized with Adobe Photoshop CS. Results Growth and sporulation Deletion of sigB had a minor effect on the specific growth-rate in the exponential phase. Glucose consumption during exponential growth was slightly retarded in the sigB null mutant (Fig. 1). The consumption of amino acids from the medium was not different between the wild-type and the sigB null mutant (Fig. 2). During exponential growth, the pH of the medium decreased because of the fermentation of glucose resulting in the production of acids (Chapter 2). The sigB null mutant caused a smaller pH drop during exponential growth, presumably due to the slightly slower glucose consumption. From the early stationary phase onwards, which is the moment of glucose depletion and sigB transcription activation (Chapter 2), the growth and medium acidification of the sigB null

60

Deletion of sigB in Bacillus cereus affects spore properties

mutant and the parental strain differed. The sigB null mutant had a slightly lower optical density in the early stationary phase, and the pH rise was not as pronounced as that of the parental strain (Fig. 1). These data indicate that σB is of minor importance for exponential growth, but σB may be important for metabolism during stationary phase. The deletion of sigB did not cause delay in sporulation, as witnessed by the accumulation of DPA (Fig. 1).

Fig. 1. Growth and sporulation of B. cereus ATCC14579 parent strain (closed symbols) vs. the sigB mutant (open symbols). (A). A600 (circles) and pH (squares). (B). Glucose (triangles) and DPA (diamonds). The arrow indicates the moment of the peak in sigB transcription in the parent strain, as observed in the parent strain (see Fig. 3A).

Fig. 2. Amino acid consumption of B. cereus ATCC14579 parent strain (closed symbols) vs. the sigB mutant (open symbols). (A). Threonine (triangles up), Valine (triangles down), and Methionine (diamonds). (B). Histidine (squares), Leucine (hexagons), and Glutamate (circles).

DPA synthesis is regulated by σK, and coincides with the phase-brightening of the fore-spores, which is indicative of the final stages of sporulation (Errington, 1993; Piggot and Losick, 2002). While glutamate consumption during sporulation was slightly retarded in the sigB mutant (Fig. 2B), the expression of sporulation sigma-factor genes sigF and sigG was largely parallel in both the sigB null mutant and the parental strain (Fig. 3).

61

Chapter 4

Fig. 3. Sigma-factor expression level (calculated as described in the text) of B. cereus ATCC14579 parent strain (closed symbols) vs. the sigB mutant (open symbols). (A) Expression of sigA (circles) and sigB (triangles down); DPA concentration (diamonds). (B) Expression of sporulation sigma-factors sigF (triangles up) and sigG (squares).

These findings suggest that the sigB deletion did not affect the temporal kinetics of the sporulation process. However, the fore-spores produced by the sigB null mutant had a clear spheroid appearance, in contrast to the ellipsoid appearance of parental fore-spores (Fig. 4). After release from the mother-cells, the sigB null mutant spores remained spheroid (Fig. 5). Also, when cells were allowed to sporulate in CCY medium (Stewart et al., 1981) and modified G medium (Nakata, 1963), the resulting spores of the sigB deletion mutant were clearly more spheroid than those of the parental strain (data not shown).

Fig. 4. Phase-contrast micrographs of sporulating cells from the B. cereus ATCC14579 parent strain (A) and the sigB mutant (B). Bars indicate 5 µm.

62

Deletion of sigB in Bacillus cereus affects spore properties

Fig. 5. Scanning Electron Micrographs of spores from the B. cereus ATCC14579 parent strain (left) and the sigB mutant (right). Bars indicate 1 µm.

The resistance of spores to wet heat is an important parameter because it contributes to the survival of B. cereus during industrial food production processes such as pasteurization (Brown et al., 2000; Nicholson et al., 2000). The sigB null mutant spores had a slightly lower heat resistance than parental spores. Exposure to 95°C for five min caused a reduction of 3.10 ± 0.11 logs in the sigB deletion mutant, compared to 2.27 ± 0.10 logs in the parental strain. This suggests that the alternative sigma factor σB is needed for optimal morphogenesis of the spore, with the absence of the sigB gene leading to a spore with diminished resistance to wet heat. Bacterial spores are metabolically dormant, and in order to grow and proliferate spores undergo dramatic changes in a process called germination (reviewed in Moir et al., 2002; Setlow, 2003). The germination response is essential, as it is through germination that spores ultimately cause food spoilage and disease (Setlow, 2003). Several compounds are known to invoke germination of B. cereus spores. The responses to L-alanine and inosine are best studied, and a combination of L-alanine and inosine is the most powerful germinant for B. cereus (Barlass et al., 2002; Clements et al., 1998; Hornstra et al., 2005). Germination in response to 10 mM of L-alanine was almost completely abolished in the sigB null mutant, and severely impeded at 100 mM (Fig. 6A). Furthermore, the germination in response to inosine, and to a combination of inosine and Lalanine, was affected in the sigB null mutant (Fig. 6B). In addition to the loss of optical density upon the addition of the germinants, the excretion of DPA (an early event in spore germination) was measured and was found to be affected by the deletion of sigB in a similar way as the loss in optical density (data not shown). Discussion In B. subtilis σB was first described upon its isolation from sporulating cells, suggesting that σB had a role in sporulation (Haldenwang and Losick, 1979). However, after a sigB deletion mutant in B. subtilis was constructed, no differences in the sporulation properties were observed between this mutant and the parental strain (Binnie et al., 1986). Recently, however, a study showed that in B. subtilis σB activity is required for efficient sporulation at low temperatures (Mendez et al., 2004). In this study, we did not observe a clear effect of the deletion of sigB on the temporal frame of the sporulation process in B. cereus. However, we have only tested sporulation under favorable conditions and we cannot rule out the possibility that under sub optimal conditions sporulation is affected in the sigB null mutant.

63

Chapter 4

Fig. 6. Germination of spores from B. cereus ATCC14579 parent strain (closed symbols) vs. the sigB mutant (open symbols) (A). in response to alanine 10 mM (triangles); 100 mM (squares). (B). in response to inosine 5 mM (diamonds); and inosine in combination with alanine (0.5 mM each) (hexagons).

Although the temporal frame of sporulation was not influenced by the deletion of the sigB gene, spore properties were clearly affected. We observed that spores of the sigB deletion mutant appeared less resistant to wet heat than spores of the parental strain. Other resistance properties (e.g. to acid, high pressure or UV) may also be affected by the deletion of sigB but were not tested in this study. Another notable effect was the altered gross morphology of the spores of the sigB mutant. As σB of B. cereus is activated during the stationary phase of growth (Chapter 2), it is likely that σB may contribute to determining spore properties. Indeed, ongoing research in our laboratory has revealed that a number of genes coding for proteins, which are directly involved in sporulation and germination, are expressed in a σB-dependent fashion (W. van Schaik, personal communication). Furthermore, a significant number of σB-regulated genes are involved in metabolism and co-factor synthesis (van Schaik et al., 2004b). Possibly, important metabolic pathways that contribute to the process of sporulation are affected in the sigB deletion mutant, which in its turn may lead to the poor spore properties observed in this study. A major outcome of this work is the deleterious effect of a sigB deletion on B. cereus spore germination. Interestingly, Fouet et al. (2000) reported that in B. anthracis a sigB null mutation affects virulence. These authors tested virulence by injecting mice with B. anthracis spores. Efficient germination of the B. anthracis spores is crucial for causing illness and although Fouet et al. did not test the germination response of the B. anthracis sigB null mutant, it is tempting to

64

Deletion of sigB in Bacillus cereus affects spore properties

speculate that also in B. anthracis the germination response is affected by the deletion of sigB, thus leading to a decreased virulence. In conclusion, we have shown that a sigB deletion affects spore properties of B. cereus. Our findings add evidence for a relation between sporulation and σB dependent stress adaptation. Further elucidation of the mechanisms involved will be facilitated by the analysis of global transcriptional responses in the sigB null mutant and the wild-type by the use of B. cereus DNA microarrays. These and other comparative studies with the related bacteria B. thuringiensis, B. anthracis, and the model system B. subtilis should also reveal how widespread the coupling is between σB, spore formation and spore properties. Acknowledgements The authors wish to express their gratitude towards Adriaan van Aelst, for performing the cryo-SEM experiments. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Barlass, P. J., C. W. Houston, M. O. Clements, and A. Moir. 2002. Germination of Bacillus cereus spores in response to L-alanine and to inosine: the roles of gerL and gerQ operons. Microbiology 148:2089-2095. Binnie, C., M. Lampe, and R. Losick. 1986. Gene encoding the sigma 37 species of RNA polymerase sigma factor from Bacillus subtilis. Proc Natl Acad Sci U S A 83:5943-5947. Brown, K. L. 2000. Control of bacterial spores. Br Med Bull 56:158-71. Clements, M. O., and A. Moir. 1998. Role of the gerI operon of Bacillus cereus 569 in the response of spores to germinants. J Bacteriol 180:6729-6735. de Vries, Y. P., R. D. Atmadja, L. M. Hornstra, W. M. De Vos, and T. Abee. 2005. Influence of glutamate on growth, sporulation and spore properties of Bacillus cereus ATCC14579 in a defined medium. Appl Env Microbiol 71:3248-3254. de Vries, Y. P., L. M. Hornstra, W. M. de Vos, and T. Abee. 2004. Growth and sporulation of Bacillus cereus ATCC 14579 under defined conditions: temporal expression of genes for key sigma factors. Appl Environ Microbiol 70:2514-2519. Errington, J. 1993. Bacillus subtilis sporulation: regulation of gene expression and control of morphogenesis. Microbiol Rev 57:1-33. Fouet, A., O. Namy, and G. Lambert. 2000. Characterization of the operon encoding the alternative sigma(B) factor from Bacillus anthracis and its role in virulence. J Bacteriol 182:5036-5045. Haldenwang, W. G., and R. Losick. 1979. A modified RNA polymerase transcribes a cloned gene under sporulation control in Bacillus subtilis. Nature 282:256-260. Hornstra, L. M., Y. P. de Vries, W. M. de Vos, T. Abee, and M. H. Wells-Bennik. 2005. gerR, a novel ger operon involved in L-alanine- and inosine-initiated germination of Bacillus cereus ATCC 14579. Appl Environ Microbiol 71:774-781. Mendez, M. B., L. M. Orsaria, V. Philippe, M. E. Pedrido, and R. R. Grau. 2004. Novel roles of the master transcription factors Spo0A and sigmaB for survival and sporulation of Bacillus subtilis at low growth temperature. J Bacteriol 186:989-1000. Moir, A., B. M. Corfe, and J. Behravan. 2002. Spore germination. Cell Mol Life Sci 59:403-409. Nakata, H. M. 1963. Effect of pH on intermediates produced during growth and sporulation of Bacillus cereus. J Bacteriol 86:577-581.

65

Chapter 4

14. 15. 16. 17. 18. 19. 20. 21.

66

Nicholson, W. L., N. Munakata, G. Horneck, H. J. Melosh, and P. Setlow. 2000. Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiol Mol Biol Rev 64:548-572. Nijsse, J., and A. C. van Aelst. 1999. Cryo-planing for cryo-scanning electron microscopy. Scanning 21:372-378. Piggot, P. J., and R. Losick. 2002. Sporulation genes and intercompartmental regulation, p. 483-519. In A. L. Sonenshein, J. A. Hoch, and R. Losick (ed.), Bacillus subtilis and its closest relatives: from genes to cells. ASM Press, Washington D. C. Setlow, P. 2000. Resistance of bacterial spores, p. 217-230. In G. Storz and R. HenggeAronis (ed.), Bacterial stress responses. ASM press, Washington, D.C. Setlow, P. 2003. Spore germination. Curr Opin Microbiol 6:1-7. Stewart, G. S., K. Johnstone, E. Hagelberg, and D. J. Ellar. 1981. Commitment of bacterial spores to germinate. A measure of the trigger reaction. Biochem J 198:101-106. van Schaik, W., M. H. Tempelaars, J. A. Wouters, W. M. de Vos, and T. Abee. 2004. The alternative sigma factor sigmaB of Bacillus cereus: response to stress and role in heat adaptation. J Bacteriol 186:316-325. van Schaik, W., M. H. Zwietering, W. M. de Vos, and T. Abee. 2004b. Identification of sigmaB-dependent genes in Bacillus cereus by proteome and in vitro transcription analysis. J Bacteriol 186:4100-4109.

5. Water in the Core of Bacillus Spores is not in a Glassy State: a Spin-Probe Study Ynte P. de Vries, Elena A. Golovina, Luc M. Hornstra, Marjon Wells-Bennik, Willem M. de Vos, Tjakko Abee and Folkert A. Hoekstra. To be submitted for publication

ABSTRACT Bacterial spores are characterized by extreme dormancy and resistance. These features have been attributed to a supposed glassy state of the spore cytoplasm. We applied non-destructive, spin-probe-based electron spin resonance (ESR) for the selective study of the aqueous phase in the different compartments of Bacillus subtilis and Bacillus cereus spores. Spectra derived specifically from the core and from the cortex of dormant spores revealed not a glassy state, but a structured environment with a micro-viscosity that was higher in the core than in the cortex. Germination led to a marked increase in cytoplasmic volume and a considerable reduction in viscosity. Such changes were not observed in germinating spores of the B. subtilis CwlD and FB113 mutants, from which most of the dipicolinic acid and cations are excreted, while cortex degradation and core rehydration do not occur. The proportion of bulk water in the core and the cortex correlated with the respective volumes of these two spore compartments.

67

Chapter 5

Introduction Bacterial spores have a unique structure (Tipper and Gauthier, 1972; Warth, 1978) that endows them with unique properties, such as metabolic dormancy (Keynan, 1972; Lewis, 1969) and a remarkable resistance to heat, radiation, toxic chemicals, pH extremes, oxidative agents, lytic enzymes, desiccation, mechanical disruption and high pressure (Setlow, 2000). In the autumn of 2001, Bacillus anthracis (anthrax) spores were applied in a bioterrorism attack in Washington, D. C. (Higgins et al., 2003). The resistance and dormancy of anthrax spores are of key importance to their use as biological weapons (Mock and Fouet, 2001), and to decontamination efforts after an attack. Furthermore, dormancy and resistance enable spores from a variety of species to cause serious problems in the food-, medicare-, paper-, and space-industry. Therefore, spore dormancy and resistance properties are the subject of intense research efforts. A key resistance property in relation to decontamination and food processing is the resistance of spores to wet heat (Brown, 1994; Newsome, 2003), however, the mechanism behind spore wet heat resistance is complex, multifactorial and may depend on the applied killing temperature (Atrih and Foster, 2001; Movahedi and Waites, 2002). It is generally agreed upon that the most important factor contributing to wet heat resistance is the dehydrated state of the core (Beaman et al., 1982; 1984; Gould, 1986; Nakashio et al., 1985; Setlow, 2000), but the exact mechanism of spore wet heat resistance is still far from resolved (Newsome, 2003; Setlow, 2000). It has been suggested that the dehydration of the core results in a glassy state of the spore cytoplasm (Ablett et al., 1999; Gould, 1986; Sapru and Labuza, 1993). A glass is a semiequilibrium solid liquid with an extremely high viscosity (Franks et al., 1991). Glasses occur at low water contents and/or temperatures, and are characterized by the absence of crystals and a specific melting point, resulting in a glass transition peak in thermograms. The high viscosity of intracellular glasses decreases molecular mobility and impedes molecular diffusion, thus slowing deleterious reactions and changes in structure and chemical composition during ageing. In desiccated pollen and seeds, a glassy state is present, and thought to be responsible for metabolic inactivity and long-term survival (Aguilera 1997; Buitink et al., 2000). The proposed glassy state in the core could thus contribute to the resistance and dormancy of bacterial spores (Cowan et al., 2003). The hypothesis of a glassy state in the core of bacterial spores has been based on the observation of glass transition peaks in thermograms, obtained by differential scanning calorimetry (DSC) (Sapru and Labuza, 1993), and further supported by a combination of DSC and nuclear magnetic resonance spectroscopy (NMR) (Ablett et al., 1999). However, these techniques cannot separate information from the different spore compartments. Recent results from Leuschner and Lillford (2003) indicated that the supposed glass transition peaks in thermograms from dormant bacterial spores could have been caused by denaturation of coat proteins rather than a glass transition in the core. Furthermore, several studies have suggested that water in the core is, to a large extent, freely exchangeable (Black and Gerhardt, 1962b; Leuschner and Lillford, 2000; Rode and Foster, 1960; Westphal et al., 2003), which contradicts the presence of a glassy state. Nevertheless, several other investigations indicated that other key compounds in the spore core are immobilized, such as ions (Carstensen et al., 1971; 1979; Gerhardt et al., 1971), dipicolinic acid (Ablett et al., 1999; Leuschner and Lillford, 2000; 2001), cytoplasmic proteins (Cowan et al., 2003) and lipids of the inner membrane (Cowan et al., 2004; Stewart et al., 1980). The question remains as to whether or not this is the result of a glassy state in the core (Cowan et al., 2003; Gould, in: Newsome, 2003). Here we applied a non-destructive ESR spin-probe method, which allows the different spore compartments and their physical state to be measured in vivo. The crux of the method is the combined use of a water-soluble spin probe and a polar paramagnetic ion that selectively removes spin-probe signals from particular compartments because of paramagnetic interactions, leaving the signals from the other compartments unaffected (Keith et al., 1977). Thus, we determined in vivo

68

Water in the core of Bacillus spores is not in a glassy state: a spin-probe study

the rotational correlation time, anisotropy and viscosity in the core of dormant Bacillus cereus and Bacillus subtilis spores. It was shown that the cytoplasm in the core of dormant bacterial spores is not in a glassy state. Rather, the core contains a highly ordered three-dimensional matrix enclosing highly viscous bulk water. Through spectral integration, the relative proportion of this bulk water in the core and the cortex was estimated. Materials and Methods Bacterial strains, cultivation and spore handling Bacillus cereus strain ATCC 14579 was cultivated in defined liquid medium. Spores were harvested and purified as described in Chapter 2. Repeated Nycodenz density gradient purification provided more than 99.9% phase-bright spores, as determined by phase-contrast microscopy (Zeiss axioskop). Bacillus subtilis mutant strains FB113 and CwlD (Setlow et al., 2001), which are blocked in germination stage I, and their isogenic parent strain PS832 were kindly provided by Dr. Setlow (UConn Health Center, Farmington, CT). B. subtilis spores were produced from liquid Schaefer’s sporulation medium at 37 °C as described in Nicholson and Setlow, 1990. B. subtilis spores were harvested by centrifugation, washed 6 times with sterile Millipore water and purified on a Nycodenz density gradient to obtain >99.9% phase-bright spores. Sample preparation Spores were suspended in Tris-HCl buffer (10 mM, pH 7.5), which for B. cereus contained an additional 0.1% of Tween80, and pelleted in an Eppendorf centrifuge at maximum speed for one min. The supernatant was removed, and equal volumes of the spore pellet and a solution that contained 2 mM perdeuterated TEMPONE (PDT) and 240 mM potassium-ferricyanide (FC; = K3Fe(CN)6) were combined, giving a final concentration of the spin probe and the broadening agent of 1 mM and 120 mM, respectively. The sample was carefully mixed by stirring and incubated at room temperature for at least 15 min to promote the diffusion of PDT and FC, and subsequently placed in a glass capillary (approx. 1 mm diameter) that was sealed from the bottom side. After centrifugation and removal of the supernatant, the capillary was placed in a quartz tube and fixed in the resonator of the ESR spectrometer. We measured the samples containing dormant spores for a second time after approximately 80 min to investigate whether the signal would change with time due to delayed diffusion. There was no change, indicating that the diffusion process was fast and completed before the initial measurement. Spore heat-activation and germination B. subtilis spores were heat-activated by incubation in Millipore water at 70 °C for 30 min. For germination, heat-activated spores were suspended at 37 °C in 10 mM Tris-HCl buffer (pH 7.5) with 10 mM NaCl and 50 mM L-alanine until >95 % of their DPA was released (50, 90 and 40 minutes for CwlD, FB113 and PS832, respectively; Fig. 1). In the mutants, this treatment slightly decreased spore refractivity and spore density. DPA was measured by the Tb-fluorescent method, as described in Chapter 3. B. cereus spores were heat-activated in 10 mM Tris-HCl buffer (pH 7.5) with 10 mM NaCl and 0.1% Tween80 to prevent clumping and attachment of spores to plastic laboratory equipment. Heat activated spores were incubated at 30 °C, and germination was started by the addition of L-alanine and inosine, to a final concentration of 25 and 1 mM, respectively. This treatment caused >95% DPA release within 10 minutes.

69

Chapter 5

Fig. 1. DPA excretion by germinating spores of B. subtilis PS832 (circles), CwlD (squares), and FB113 (triangles). Closed symbols: L-alanine 50 mM; Open symbols: Blanc. Curves are averages from duplicate experiments.

Disruption of spore membranes The outer membrane of the spores was disrupted by decoating according to Vary (1973) and Fitz-James (1971). Decoating removes parts of the spore-coat and disrupts the outer membrane. The cortical peptidoglycan is thus exposed, but the inner membrane remains intact, and viability is retained (Koshikawa et al., 1984). Briefly, spores at an OD600 of ~15 units were suspended in a decoating solution (containing 0.1 M NaCl, 0.5 % SDS, 0.1 M DTT, pH adjusted to 10.5 with NaOH) and shaken for 2 h at 37°C. The decoated spores were washed 5 times by pelleting (at low speed to prevent aggregation) and resuspension in Millipore water, and suspended in 10 mM TrisHCl buffer (pH 7.5) with 10 mM NaCl. For disruption of both membranes, we disrupted the entire spores by bead-beating. 0.6 ml of spores was bead-beaten (3 x 5 min at 30 Hz with 3 min intervals for cooling) with 0.6 grams of zirkonia silica beads (0.1 mm diameter) from Biospec Products (Bartlesville, OK) in 2 ml Eppendorf vials. To prevent degradation of the cortex by lytic enzymes after spore breakage (Warth, 1972), we used the B. subtilis mutants FB113 and CwlD, whose cortex-lytic enzymes are absent or unable to degrade the cortex peptidoglycan, respectively (Setlow et al., 2001). Microscopic observation confirmed that this treatment disrupted >99.9% of the spores. Spectra recording and analysis Spectra were recorded on an X-band ESR spectrometer (Bruker Analytik, Rheinstetten, Germany, Model Elexys 500). Spectra were recorded in the range of 100 Gauss, and the modulation amplitude was kept at 0.25 G to avoid signal over-modulation; a low power of 2 mW (20 dB) was used to prevent saturation effects. The rotation correlation time (τR) was calculated from the line shape of the narrow component according to: τR=6.5x10-10 ∆W0 (√h0/h -1 -1), where ∆W0 is the peak-to-peak width of the central line in Gauss, and h0 and h–1 are the line-heights of the central and high-field lines, respectively. The viscosity was calculated from the Stokes-Einstein relationship η= τR kT /4πr3, where η is the viscosity, r the effective radius of the PDT molecule, K the Bolzmann constant, T the absolute temperature, and τR the rotation correlation time. For the TEMPONE molecule (Mw = 179), r = 3*10-10m, and we assume PDT to approximate this value. The anisotropy of the PDT rotation was calculated as: │C/B│=[√(h0/h +1)+√(h0/h –1) -2]/ [√(h0/h +1)-√(h0/h –1)], where h+1, h0 and h-1 are the line-heights of the low-field (left-hand side), central, and high-field (right-hand side) lines, respectively (Schobert and Marsh, 1982). If the motion is isotropic (nonstructured environment, free rotation), then │C/B│=1; anisotropy (structured environment, restricted rotation) causes the │C/B│≠ 1.

70

Water in the core of Bacillus spores is not in a glassy state: a spin-probe study

PDT was obtained from Prof. I. Grigor’ev (Institute of Organic Chemistry, Russian Academy of Sciences, Novosibirsk, Russia). The PDT-ferricyanide system for the selective study of compartments The spectrum of 1 mM PDT in water contains 3 equally distant, narrow lines (Fig. 2A). The isotropic hyperfine splitting constant (the distance between lines) aiso was 16.02 G, and τR calculated from the spectral shape was 0.22*10-10 s (Table 1). The calculated viscosity of water, based on τR, was 0.85 cP. When FC is added to a solution of spin probe, the peak-to-peak line-width increases with concentration because of the broadening effect of paramagnetic Fe(CN)6−3 ions via spin-spin exchange (Keith et al., 1977).

Fig. 2. ESR spectrum of 1 mM PDT in water (A); ESR spectra of a broadened (120 mM FC) solution of 1 mM PDT containing dormant B. subtilis spores (B); and the interstitial fluid from the spore suspension (C).

The broadening effect of FC allows the signal from spin probe molecules located inside cells to be separated from that in the outside solution. This is possible because intact cellular membranes are permeable to PDT, but impermeable to FC ions (see Golovina et al., 2001). Thus, if living cells are placed in such a broadened solution, the resulting spectrum will originate from PDT located both inside and outside cells. However, because of the selective broadening, the spectrum from the outside solution can be distinguished from the spectrum originating from PDT inside the cells. By subtracting the signal that originated from the outside solution from the total signal, the signal from inside the cells is obtained.

71

Chapter 5

Results Spectra from dormant B. subtilis and B. cereus spores Placing a suspension of dormant spores from either B. subtilis or B. cereus in an equal volume of [2mM PDT + 240 mM FC] solution changed the broadened spectrum into one consisting of broad and narrow triplets (Fig. 2B). Some interstitial fluid was present in the sample, giving rise to a partially broadened signal, which was identified by recording the spectrum from the supernatant that was removed from the sample capillary during sample preparation (Fig. 2C). Subtraction of this supernatant signal from the signal of the spore samples (Fig. 3A) yielded the spectrum derived exclusively from the spores, which consisted of narrow and partially broadened triplets (Fig. 3B). Table 1. The spectral parameters of the narrow components of ESR spectra of PDT in dormant, heat-activated and germinating (mutant) spores of B. cereus and B. subtilis. For comparison, the parameters of PDT in water are given. The following parameters are indicated: width of the central line, the anisotropy parameter, │C/B│, the rotational correlation time, τR, and the micro viscosity, η. │C/B│ τR, 10–10 s Viscosity η, cP Specimen Width, G _________________________________________________________________________ Water 0.39 1.04 0.23 0.85

B. cereus Dormant Germinated

0.49 0.39

1.46 1.10

1.98 0.54

7.25 1.99

B. subtilis PS832 (wild-type) Dormant Heat-activated Germinated Core of dormant spores Cortex of dormant spores

0.49 0.49 0.39 0.68 0.49

1.65 1.69 1.53 1.41 1.37

1.52 1.53 0.54 2.36 1.24

5.56 5.58 2.00 8.70 4.53

B. subtilis FB113 Dormant Heat-activated Germinated to stage I

0.49 0.49 0.49

1.70 1.71 1.51

1.55 1.50 1.46

5.68 5.49 5.32

B. subtilis CwlD Dormant Heat-activated Germinated to stage I

0.49 0.49 0.49

1.59 1.68 1.43

1.39 1.38 1.37

5.10 5.06 5.00

LSD (P= 0.05)

-

0.17

0.14

0.51

72

Water in the core of Bacillus spores is not in a glassy state: a spin-probe study

Fig. 3. Decomposition of ESR PDT spectra from dormant B. subtilis spores. Fitting of the supernatant spectrum as shown in Fig. 2C to the spectrum from dormant spores as shown in Fig. 2B (A); the supernatant-free spectrum after subtraction of the two spectra in A (B); fitting of the supernatant spectrum to the spectrum from bead-beaten spores (C); the result of subtraction of the two spectra under C (intermediate component, D); fitting of the intermediate component spectrum as shown in D to the supernatant-free spectrum in B (E); subtraction of the two spectra under E results in the narrow component (F).

Assignment of the spectral components to spore structures The emergence of the narrow and partially broadened triplets indicated that the spin-probe PDT had penetrated into compartments of the spores, which are not accessible to FC ions. To investigate the role of the inner and outer spore-membranes in the selective permeability for PDT and FC, we disrupted the two spore-membranes by bead beating. This led to a complete disappearance of the narrow component (Fig. 3C), indicating that the narrow component originated from a membrane-enclosed spore-compartment. However, a partially broadened component still remained, which we call the intermediate component to avoid confusion with the broadened component from the interstitial fluid. After correction for the interstitial fluid, the spectrum from completely disrupted spores consisted of only the intermediate component (Fig. 3D), and this component was used to obtain the shape of the narrow component from intact spores, by subtraction (Fig. 3E, F). While broadening by FC via spin-spin interaction causes equal broadening of all three lines, the lines in the intermediate component were differentially broadened, indicating motional restriction of the PDT molecules. This means that the PDT molecules generating this signal were not in a bulk aqueous phase but in an environment within the spores, where their tumbling velocity was lower than the lifetime of the resonance state (Marsh, 1981). This environment was not

73

Chapter 5

accessible for FC ions even when both spore-membranes were disrupted. Such an environment is provided by small cavities in polymers, accessible for small amphiphilic PDT molecules, but not for large, negatively charged FC ions. The movement of spin-probe molecules in such cavities is restricted and relatively slow. The coat of bacterial spores consists of dense layers of highly crosslinked proteins (Driks, 1999; 2002), a structure prone to harbor such cavities. In addition, the cortex could harbor such cavities, because it consists of loosely cross-linked peptidoglycan polymer (Popham and Setlow, 1993), while the negative charge of the cortex polymer (Gould and Dring, 1975) could play a role in the exclusion of the FC ions from the cavities. For these reasons, we propose that the intermediate component originates from PDT molecules situated in molecular cavities of the coat and/or cortex.

Fig. 4. Decomposition procedures with ESR PDT spectra from dormant B. subtilis spores to obtain the narrow component from the core (A-C) and from the cortex (D-E). Fitting of the supernatant spectrum to the spectrum from decoated spores (A); the difference between the two spectra under A (the supernatant-free decoated spectrum, B); also shown in B is the fitting of the intermediate component spectrum to the supernatant-free decoated spectrum; the difference of the two spectra under B gives the core narrow component (C); fitting of the supernatant-free decoated spectrum (as in B) to the supernatant-free spectrum from intact spores (D); the difference of the two spectra under D gives the cortex narrow component (E); absorption (integrated) spectra of the narrow components from the core as shown in C and the cortex as shown in E (F). The areas beneath these spectra permit the relative volumes of the spore compartments to be calculated.

The narrow component contained very sharp peaks (Fig. 3F). The distance between the lines (isotropic hyperfine splitting, aiso) was the same as in the spectrum of 1 mM PDT in water (Fig. 2A). Being indicative of the polarity of the environment (reviewed in Marsh, 1981), this similar aiso

74

Water in the core of Bacillus spores is not in a glassy state: a spin-probe study

value indicates that the narrow component originated from bulk aqueous phase. We proceeded to investigate from which membrane-enclosed compartment of the spores the narrow component originated. Bacterial spores contain two membranes, which can easily be seen by electron microscopy at the early stages of spore formation (see Ellar and Lundgren, 1966; Young and FitzJames, 1959). In the dormant spore, the outer membrane is located in between the cortex and coat (Crafts-Lighty and Ellar, 1980), while the inner membrane surrounds the core (see Cowan et al.,

Inner membrane

Coat

1 Core

2

Cortex

A

Outer membrane

Inner membrane

Coat

3 Core

Cortex

B

4

Outer membrane

Fig. 5. Schematic representation of the penetration of FC and PDT into intact spores (A) and decoated spores (B). FC (dark grey area) is blocked by intact membranes, while PDT (light grey area) diffuses freely. The different ESR signals from spore samples originate from the interstitial fluid (1), the cortex-plus-core region in intact spores (2), molecular cavities in the coat/cortex in intact, decoated and disrupted spores (3), and the core in decoated spores (4).

2004). Thus, two different membrane-enclosed compartments are present: the core, and the cortexplus-core compartment. Disruption of the outer membrane resulted in a dramatic decrease of the narrow signal while the intermediate signal remained present (Fig. 4A-C). This means that, in intact spores, the outer membrane is an effective barrier to the diffusion of FC ions, and that the narrow signal from intact spores originates largely from the bulk aqueous phase in the cortex compartment. However, a reduced proportion of the narrow component remained present after decoating. The only membrane-enclosed compartment left in decoated spores is the core, and therefore, we conclude that the narrow signal from decoated spores originates from the spore core. Fig. 5 schematically represents the location of PDT molecules in the

75

Chapter 5

intact and decoated dormant spore, and the spectra generated by from these PDT molecules. The narrow component obtained from intact spores is a superposition of two non-broadened signals from bulk aqueous phase, one originating from the cortex and another one originating from the core. Characteristics of the core and cortex signals Subtraction of the intermediate component from the spectrum of decoated spores shows the shape of the narrow component originating from PDT molecules in the spore core (Fig. 4B, C). From this spectrum, spectral parameters of the core signal were calculated (Table 1). The ratios of the line width coefficients, │C/B│, were larger than 1, indicating a highly structured environment in the core. The microviscosity in the core cytoplasm was high, as derived from the rotational correlation time of PDT molecules in the core. Notably, this rotational correlation time was 6 orders of magnitude faster than in a glassy state (Buitink et al., 1999). Figure 4D shows fitting of supernatant-free spectrum of decoated spores as shown in Fig. 4B to the supernatant-free spectrum of intact spores as shown in Fig. 3B. The resulting signal (Fig. 4E) originates from the bulk aqueous phase in the cortex. The parameters of this spectrum (Table 1) were different from that of the core, indicating that the aqueous phase in the cortex had a lower viscosity than the core cytoplasm. Spore heat activation and germination Upon germination, the unique structure of bacterial spores is degraded, and resistance properties are lost. Key events are rehydration and expansion of the core, excretion of the Ca-DPA from the core, and breakdown of the cortex peptidoglycan (reviewed in Moir et al., 2002; Setlow, 2003). Furthermore, cytoplasmic proteins that are immobile in dormant spores become mobile after germination (Cowan et al., 2003). Heat activation, which is known to enhance the germination response of spores (Keynan and Evenchik, 1969), did not change the shape of the PDT spectra from either normal or decoated spores (Table 1, spectra not shown). However, placing fully germinated B. subtilis spores (>95 % of DPA released) in a broadened solution of PDT caused the emergence of an ESR spectrum that was highly different from the spectra obtained from dormant spores. The spectrum from germinated spores was characterized by a very dominant narrow component (Fig. 6A), while the intermediate component was reduced considerably. Subtraction of the supernatant and intermediate signals from the spectrum derived from germinated spores, according to procedures as outlined in Fig. 3, revealed the shape of the large narrow component (Fig. 6B). The spectral characteristics of this narrow component indicated that viscosity and anisotropy had decreased markedly upon germination (Table 1). This is consistent with core rehydration and breakdown of the core internal structure. For B. cereus, similar trends were observed (Table 1; spectra not shown). However, τR and the related micro viscosity in dormant B. cereus spores were significantly higher than in dormant B. subtilis spores, while germination resulted in reduction to values similar to that in germinated B. subtilis spores (Table 1). The role of Ca-DPA in the structure of the spore core To study the role of dipicolinic acid in the physical structure of the spore, we used two mutants of B. subtilis; FB113 and CwlD. Spores from these mutants excrete their Ca-DPA upon germination, but subsequently remain in an intermediate stage, which is characterized by a slightly increased core water content, greatly reduced enzyme activity in the core, and core proteins that remain immobile, as in dormant spores (Cowan et al., 2003; Setlow et al., 2001). The spectra from dormant FB113 spores (Fig. 6C, D) had a line-shape similar to that from the parental strain PS832 (Fig. 2B, 3F), and the spectral parameters of the narrow component from dormant FB113 spores were close to those observed for PS832 (Table 1). The release of >95% of the DPA (Fig. 1) from

76

Water in the core of Bacillus spores is not in a glassy state: a spin-probe study

the FB113 mutant spores resulted in a slight increase of the proportion of the narrow component (Fig. 6E), but not by far as substantial as in germinated PS832 spores (Fig. 6A), while the spectral parameters calculated from the shape of the narrow component did not change significantly (Compare Fig. 6D with Fig. 6F; Table 1). This indicated that the core and cortex structure had remained intact, despite the excretion of nearly all of the DPA from the core. For the CwlD mutant, similar results were obtained (Table 1; spectra not shown).

A

C

B

D

E

F

Fig. 6. ESR PDT spectra from germinated spores of wild-type B. subtilis (A) and its narrow component (B) obtained according to procedures outlined in Fig. 2. For comparison, ESR spectra from dormant (C) and germinated (E) spores of the B. subtilis mutant FB113 are shown, with their respective narrow components (D, F).

The proportion of bulk water in the core of bacterial spores A key question in relation to the resistance of spores is how much bulk water is present in the core (Setlow, 1994; 2000). The proportion of bulk water in the core was estimated on the basis of the relative proportion of PDT molecules in the core and cortex. The area beneath the absorption curve represents the number of paramagnetic centers (i.e. PDT molecules) responsible for each signal, and was calculated by double integration. Fig. 4F shows the integrated spectra from the narrow components derived from the cortex and the core. After subtraction and baseline correction, the relative amount of the core bulk water was estimated to be approximately 40% of the total (core-plus-cortex) in dormant B. subtilis spores and approximately 34-37% in dormant B. cereus spores (spectra not shown).

77

Chapter 5

Discussion Using a spin-probe-based ESR method, we have measured physical properties of the aqueous environment in the spore core in vivo in a direct manner. Other attempts employing NMR and DSC fell short because these techniques are unable to discriminate between the different spore compartments. ESR has been applied to spores before, for example for measurement of paramagnetic metal signals (Windle and Sacks, 1993). Also, hydrophobic spin-probes have been used for the estimation of spore membrane properties, however, these investigations suffered from lack of proof about the exact location of the probes employed (see Cowan et al., 2004 and references therein). From our measurements it became clear that the cytoplasm in the core of dormant bacterial spores is not in a glassy state, but organized as a highly structured matrix with entrapped bulk water, as has been proposed over forty years ago by Black and Gerhardt (1962b). The presence of bulk water in the spore compartments can be expected from thermodynamic consideration of equal water activity on both sides of the spore membranes. Glasses do exist in biological systems such as desiccated plant seeds and pollen, but at very low water contents, were bulk water is absent (Williams and Leopold, 1989). In comparison to those systems, the water content in the core of bacterial spores is relatively high. Our findings imply that dormancy, heat resistance, and molecular immobility cannot be attributed to a glassy state in the core. As pointed out by Gould (1986), mechanical entrapment in polymethacrylate gels with water contents as high as 50 % (comparable with the water content of the core of B. cereus spores [Beaman et al., 1986]), can increase the resistance of proteins to heat by factors as great as 105-fold. Thus, the entrapment of proteins in the core matrix contributing to heat resistance is an appealing alternative for the hypothesis of a glassy state. The model of a flexible three-dimensional macro-molecular matrix in the spore-core containing entrapped bulk water is appealing, because this model explains the immobility of proteins, ions, and DPA in the core, while water is still freely mobile and exchangeable. As such, this model is consistent with recent reports describing that spores can take up water and swell upon increase of relative humidity (Driks, 2003; Plomp et al., 2005; Westphal et al., 2003), but that water uptake does not result in increased mobility of DPA in the core (Leuschner and Lillford, 2000). Our findings also imply that the phenomenon of heat activation cannot be attributed to a glass transition in the core, as has been repeatedly suggested (Ablett et al., 1999; Gould, 1986; Sapru and Labuza, 1993). We observed no change in core and cortex structure upon heat activation, which supports the view that heat activation does not evoke structural changes in the cortex or core, but rather affects coat permeability to nutrient molecules or induces a structural change of the nutrient receptors, as was proposed earlier (Hashimoto and Conti, 1971; Johnstone, 1994; Keynan and Evenchik, 1969; Leuschner and Lillford, 1999). We found that the coat/outer membrane complex of dormant spores is impermeable to FC ions, but not to PDT molecules. Previously, the integrity of the outer membrane has been questioned, and the outer membrane was suggested to have a function only during sporulation (Tipper and Gauthier, 1972). However, in B. megaterium spores, the outer membrane is intact (Crafts-Lighty and Ellar, 1980). In the past, the outer membrane of dormant B. cereus spores was believed to be permeable to a large variety of molecules (Black et al., 1960; Black and Gerhardt, 1961; 1962; 1962b; Gerhardt and Black 1961; 1961b; Gerhardt et al., 1972), and although the authors have amended their views and identified the outer membrane as an important permeability barrier in later publications (see Gerhardt et al., 1982; Koshikawa et al., 1984; Nakashio et al., 1985), the early beliefs that the outer membrane is highly permeable are still propagated in current literature. PDT molecules diffuse freely over the spore membranes, as was demonstrated by the non-broadened signal that we obtained from intact dormant spores. This finding is consistent with

78

Water in the core of Bacillus spores is not in a glassy state: a spin-probe study

the amphiphilic nature of the PDT molecule, which has been shown previously to rapidly diffuse over intact membranes (Golovina et al., 2001). The inner membrane has been claimed to be in an immobile state and is supposed to be nearly impermeable, even to small molecules (Cowan et al., 2004; Swerdlow et al., 1981). Nevertheless, water and other small molecules, including various DNA damaging agents, can permeate across the inner membrane (Black and Gerhardt, 1962b; Cortezzo et al., 2005; Leuschner and Lillford, 2000; Rode and Foster, 1960; Setlow, 2000; Setlow and Setlow, 1980). The ESR spectrum we obtained from decoated spores contained a narrow component, which confirms that diffusion of PDT over the inner membrane had taken place. This was supported by the disappearance of the narrow component upon disruption of the inner membrane. Finally, spin-probe-based ESR allowed us to assess the relative proportion and state of the bulk core water for the first time. These parameters are of key importance to spore heat resistance and dormancy (Cowan et al., 2003; Setlow, 2000), and many reports have addressed spore core water content and the core-water activity (Algie, 1980; 1983; 1984; 1984b; Beaman et al., 1982; Black and Gerhardt, 1962b; Bradbury et al., 1981; Carstensen et al., 1971; Gould, 1986; Lindsay et al., 1985; Popham et al., 1995). Since a portion of the core water may be bound to the core matrix, estimations of the total core water content cannot be used to estimate the amount of bulk water in the core (Beaman et al., 1984; Setlow, 1994; 2000). Furthermore, viscosity rather than water content or water activity per se is of key importance for heat resistance (Careri, 1982). We found that the water in the core is indeed highly viscous, and that the proportion of bulk water in core and cortex is in the range of the volume relations between these compartments – approximately 30-40% for the core and 60-70% for the cortex (Algie, 1984; Beaman et al., 1982). This suggests that the relative amount of bulk water in the core is similar in both compartments, and therefore the high resistance of spores cannot be attributed to a very low amount of bulk water in the core. Conclusions In conclusion, the spin-probe-based ESR method described in this communication has provided us with a new set of parameters for spore research. It is now possible to estimate to what extent the core-water viscosity and the degree of structuring in the core contribute to spore heat resistance. Systematic study of very heat-resistant isolates is likely to provide new clues about the mechanism of their extreme resistance. Such new data will aid in the development and evaluation of more efficient methods for spore inactivation. References 1. 2. 3. 4. 5. 6.

Ablett, S., A. H. Darke, P. J. Lillford, and D. R. Martin. 1999. Glass formation and dormancy in bacterial spores. Int J Food Sci Technol 34:59-69. Aguilera, J. M., and M. Karel. 1997. Preservation of biological materials under desiccation. Crit Rev Food Sci Nutr 37:287-309. Algie, J. E. 1984. Effect of the internal water activity of bacterial spores on their heat resistance in water. Curr Microbiol 11:293-296. Algie, J. E. 1983. The heat resistance of bacterial spores and its relationship to the contraction of the forespore protoplasm during sporulation. Curr Microbiol 9:173-176. Algie, J. E. 1980. The heat resistance of bacterial spores due to their partial dehydration by reverse osmosis. Curr Microbiol 3:287-290. Algie, J. E., and I. C. Watt. 1984b. Calculation of mass and water content between core, cortex and coat of B. stearothermophilus. Curr Microbiol 10:249-254.

79

Chapter 5

7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

80

Atrih, A., and S. J. Foster. 2001. Analysis of the role of bacterial endospore cortex structure in resistance properties and demonstration of its conservation amongst species. J Appl Microbiol 91:364-372. Beaman, T. C., and P. Gerhardt. 1986. Heat resistance of bacterial spores correlated with protoplast dehydration, mineralization, and thermal adaptation. Appl Environ Microbiol 52:1242-1246. Beaman, T. C., J. T. Greenamyre, T. R. Corner, H. S. Pankratz, and P. Gerhardt. 1982. Bacterial spore heat resistance correlated with water content, wet density, and protoplast/sporoplast volume ratio. J Bacteriol 150:870-877. Beaman, T. C., T. Koshikawa, H. S. Pankratz, and P. Gerhardt. 1984. Dehydration partitioned within core protoplast accounts for heat resistance of bacterial spores. FEMS Microbiol Lett 24:47-51. Black, S. H., and P. Gerhardt. 1961. Permeability of bacterial spores. I. Characterization of glucose uptake. J Bacteriol 82:743-749. Black, S. H., and P. Gerhardt. 1962. Permeability of bacterial spores. III. Permeation relative to germination. J Bacteriol 83:301-308. Black, S. H., and P. Gerhardt. 1962b. Permeability of bacterial spores. IV. Water content, uptake, and distribution. J Bacteriol 83:960-967. Black, S. H., R. E. MacDonald, T. Hashimoto, and P. Gerhardt. 1960. Permeability of dormant bacterial spores. Nature 185:782-783. Bradbury, J. H., J. R. Foster, B. Hammer, J. Lindsay, and W. G. Murrell. 1981. The source of the heat resistance of bacterial spores. Study of water in spores by NMR. Biochim Biophys Acta 678:157-164. Brown, K. L. 1994. Spore resistance and ultra heat treatment processes. Soc Appl Bacteriol Symp Ser 23:67S-80S. Buitink, J., S. A. Dzuba, F. A. Hoekstra, and Y. D. Tsvetkov. 2000. Pulsed EPR spinprobe study of intracellular glasses in seed and pollen. J Magn Reson 142:364-368. Buitink, J., M. A. Hemminga, and F. A. Hoekstra. 1999. Characterization of molecular mobility in seed tissues: an electron paramagnetic resonance spin probe study. Biophys J 76:3315-3322. Careri, G. 1982. Molecular hydration and its possible role in enzymes, p. 58-61. In F. Franks and S. F. Mathias (ed.), Biophysics of water. John Wiley, Chichester. Carstensen, E. L., R. E. Marquis, S. Z. Child, and G. R. Bender. 1979. Dielectric properties of native and decoated spores of Bacillus megaterium. J Bacteriol 140:917-928. Carstensen, E. L., R. E. Marquis, and P. Gerhardt. 1971. Dielectric study of the physical state of electrolytes and water within Bacillus cereus spores. J Bacteriol 107:106-113. Cowan, A. E., D. E. Koppel, B. Setlow, and P. Setlow. 2003. A soluble protein is immobile in dormant spores of Bacillus subtilis but is mobile in germinated spores: implications for spore dormancy. Proc Natl Acad Sci U S A 100:4209-4214. Cowan, A. E., E. M. Olivastro, D. E. Koppel, C. A. Loshon, B. Setlow, and P. Setlow. 2004. Lipids in the inner membrane of dormant spores of Bacillus species are largely immobile. Proc Natl Acad Sci U S A 101:7733-7738. Crafts-Lighty, A., and D. J. Ellar. 1980. The structure and function of the spore outer membrane in dormant and germinating spores of Bacillus megaterium. J Appl Bacteriol 48:135-145. Driks, A. 1999. Bacillus subtilis spore coat. Microbiol Mol Biol Rev 63:1-20. Driks, A. 2003. The dynamic spore. Proc Natl Acad Sci U S A 100:3007-3009. Driks, A. 2002. Maximum shields: the assembly and function of the bacterial spore coat. Trends Microbiol 10:251-254.

Water in the core of Bacillus spores is not in a glassy state: a spin-probe study

28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40.

41. 42. 43. 44. 45. 46. 47.

Ellar, D. J., and D. G. Lundgren. 1966. Fine structure of sporulation in Bacillus cereus grown in a chemically defined medium. J Bacteriol 92:1748-1764. Fitz-James, P. C. 1971. Formation of protoplasts from resting spores. J Bacteriol 105:11191136. Franks, F., R. H. M. Hatley, and S. Mathias. 1991. Materials science and the production of shelf-stable biologicals. BioPharm 4:38-42, 55. Gerhardt, P., T. C. Beaman, T. R. Corner, J. T. Greenamyre, and L. S. Tisa. 1982. Photometric immersion refractometry of bacterial spores. J Bacteriol 150:643-648. Gerhardt, P., and S. H. Black. 1961b. Permeability of bacterial spores, p. 218-229. In H. O. Halvorson (ed.), Spores, vol. II. Burgess Publishing Company, Minneapolis. Gerhardt, P., and S. H. Black. 1961. Permeability of bacterial spores. II. Molecular variables affecting solute permeation. J Bacteriol 82:750-760. Gerhardt, P., R. Scherrer, and S. H. Black. 1972. Melecular sieving by dormant spore structures, p. 68-77. In H. O. Halvorson, R. Hanson, and L. L. Campbell (ed.), Spores, vol. 5. ASM, Fontana, Wisconsin. Gerhardt, P., R. Scherrer, E. L. Carstensen, and R. E. Marquis. 1971. Electric probes of the physical state within dormant spores, p. 341. In A. N. Barker, G. W. Gould, and J. Wolf (ed.), Spore Research 1971. Academic Press, London. Golovina, E. A., F. A. Hoekstra, and A. C. Van Aelst. 2001. The competence to acquire cellular desiccation tolerance is independent of seed morphological development. J Exp Bot 52:1015-1027. Gould, G. W. 1986. Water and the survival of bacterial spores, p. 143-156. In A. C. Leopold (ed.), Membranes, Metabolism, and Dry Organisms. Cornell University Press, Ithaca, USA. Gould, G. W., and G. J. Dring. 1975. Heat resistance of bacterial endospores and concept of an expanded osmoregulatory cortex. Nature 258:402-405. Hashimoto, T., and S. F. Conti. 1971. Ultrastructural changes associated with activation and germination of Bacillus cereus T spores. J Bacteriol 105:361-368. Higgins, J. A., M. Cooper, L. Schroeder-Tucker, S. Black, D. Miller, J. S. Karns, E. Manthey, R. Breeze, and M. L. Perdue. 2003. A field investigation of Bacillus anthracis contamination of U.S. Department of Agriculture and other Washington, D.C., buildings during the anthrax attack of october 2001. Appl Env Microbiol 69:593-599. Johnstone, K. 1994. The trigger mechanism of spore germination: current concepts. Soc Appl Bacteriol Symp Ser 23:17S-24S. Keith, A. D., W. Snipes, and D. Chapman. 1977. Spin-label studies on the aqueous regions of phospholipid multilayers. Biochemistry 16:634-641. Keynan, A. 1972. Cryptobiosis: a review of the mechanisms of the ametabolic state in bacterial spores, p. 355-363. In H. O. Halvorson, R. Hanson, and L. L. Campbell (ed.), Spores, vol. 5. ASM, Fontana, Wisconsin. Keynan, A., and Z. Evenchik. 1969. Activation, p. 359-396. In G. W. Gould and A. Hurst (ed.), The Bacterial Spore, vol. 1. Academic Press, London and New York. Koshikawa, T., T. C. Beaman, H. S. Pankratz, S. Nakashio, T. R. Corner, and P. Gerhardt. 1984. Resistance, germination, and permeability correlates of Bacillus megaterium spores successively divested of integument layers. J Bacteriol 159:624-632. Leuschner, R. G., and P. J. Lillford. 2000. Effects of hydration on molecular mobility in phase-bright Bacillus subtilis spores. Microbiology 146 ( Pt 1):49-55. Leuschner, R. G., and P. J. Lillford. 1999. Effects of temperature and heat activation on germination of individual spores of Bacillus subtilis. Lett Appl Microbiol 29:228-232.

81

Chapter 5

48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66.

67. 68. 69. 70.

82

Leuschner, R. G., and P. J. Lillford. 2001. Investigation of bacterial spore structure by high resolution solid-state nuclear magnetic resonance spectroscopy and transmission electron microscopy. Int J Food Microbiol 63:35-50. Leuschner, R. G., and P. J. Lillford. 2003. Thermal properties of bacterial spores and biopolymers. Int J Food Microbiol 80:131-143. Lewis, J. C. 1969. Dormancy, p. 301-358. In G. W. Gould and A. Hurst (ed.), The Bacterial Spore, vol. 1. Academic Press, London and New York. Lindsay, J. A., T. C. Beaman, and P. Gerhardt. 1985. Protoplast water content of bacterial spores determined by buoyant density sedimentation. J Bacteriol 163:735-737. Marsh, D. 1981. Electron spin resonance: spin labels. Mol Biol Biochem Biophys 31:51142. Moir, A., B. M. Corfe, and J. Behravan. 2002. Spore germination. Cell Mol Life Sci 59:403-409. Movahedi, S., and W. Waites. 2002. Cold shock response in sporulating Bacillus subtilis and its effect on spore heat resistance. J Bacteriol 184:5275-5281. Nakashio, S., and P. Gerhardt. 1985. Protoplast dehydration correlated with heat resistance of bacterial spores. J Bacteriol 162:571-578. Newsome, R. 2003. Dormant microbes: research needs. Food Tech. 57:38-42. Nicholson, W. L., and P. Setlow. 1990. Sporulation, germination and outgrowth, p. 391451. In C. R. Harwood and S. M. Cutting (ed.), Molecular Biological Methods for Bacillus. John Wiley and Sons, Chichester. Plomp, M., T. J. Leighton, K. E. Wheeler, and A. J. Malkin. 2005. The high-resolution architecture and structural dynamics of Bacillus spores. Biophys J 88:603-608. Popham, D. L., S. Sengupta, and P. Setlow. 1995. Heat, hydrogen peroxide, and UV resistance of Bacillus subtilis spores with increased core water content and with or without major DNA-binding proteins. Appl Environ Microbiol 61:3633-3638. Popham, D. L., and P. Setlow. 1993. The cortical peptidoglycan from spores of Bacillus megaterium and Bacillus subtilis is not highly cross-linked. J Bacteriol 175:2767-2769. Sapru, V., and T. P. Labuza. 1993. Glassy state in bacterial spores predicted by polymer glass-transition theory. J Food Sci 58:445-448. Setlow, B., E. Melly, and P. Setlow. 2001. Properties of spores of Bacillus subtilis blocked at an intermediate stage in spore germination. J Bacteriol 183:4894-4899. Setlow, B., and P. Setlow. 1980. Measurements of the pH within dormant and germinated bacterial spores. Proc Natl Acad Sci U S A 77:2474-2476. Setlow, P. 2000. Resistance of bacterial spores, p. 217-230. In G. Storz and R. HenggeAronis (ed.), Bacterial stress responses. ASM press, Washington, D.C. Setlow, P. 2003. Spore germination. Curr Opin Microbiol 6:1-7. Stewart, G. S., M. W. Eaton, K. Johnstone, M. D. Barrett, and D. J. Ellar. 1980. An investigation of membrane fluidity changes during sporulation and germination of Bacillus megaterium K.M. measured by electron spin and nuclear magnetic resonance spectroscopy. Biochim Biophys Acta 600:270-290. Swerdlow, B. M., B. Setlow, and P. Setlow. 1981. Levels of H+ and other monovalent cations in dormant and germinating spores of Bacillus megaterium. J Bacteriol 148:20-29. Tipper, D. J., and J. J. Gauthier. 1972. Structure of the bacterial endospore, p. 3-13. In H. O. Halvorson, R. Hanson, and L. L. Campbell (ed.), Spores, vol. 5. ASM, Fontana, Wisconsin. Vary, J. C. 1973. Germination of Bacillus megaterium spores after various extraction procedures. J Bacteriol 116:797-802. Warth, A. D. 1978. Molecular structure of the bacterial spore. Adv Microb Physiol 17:1-45.

Water in the core of Bacillus spores is not in a glassy state: a spin-probe study

71. 72. 73.

Westphal, A. J., P. B. Price, T. J. Leighton, and K. E. Wheeler. 2003. Kinetics of size changes of individual Bacillus thuringiensis spores in response to changes in relative humidity. Proc Natl Acad Sci U S A 100:3461-3466. Williams, R. J., and A. C. Leopold. 1989. The glassy state in corn embryos. Plant Physiol 89:977-981. Young, I. E., and P. C. Fitz-James. 1959. Chemical and morphological studies of bacterial spore formation. II. Spore and parasporal protein formation in Bacillus cereus var. alesti. J Biophys Biochem Cytol 6:483-498.

83

6. Bacillus cereus Spore Germination Quantified by Flow-Cytometry Ynte P. de Vries, Kaouther Ben Amor, Luc M. Hornstra, Willem M. de Vos, Marcel Zwietering and Tjakko Abee Manuscript in preparation

ABSTRACT Individual spores from Bacillus cereus were monitored during germination with flow cytometry (FCM). Dormant spores were not stainable by fluorescent dyes, but after addition of combinations of L-alanine and inosine the majority of the spores quickly became stainable by fluorescent esterase markers and DNA-binding dyes. This indicated activation of lytic enzymes and an increase in permeability of the spore membranes, both of which are events in the process of spore germination. Our study demonstrates that FCM is a powerful method for the quantification of individual germination events and the assessment of heterogeneity in germinating spore populations. Furthermore, the use of different fluorescent dyes in combination with FCM allowed for quantification of various specific stadia of germination in spore populations.

85

Chapter 6

Introduction Spore germination is the process whereby spores change from the dormant state to a metabolically active state. As mentioned in Chapter 1, the process of spore germination involves a rapid sequence of events in which the structure of the spore is degraded. It is through germination and subsequent growth that spores cause food spoilage and potential toxin formation, which may ultimately lead to food-borne disease. Furthermore, because resistance properties of the spore are concomitantly lost, the mechanism of germination may hold clues to more efficient methods for spore inactivation. Therefore, apart from being scientifically interesting, the process of spore germination is of great importance from an applied perspective. In the first stages of spore germination, the large depot of dipicolinic acid (DPA) is excreted from the spore core while water is taken up (Moir et al., 2002; Setlow, 2003). These processes lead to a loss of spore refractility, and as a result, a decrease of the absorbance is observed when germination occurs in a spore suspension. This is the basis of the most commonly employed method for measuring spore germination, which involves real-time measurement of the absorbance. Alternatively, the increase of DPA in the supernatant can be measured with a fluorescence-based technique, as used in Chapters 3 and 5. Because the curves obtained with these methods are superpositions of 107-108 individual germination events, data on the behavior of individual spores are lost. These data are important, because the heterogeneous distribution of dormancy in spore populations may result in a number of spores that, in response to germination stimuli, do not germinate, and retain their resistance properties. This causes difficulties in the application of germination as a spore control procedure (Gould et al., 1968; Gould, 1970; Gould and Sale, 1970). Therefore, a method for the study of the individual germination responses and assessment of heterogeneity in germinating spore populations is needed. Phase-contrast microscopic studies have indicated that the germination response of a single B. cereus spore consists of a lag-phase (microlag) and a biphasic event in which the actual germination reactions take place (Hashimoto et al., 1969; 1969b; 1972; Vary and Halvorson, 1965). Heat activation stimulates the germination of spores primarily by reducing the microlag times, and the kinetics of germination of spore suspensions are most critically influenced by the microlag time of each member of that population (Hashimoto et al., 1972). More recent analysis confirmed that the microlag was affected by heat activation treatment and indicated that the germination phase varied considerably with germination temperature (Leuschner and Lillford, 1999). Unfortunately, the methods employed in these studies allow only the phase-change of the spores to be monitored, and are very time-consuming, as each individual spore is analyzed manually. Advances in fluorescence-based flow cytometry (FCM) allow for semi-automated analysis of the behavior of individual spores and estimation of heterogeneity in large spore populations. Recently, the membrane potential of germinating spores was monitored with FCM (Laflamme et al., 2005), and earlier, FCM has been successfully applied in combination with fluorescent labeling for the detection of spores from Bacillus anthracis (Stopa, 2000), and for the enumeration of spores in sporulating cultures of Peanibacillus polymyxa (Comas-Riu and Vives-Rego, 2002). In this study we describe an FCM approach for real-time quantification of germination events in suspensions of germinating spores from the important food-borne pathogen Bacillus cereus. Materials and methods Spore preparation and germination measurements The spores used in this study were prepared on defined medium containing glucose, and harvested, washed and stored as described in Chapter 2. For FCM analysis, spores (107/ml) were washed by spinning at 4,000 x g for 5 min and then resuspended in germination buffer (10 mM Tris/HCl, pH 7.4, 10 mM NaCl and 0.1% TWEEN80). Germination was performed without heat

86

Bacillus cereus spore germination quantified by flow-cytometry

activation, and initiated by the addition of 0.1 mM L-alanine in combination with different concentrations of inosine (0, 0.01, 0.05 and 0.1mM). Samples were incubated at 37 °C and aliquots were taken at different time intervals and centrifuged at 5,000 x g for 5 min. The pellet was suspended in phosphate buffer saline (PBS) to a concentration of 106 to 5 x 106 spores/ml and then stained as described previously (Ben Amor et al., 2002) with 5,(and 6)-Carboxyfluorescein diacetate (cFDA), Propidium iodide (PI) or SYTO BC, obtained from the bacterial viability kit (Molecular Probes Europe BV, Leiden, The Netherlands). SYTO BC was used as described by the manufacturer. Control samples consisting of non-induced spores were included, and all samples were thoroughly vortexed prior to FCM analysis. FCM analysis FCM analysis was carried out with a FACSCalibur flow cytometer (Beckton Dickinson Immunocytometry Systems, San Jose, California, USA) equipped with an air-cooled argon ion laser emitting 15 mW of blue light at 488 nm and with the standard filter set-up. SYTO BC and cFDA fluorescence were collected using a 530 ± 30 nm bandpass filter (FL1 channel); the red fluorescence emitted from PI was collected by a 650 ± 13 nm bandpass filter (FL3-channel). All analyses were performed at low flow-rate settings (~12 µl/min) while the count was lower than 1000 events/s. Data were collected in list mode as pulse height signals (four decades in logarithmic scale each) and analyzed with CellQuestPro software. Windows Multiple Document Interface software (WinMDI, Joseph Totter, Salk Institute for Biological Studies, La Jolla, CA, USA. http//:facs.Scripps.edu/software.html) was also used to analyze the data. Control samples as well as beads (CaliBRITE, Becton Dickinson) were used for the instrument calibration (voltage of the detectors and threshold) and for checking the instrument sensitivity over time. Spores were discriminated from background using a double threshold set on both side scatter (SSC) and forward scatter (FSC), with FSC set on E01 and SSC on 300V. A dual dot plot FSC versus SSC in combination with a one parameter histogram representing the green fluorescence (FL1) was used to back gate spores and distinguish them from the background.

Fig. 1. Changes in scatter parameters caused by germination. Left: ungerminated control; Right: spores germinated (>99% loss of heat resistance) in a mixture of L-alanine and inosine (0.1mM each) for 10 minutes at 37 ºC. Region R1 is depicted for reference at the same position in both plots.

87

Chapter 6

Results and Discussion We developed and applied flow-cytometry approaches to monitor individual spores of B. cereus during germination. A large number (several thousands) of individual spores in germinating suspensions was analyzed and the kinetics of the response to the germinants was determined. Fig. 1 shows the changes in the scatter parameters that occur upon germination. Each dot in the plot represents one single spore, and the spreading of the dots in the scatter plot reflects the heterogeneity in the population. The signals of dormant spores were clustered, and the germination event caused a major shift towards lower scatter values (Fig. 1). This is likely to be associated with swelling of the spores or the phase-change that occurs in the spores during germination.

A

B

C

Fig. 2. Changes in SYTO staining upon germination. Ungerminated control (A); Spores germinated for 10 minutes at 37 ºC in L-alanine (0.1 mM; >74% loss of heat resistance) (B); or in a mixture of L-alanine and inosine (1mM each; >99% loss of heat resistance) (C). Regions R2 and R3 are depicted for reference at the same position in all three plots. Region R2: stained spores; Region R3: unstained spores. Between R2 and R3 the intermediately stained spores are clearly visible in B.

Staining with the fluorescent DNA-binding dyes SYTO and propidium iodide (PI) allowed the accessibility of the spore DNA and the status of the cytoplasmic membrane to be assessed (see Amor et al., 2002). Dormant spores were not stained (Fig. 2A), confirming the previously reported impermeability of dormant spores to DNA-binding dyes (Setlow et al., 2002; Welkos et al., 2004). During germination, however, the spores became stainable by SYTO (Fig. 2B,C), but not by PI (data not shown). SYTO can permeate across intact membranes, and stains living and dead cells, while PI can only permeate over damaged membranes, specifically staining damaged and dead cells (see Amor et al., 2002). Over a 60 minute time-course, none of the spores became stainable by PI (data not shown), indicating that the freshly germinated spores remained viable during this time course, even while suspended in nutrient-poor germination buffer. Furthermore, this confirmed that the small amount of Tween80, which was added to the buffer to prevent spore clumping, did not damage the cytoplasmic membrane of dormant or germinated spores. The number of spores that remained unstained depended on the time of incubation with the germinants, and on the concentration of the germinants. Early in germination, spore suspensions exhibit esterase activity that can be measured with fluorogenic substrates. This esterase activity is believed to be caused by the spore lytic enzyme system that is activated during germination (Ferencko et al., 2004). By using the substrate cFDA in combination with FCM, we found that the esterase activity in germinating spore suspensions was specifically associated with the spores themselves. The number of spores that showed esterase activity corresponded to the number of spores that became stainable with SYTO over time. Interestingly, a significant group of intermediately stained spores was observed, both with SYTO staining (Fig. 2B) and with cFDA staining (Fig. 3).

88

Bacillus cereus spore germination quantified by flow-cytometry

Fig. 3. Changes in the percentage of spores that is stained with cFDA in germinating spore populations, in response to different germinants. A. L-alanine (0.1 mM). B. Inosine (1 mM). C. Inosine (5 mM). D. L-alanine combined with inosine (0.1 mM each). Black: Unstained spores; Dark grey: Stained spores; Light grey: Intermediately stained spores.

This group of intermediately stained spores was present in nearly all germinating spore suspensions, and fluctuated with germinant type and incubation time (Fig. 3). This group possibly represents a transient stage in germination, and may consist of spores that for example have excreted their DPA and taken up some water, but whose DNA is not yet fully accessible to SYTO because it is still condensed and complexed with SASP proteins, while their lytic enzyme system is only partly activated. This would correspond to stage I of a recently proposed germination pathway (Fig. 4). Mutants blocked in cortex degradation cannot complete germination, and remain in stage I (Setlow et al., 2001). Inclusion of such mutants in future experiments may shed more light on this issue.

89

Chapter 6

Fig. 4. Sequence of events during the process of spore germination. The stainability with SYTO and cFDA of the spores in the proposed different stages is indicated. Adapted from Setlow, 2003.

Incubation with a mixture of L-alanine and inosine, the strongest germinant for B. cereus (Barlass et al., 2002), caused staining of nearly 100% of the spores within 10 minutes with cFDA (Fig. 3D) and SYTO (Fig. 2), while the heat resistance of the spores (not shown) was concomitantly lost. Notably, when used as a single germinant, 5 mM of inosine led to a lower number of germinating spores than 1 mM of inosine (Fig. 3B, C). This inverse correlation between germination induction and inosine concentration is in contrast to L-alanine induced germination, and was also observed when germination was measured by decrease in absorbance (L. M. Hornstra, unpublished results). Furthermore, previous experiments indicated that inosine-induced germination is inhibited by exogenous Ca2+ en Mg2+, while L-alanine induced germination is not (Y. de Vries, unpublished results). These results suggest that the inosine-induced germination of B. cereus spores is mechanistically different from L-alanine induced germination. Indeed, it is known that inosineinduced germination is mediated by more than one germination receptor (Barlass et al., 2002; Clements et al., 1998). Furthermore, the spore coats seem to be involved in inosine induced germination but not so much in L-alanine induced germination (Senesi et al., 1992; Shibata et al., 1992; 1993). Finally, the kinetics of inosine-mediated germination differ from those of L-alanine induced germination (Preston and Douthit, 1984; 1988). The exact mechanism whereby inosine induces germination in B. cereus spores remains an interesting topic for future germination studies. Conclusions In conclusion, we have shown that FCM allows automated analysis of a large number of spores, and is highly suitable for the assessment and quantification of germination events in germinating spore suspensions. The combination with specific fluorescent dyes and labels enabled

90

Bacillus cereus spore germination quantified by flow-cytometry

quantification of important germination parameters, such as specific enzyme activity and membrane permeability. This method is ideally suitable for investigation of heat-damaged spores, which have a very heterogeneous germination response and often show delayed colony formation capacity (Y. de Vries, unpublished data). Large amounts of quantitative data on the behavior of such stressed spores, which is easily generated with FCM, can be readily applied in risk assessments with respect to food processing. Acknowledgements The authors wish to express their gratitude towards Rakel Conesa for performing the heat resistance assays. References: 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Amor, K. B., P. Breeuwer, P. Verbaarschot, F. M. Rombouts, A. D. Akkermans, W. M. De Vos, and T. Abee. 2002. Multiparametric flow cytometry and cell sorting for the assessment of viable, injured, and dead bifidobacterium cells during bile salt stress. Appl Environ Microbiol 68:5209-5216. Barlass, P. J., C. W. Houston, M. O. Clements, and A. Moir. 2002. Germination of Bacillus cereus spores in response to L-alanine and to inosine: the roles of gerL and gerQ operons. Microbiology 148:2089-2095. Clements, M. O., and A. Moir. 1998. Role of the gerI operon of Bacillus cereus 569 in the response of spores to germinants. J Bacteriol 180:6729-6735. Comas-Riu, J., and J. Vives-Rego. 2002. Cytometric monitoring of growth, sporogenesis and spore cell sorting in Paenibacillus polymyxa (formerly Bacillus polymyxa). J Appl Microbiol 92:475-481. Ferencko, L., M. A. Cote, and B. Rotman. 2004. Esterase activity as a novel parameter of spore germination in Bacillus anthracis. Biochem Biophys Res Commun 319:854-858. Gould, G. W. 1970. Symposium on bacterial spores: IV. Germination and the problem of dormancy. J Appl Bacteriol 33:34-49. Gould, G. W., A. Jones, and C. Wrighton. 1968. Limitations of the initiation of germination of bacterial spores as a spore control procedure. J Appl Bacteriol 31:357-366. Gould, G. W., and A. J. Sale. 1970. Initiation of germination of bacterial spores by hydrostatic pressure. J Gen Microbiol 60:335-346. Hashimoto, T., W. R. Frieben, and S. F. Conti. 1969. Germination of single bacterial spores. J Bacteriol 98:1011-1020. Hashimoto, T., W. R. Frieben, and S. F. Conti. 1972. Kinetics of germination of heatinjured Bacillus cereus spores, p. 409-416. In H. O. Halvorson, R. Hanson, and L. L. Campbell (ed.), Spores, vol. 5. ASM, Fontana, Wisconsin. Hashimoto, T., W. R. Frieben, and S. F. Conti. 1969. Microgermination of Bacillus cereus spores. J Bacteriol 100:1385-1392. Helgason, E., O. A. Okstad, D. A. Caugant, H. A. Johansen, A. Fouet, M. Mock, I. Hegna, and Kolsto. 2000. Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis-one species on the basis of genetic evidence. Appl Environ Microbiol 66:2627-2630. Laflamme, C., J. Ho, M. Veillette, M. C. de Latremoille, D. Verreault, A. Meriaux, and C. Duchaine. 2005. Flow cytometry analysis of germinating Bacillus spores, using membrane potential dye. Arch Microbiol 183:107-112. Leuschner, R. G., and P. J. Lillford. 1999. Effects of temperature and heat activation on germination of individual spores of Bacillus subtilis. Lett Appl Microbiol 29:228-232.

91

Chapter 6

15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

92

Moir, A., B. M. Corfe, and J. Behravan. 2002. Spore germination. Cell Mol Life Sci 59:403-409. Preston, R. A., and H. A. Douthit. 1988. Functional relationships between L- and Dalanine, inosine and NH4Cl during germination of spores of Bacillus cereus T. J Gen Microbiol 134 ( Pt 11):3001-3010. Preston, R. A., and H. A. Douthit. 1984. Stimulation of germination of unactivated Bacillus cereus spores by ammonia. J Gen Microbiol 130 ( Pt 5):1041-1050. Senesi, S., G. Freer, G. Batoni, S. Barnini, A. Capaccioli, and G. Cercignani. 1992. Role of spore coats in the germinative response of Bacillus cereus to adenosine and its analogues. Canadian Journal of Microbiology 38:38-44. Setlow, B., C. A. Loshon, P. C. Genest, A. E. Cowan, C. Setlow, and P. Setlow. 2002. Mechanisms of killing spores of Bacillus subtilis by acid, alkali and ethanol. J Appl Microbiol 92:362-375. Setlow, B., E. Melly, and P. Setlow. 2001. Properties of spores of Bacillus subtilis blocked at an intermediate stage in spore germination. J Bacteriol 183:4894-4899. Setlow, P. 2003. Spore germination. Curr Opin Microbiol 6:1-7. Shibata, H., S. Adachi, Y. Hirose, M. Ike, I. Tani, and T. Hashimoto. 1993. Role of calcium in biphasic germination of Bacillus cereus T spores sensitized to lysozyme. Microbiology and Immunology 37:187-194. Shibata, H., S. Miyoshi, T. Osato, I. Tani, and T. Hashimoto. 1992. Involvement of calcium in germination of coat-modified spores of Bacillus cereus T. Microbiology and Immunology 36:935-946. Stopa, P. J. 2000. The flow cytometry of Bacillus anthracis spores revisited. Cytometry 41:237-244. Vary, J. C., and H. O. Halvorson. 1965. Kinetics of germination of Bacillus spores. J Bacteriol 89:1340-1347. Welkos, S. L., C. K. Cote, K. M. Rea, and P. H. Gibbs. 2004. A microtiter fluorometric assay to detect the germination of Bacillus anthracis spores and the germination inhibitory effects of antibodies. J Microbiol Methods 56:253-265.

7. Germination Capacity and Heat Resistance of Spores from Naturally occurring Bacillus cereus Strains Ynte P. de Vries, George Aboagye, Luc M. Hornstra, Willem M. de Vos, Tjakko Abee and Marcel Zwietering. Manuscript in preparation

ABSTRACT Insight into specific properties of spores occurring in foods is of great importance in risk assessments and for the optimization of spore inactivation treatments during food processing. We tested spores from several B. cereus strains isolated from food-stuffs and foodpoisoning outbreaks for their heat resistance, and compared their characteristics with the laboratory model strain ATCC14579. In addition, we performed a comprehensive investigation into their germination properties. Spores from the isolates showed a large variation in heat resistance, and the majority had a higher heat resistance than the laboratory model strain. With respect to germination, many of the isolates were less sensitive than the laboratory model strain to the germinants tested. A clear link between germination capacity and heat resistance could not be established. Heat activation and ageing enhanced germination to a variable extent. The possibility to use stimulation of in situ spore germination for improvement of current Cleaning In Place (CIP) procedures is discussed.

93

Chapter 7

Introduction Considerable genomic variation exists between strains of several pathogenic bacterial species, including Bacillus cereus (see Fux et al., 2005; Helgason et al., 1998). Bacillus cereus strain ATCC14579, which is used as a model strain throughout this thesis, was isolated from the air in a cowshed, in 1887 (Frankland and Frankland, 1887). Thus, since its isolation, this strain has been cultivated in laboratory conditions for over a century. It is generally known that prolonged cultivation of bacteria in laboratory conditions can cause changes in gene expression patterns and loss of several properties that are of key importance for the survival of the bacteria in their natural habitat (See Branda et al., 2001; Cooper et al., 2003; Fux et al., 2005). Furthermore, during cultivation in laboratory conditions, specific properties are selected for, such as rapid growth in laboratory growth-media, and, in the case of spore-formers, rapid spore germination. For these reasons, it is highly conceivable that several key properties of naturally occurring B. cereus species are different from that of laboratory strains such as B. cereus ATCC14579. With respect to processing and modeling, it is of prime importance to know the variability of key spore properties such as heat resistance and germination in the natural population (Nauta et al., 2003). The wet heat resistance has been documented for a variety of natural B. cereus isolates, and major differences among the isolates and laboratory strains were revealed (Sarrías et al., 2002; te Giffel et al., 2002). However, the germination behavior of natural isolates has been investigated only in the far past and to a limited extent. Foerster and Foster (1966) described the response of several Bacillus species to germinative compounds, and reported marked differences in germination patterns among strains of the same species. A low germination efficiency was observed frequently, as well as “fractional germination”, i.e. germination of only a minor part of the spore population. Gould et al. (1968) investigated the germination behavior of crude spores isolated directly from soil, and here again a significant proportion of the spores did not respond to any germinative compound. Spore germination can be used in spore control procedures, because germination is accompanied by loss of resistance properties. Spore control procedures exploiting spore germination may involve exposure of spores to strong germinative compounds, followed by inactivation of germinated spores by a relatively mild heat treatment. However, the applicability of such procedures is believed to be limited by the incomplete germination responses that are commonly observed in natural spore populations (Gould et al., 1968). Nevertheless, more detailed insight into the variability of the germination responses of natural isolates may yield clues to the applicability of controlled germination in specific processes, such as enhanced cleaning of B. cereus spores from processing lines and equipment in the dairy industry. Furthermore, comparison of the natural B. cereus isolates to the ATCC14579 strain provides an indication to what extent this strain can be used as a representative model for other B. cereus strains. Therefore, we have selected a number of B. cereus strains isolated from industrial and environmental sources, including food-poisoning outbreaks, and analyzed several of their key spore properties in comparison to the ATCC14579 laboratory strain, with special emphasis on spore germination. Materials and Methods Strains and cultivation The strains used in this study (Table 1) were collected from various sources and generously supplied by Ir. Janneke Wijman, from the Food Microbiology group of Wageningen University. Strain purity and identity was confirmed by streaking on PEMBA (Polymyxin Egg-yolk Mannitol Bromothymol blue Agar; Difco) plates. All strains were grown and sporulated in liquid chemically defined medium (described in Chapter 2), in triplicate in 100 ml Erlenmeyer flasks at 30 ºC, in a rotary shaker set at 140 rpm. After 4-7 days, depending on the strain, 60-90 % sporulation was

94

Germination capacity and heat resistance of spores from naturally occurring Bacillus cereus strains

observed. After the majority of the sporangia had lysed, the released spores were harvested by centrifugation, and washed, purified, and stored as described in Chapter 3. Analytical procedures Heat resistance assays, DPA analyses and germination assays were as described in Chapter 3. Germination assays were always performed in duplicate. Special care was taken to minimize experimental error: for all experiments, the same stock solutions and pipettes were used. For testing the colony-forming capacity, spores were incubated in 50 mM Ca-DPA for 30 min. at room temperature prior to spread plating. This treatment is known to increase the colony forming capacity of spores (Keynan and Evenchik, 1969), by inducing germination through activation of the cortex lytic enzymes (Paidhungat et al., 2001). Results Growth and sporulation The strains grew and sporulated well in the chemically defined medium we employed. The sporulation frequency was nearly 100 % for strains PAL5 and ATCC14579, while in strains NIZO B439, PAL2, PAL27 and 72 slightly more than 60% of the cells formed a spore. The other strains had an intermediate sporulation frequency (Table 1). The spores produced from the chemically defined medium were readily released from the sporangia and stable in time. Table 1. The origin of the strains used in this study with their growth and sporulation efficiency on the chemically defined medium

Strain ID NIZO B436 NIZO B439 PAL2 PAL3 PAL5 PAL7 PAL17 PAL18 PAL20 PAL22 PAL25 PAL27 61 72 43-92 1230-88 F450183 ATCC10987 ATCC14579

Origin and reference Pasteurized milk Pasteurized milk Sensor project Sensor project Sensor project Sensor project Sensor project Sensor project Avignon Avignon DSMZ Cream; Stenfors et al., 2001 Semi-skimmed milk; Stenfors et al., 2001 Milk; Stenfors et al., 2001 Stew (food poisoning); Stenfors et al., 2001 Clinical (PHLS); Stenfors et al., 2001 Rasko et al., 2004 Frankland, 1887

Incubation (days)

% Sporulation

7 6 4 4 4 4 4 4 4 4 4 4

>90 >60 >60 >70 >95 >80 >80 >80 >80 >80 >85 >60

4

>90

4

>60

4

>90

4

>90

4 4 7

>90 >90 >95

95

Chapter 7

Fig. 1. Survival of spores exposed to 95 ºC for 0, 5, 10 and 15 minutes.

Heat resistance Fig. 1 shows the survival op spores challenged with exposure to 95 °C. We calculated the decimal reduction time (D-value; time needed to cause 1 log reduction) by linear regression, from the four time-points shown in Fig. 1. Comparison of the D-values clearly illustrated the large differences in heat resistance among the isolates and the model strain (Fig. 2). More than half of the isolates had a clearly higher D-value than the laboratory model strain ATCC14579 (i.e. approximately 5 min). Eight strains had a D-value higher than 10 minutes, and for three of these strains the D-value exceeded 30 min. The most resistant was strain PAL22, with a D-value of 80 minutes. These more than 15-fold differences between the calculated D-values could not be linked to spore DPA content or spore core density (data not shown). All spore-suspensions used for the heat-resistance assays had an initial A600 of ~1.0, but nevertheless a considerable variance (over 30-fold) in cfu/ml was observed at time 0 of the heat treatment (Fig. 1). Notably, some of the strains with a high D-value had relatively low initial counts. It is known that some spores need an activation treatment to optimally germinate and form a colony. In such cases, plating of a non-activated spore suspension results in an underestimation of the number of viable spores (Keynan and Evenchik, 1969). We investigated this possibility in two strains with a low initial count, strains PAL22 and PAL25, by incubating the spores in the presence of Ca-DPA prior to plating. This treatment enhances colony formation without the need of additional activation (Keynan and Evenchik, 1969). The results suggested that approximately half of the spores, although viable, was highly dormant and did not form colonies without an activation

96

Germination capacity and heat resistance of spores from naturally occurring Bacillus cereus strains

treatment (data not shown). Heat is also an activating agent, known to enhance colony formation (Keynan and Evenchik, 1969). Therefore, in the heat-resistance assay two opposing processes occur: spores are inactivated due to killing by heat, resulting in a reduction of the efficiency of plating, while on the other hand highly dormant spores are activated, resulting in an increase of the plating efficiency. The larger the number of highly dormant spores in the initial population, the more important the activation process will be and the more biased the apparent D-value. The low initial counts for the strains with the highest D-values, notably strains PAL22 and PAL25, suggest that these spore preparations contain many highly dormant spores, which implies that the high Dvalues found for these strains may be biased to a considerable extent.

Fig. 2. Decimal reduction times (D-values) of the strains treated at 95 ºC.

Germination We investigated the germination capacity of fifteen B. cereus isolates, including several psychrotolerant isolates, and the ATCC14579 model strain. Germination was measured 20-50 days after harvesting, and we used the best-described germinants for B. cereus, L-alanine and inosine, as a single germinant or in concert. A general overview of the results is presented in Table 2. In these experiments, a decrease in A600 to 45-50% generally corresponded to ~99% germination as

97

Chapter 7

observed with a phasecontrast microscope. As anticipated, the laboratory strain ATCC14579 responded well to all of the germinants tested (Fig. 3A). Heat activation enhanced the germination response, by increasing the specific germination rate (slope of the curve) and decreasing the germination lag-time (Fig. 3B). The other strains showed highly variable responses; five strains did not respond to any of the germinants, six strains responded only to the combination of alanine and inosine, and four strains showed an additional response to L-alanine as a single germinant (Table 2). Heat-activation resulted in efficient germination of twelve out of fifteen isolates. Four strains still responded relatively poorly, even after heat activation. Interestingly, one strain, strain PAL22, was exceptionally sensitive specifically to L-alanine. Table 2. Response of strains to several germinants. Ala; L-alanine. Ino; Inosine. Designations, in % decrease of OD600: (-), 45 in 40 min. Strain ID Untreated Heat Activated Ala Ala Ino Ala/ino Ala/ino Ala Ala Ino Ala/ino Ala/ino 1 50 1 1mM/0.1 0.1mM 1 50 1 1mM/0.1 0.1mM/1 mM mM mM mM /1mM mM mM mM mM mM +/++ +++ +++ +++ +++ +++ +++ +++ NIZO B436 ++ ++ +++ +++ NIZO B439 +/++ +++ +/++ +++ +++ PAL2 +/+/+++ +++ PAL3 +++ +++ PAL5 + + PAL17 +/+/PAL18 +/+/+++ +++ PAL22 ++ + +++ + ++ + PAL25 +++ +++ PAL27 61 ++ ++ ++ +++ +++ 72 +/+/+ ++ 43-92 ++ ++ ++ +++ +++ 1230-88 + +++ +++ + ++ +++ +++ +++ F450183 +++ +++ +++ +++ + +++ +++ +++ +++ ATCC1457959

We tested a number of strains several times with a large time-interval, to estimate the influence of ageing on spore germination. Just as heat activation and a Ca-DPA treatment, ageing enhances germination (Keynan and Evenchik, 1969). The enhancement of germination by ageing was strain-specific and depended on the type of germinant (data not shown). For example, after storage for 82 days, the germination response of spores from strain F450183 to L-alanine and inosine was clearly enhanced when compared to the same spores stored for 38 days, while the response to the alanine/inosine combinations had not changed significantly (Fig. 4). As noted above, several strains with a relatively high D-value had a low colony forming capacity, which may be due to a low germination capacity. However, when these strains were tested in a germination assay, no clear correlation between germination capacity and colony forming capacity was apparent. For example, strains NIZO B439, PAL22 and 72 had a low colony forming capacity but germinated quite well, while strains PAL3, PAL5 and PAL18 with a very high colony forming capacity showed a poor response in the germination assay. Furthermore, a clear link between germination capacity and heat resistance was not found.

98

Germination capacity and heat resistance of spores from naturally occurring Bacillus cereus strains

Fig. 3. The effect of heat activation on germination of laboratory model strain ATCC14579, measured by drop in A600. A. Without heat activation. B. With heat activation. Symbols: Blanc (closed triangle down); Lalanine 1 mM (closed squares); L-alanine 50 mM (closed triangles up); Inosine 1 mM (closed circles); Lalanine 1mM combined with inosine 0.1 mM (open squares); L-alanine 0.1 mM combined with inosine 1 mM (open circles).

Fig. 4. The effect of ageing on germination of isolate strain F450183, measured by decrease of A600. A. 37 days after harvest. B. 82 days after harvest. Symbols: Blanc (closed triangle down); L-alanine 1 mM (closed squares); L-alanine 50 mM (closed triangles up); Inosine 1 mM (closed circles); L-alanine 1mM combined with inosine 0.1 mM (open squares); Lalanine 0.1 mM combined with inosine 1 mM (open circles).

Discussion Spore germination is essential to the proliferation of spore formers, and a key step to spoilage and poisoning caused by spore-formers (Setlow, 2003). Most of our knowledge of germination is based on studies on derivatives of the 168 laboratory strain of Bacillus subtilis. Germination has also been studied in B. cereus, using the ATCC14579 and the 569 (ATCC10876) laboratory strains (Barlass et al., 2002; Behravan et al., 2000; Clements et al., 1998; Hornstra et al., 2005; Thackray et al., 2001). In the present study we provide a comprehensive comparison of the germination capacity of spores from naturally occurring B. cereus isolates, and in addition, we studied their heat resistance. As anticipated, we observed a large variation in heat resistance among the different strains, and more than half of the isolates were more resistant than the ATCC14579 laboratory strain. Such variability in heat resistance among B. cereus isolates has been reported before (Dufrenne et al., 1994; 1995; Parry and Gilbert, 1980; Sarrias et al., 2002). Indeed, although B. cereus is generally not considered as very heat-resistant in comparison with other mesophilic spore-formers, highly heat-resistant B. cereus strains have been described, including an isolate that was able to survive a

99

Chapter 7

treatment of 30s at 125 °C (te Giffel et al., 2002) and isolates with D-values exceeding 100 and 200 min. at 90 °C (Dufrenne et al., 1995; 1994). We found large differences in germination capacity. Spore germination in response to nutrients is initiated by the binding of germinants to germination receptors. Subsequently, the spore changes from metabolically dormant to metabolically active in a number of steps (see Setlow, 2003). All of these steps can be affected by genetic variation between the different strains. The genetic variation among B. cereus isolates is extensive (Helgason et al., 1998), and thus, there may be differences in for example the germination receptor constitution among the strains. Preliminary data from genomic comparisons between several B. cereus strains indicates that this indeed is the case (Luc M. Hornstra, pers. comm.). In addition, differences between isolates with respect to coat proteins that facilitate passage of germinants to the germinant receptors (Behravan et al., 2000) would increase variability of their germination response. An important factor affecting the germination response was the age of the spores. Prolonged storage enhanced germination, a phenomenon that is known as ageing activation (Keynan and Evenchik, 1969). Therefore, care should be taken to make sure that all the spore-batches have approximately the same age, when performing comparative analyses. Furthermore, in some cases inconsistency and variability was observed in the germination response of spores from the same batch measured at the same time (data not shown). Such findings were reported previously (Welkos et al., 2004), and may be avoided by measuring germination parameters other than the decrease in A600, such as the rise in esterase activity (Chapter 6; Ferencko et al., 2004), increase in stainability with fluorescent DNA-binding dyes (Chapter 6; Welkos et al., 2004) or DPA excretion. Overall, many of the industrial isolates germinated relatively poorly without heat activation. Nevertheless, we clearly showed that after heat activation and with a strong germinant, all of the strains germinated, albeit in a few cases to a relatively small extent. Obviously, the applicability of germination in spore control procedures would be enhanced by a high degree of germination in a broad range of spore formers. Important areas of improvement are further optimization of germination and activation conditions, and of the germinant mixture. Apart from nutrient germinants, cationic surfactants may be applied, of which the best studied example is dodecylamine (Setlow et al., 2003). Furthermore, ammonia may be included in the mixture, as it has been reported that ammonia can stimulate germination of B. cereus spores without the need for heatactivation (Preston and Douthit, 1984). Based on our results, we expect that such an optimized combination of germinants, heat activation, and other activating compounds, will significantly reduce the number of heat resistant spores in naturally occurring populations. That would be a valuable attribute to commonly employed cleaning-in-place (CIP) treatments, in which currently harsh conditions that may damage processing equipment are employed to destroy resistant spores. Conclusions The isolates tested showed a large variation in heat resistance, and the majority had a higher heat resistance than the laboratory model strain. With respect to germination, many of the isolates were less sensitive to the germinants tested as compared to the laboratory model strain. A clear link between germination capacity and heat resistance could not be established. Heat activation and ageing enhanced germination in response to several germinants in various isolates. References 1.

100

Barlass, P. J., C. W. Houston, M. O. Clements, and A. Moir. 2002. Germination of Bacillus cereus spores in response to L-alanine and to inosine: the roles of gerL and gerQ operons. Microbiology 148:2089-2095.

Germination capacity and heat resistance of spores from naturally occurring Bacillus cereus strains

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

Behravan, J., H. Chirakkal, A. Masson, and A. Moir. 2000. Mutations in the gerP locus of Bacillus subtilis and Bacillus cereus affect access of germinants to their targets in spores. J Bacteriol 182:1987-1994. Branda, S. S., J. E. Gonzalez-Pastor, S. Ben-Yehuda, R. Losick, and R. Kolter. 2001. Fruiting body formation by Bacillus subtilis. Proc Natl Acad Sci U S A 98:11621-11626. Clements, M. O., and A. Moir. 1998. Role of the gerI operon of Bacillus cereus 569 in the response of spores to germinants. J Bacteriol 180:6729-6735. Cooper, T. F., D. E. Rozen, and R. E. Lenski. 2003. Parallel changes in gene expression after 20,000 generations of evolution in Escherichia coli. Proc Natl Acad Sci U S A 100:1072-1077. Dufrenne, J., M. Bijwaard, M. te Giffel, R. Beumer, and S. Notermans. 1995. Characteristics of some psychrotrophic Bacillus cereus isolates. Int J Food Microbiol 27:175-183. Dufrenne, J., P. Soentoro, S. Tatini, T. Day, and S. Notermans. 1994. Characteristics of Bacillus cereus related to safe food production. Int J Food Microbiol 23:99-109. Ferencko, L., M. A. Cote, and B. Rotman. 2004. Esterase activity as a novel parameter of spore germination in Bacillus anthracis. Biochem Biophys Res Commun 319:854-858. Foerster, H. F., and J. W. Foster. 1966. Response of Bacillus spores to combinations of germinative compounds. J Bacteriol 91:1168-1177. Fux, C. A., M. Shirtliff, P. Stoodley, and J. W. Costerton. 2005. Can laboratory reference strains mirror 'real-world' pathogenesis? Trends Microbiol 13:58-63. Gould, G. W., A. Jones, and C. Wrighton. 1968. Limitations of the initiation of germination of bacterial spores as a spore control procedure. J Appl Bacteriol 31:357-366. Helgason, E., D. A. Caugant, M. M. Lecadet, Y. Chen, J. Mahillon, A. Lovgren, I. Hegna, K. Kvaloy, and A. B. Kolsto. 1998. Genetic diversity of Bacillus cereus/B. thuringiensis isolates from natural sources. Current Microbiology 37:80-87. Hornstra, L. M., Y. P. de Vries, W. M. de Vos, T. Abee, and M. H. Wells-Bennik. 2005. gerR, a novel ger operon involved in L-alanine- and inosine-initiated germination of Bacillus cereus ATCC 14579. Appl Environ Microbiol 71:774-781. Keynan, A., and Z. Evenchik. 1969. Activation, p. 359-396. In G. W. Gould and A. Hurst (ed.), The Bacterial Spore, vol. 1. Academic Press, London and New York. Nauta, M. J., S. Litman, G. C. Barker, and F. Carlin. 2003. A retail and consumer phase model for exposure assessment of Bacillus cereus. Int. J. Food Microbiol. 83:205-218. Paidhungat, M., K. Ragkousi, and P. Setlow. 2001. Genetic requirements for induction of germination of spores of Bacillus subtilis by Ca(2+)-dipicolinate. J Bacteriol 183:48864893. Parry, J. M., and R. J. Gilbert. 1980. Studies on the heat resistance of Bacillus cereus spores and growth of the organism in boiled rice. J Hyg (Lond) 84:77-82. Preston, R. A., and H. A. Douthit. 1984. Stimulation of germination of unactivated Bacillus cereus spores by ammonia. J Gen Microbiol 130 ( Pt 5):1041-1050. Sarrías, J. A., M. Valero, and M. C. Salmerón. 2002. Enumeration, isolation and characterization of Bacillus cereus strains from Spanish raw rice. Food Microbiol 19:589595. Setlow, B., A. E. Cowan, and P. Setlow. 2003. Germination of spores of Bacillus subtilis with dodecylamine. J Appl Microbiol 95:637-648. Setlow, P. 2003. Spore germination. Curr Opin Microbiol 6:1-7. te Giffel, M. C., A. Wagendorp, A. Herrewegh, and F. Driehuis. 2002. Bacterial spores in silage and raw milk. Antonie van Leeuwenhoek 81:625-630.

101

Chapter 7

23. 24.

102

Thackray, P. D., J. Behravan, T. W. Southworth, and A. Moir. 2001. GerN, an antiporter homologue important in germination of Bacillus cereus endospores. J Bacteriol 183:476-482. Welkos, S. L., C. K. Cote, K. M. Rea, and P. H. Gibbs. 2004. A microtiter fluorometric assay to detect the germination of Bacillus anthracis spores and the germination inhibitory effects of antibodies. J Microbiol Methods 56:253-265.

8. Summary, future Perspectives and Practical Applicability of the Research described in this Thesis

103

Chapter 8

Summary Introduction Bacterial spores are highly differentiated cell types, specifically designed for the survival of adverse conditions. Their structure is very different from the structure of normal vegetative bacterial cells. Spores cause massive problems in the food industry, because their remarkable resistance allows them to survive food processing and conservation methods. The spore-forming Bacillus cereus is an important food-borne pathogen. Together with 5 other Bacilli, B. cereus belongs to the Bacillus cereus group, a group of bacteria so closely related that distinction between them is often problematic. Besides B. cereus, the best known and studied members of the B. cereus group are Bacillus thuringiensis, an insect pathogen that is applied as a bio pesticide, and Bacillus anthracis, which is a notoriously potent pathogen, being the etiological agent of the anthrax disease. B. cereus is famous for its ability to cause food-poisoning, and is an important spoilage organism in pasteurized dairy products. The work presented in this thesis has focused on the formation, structure and germination of Bacillus cereus spores (Fig. 1). In this final chapter, the most important results obtained in this thesis are summarized, future perspectives of the spore research field are provided, and the applicability of the results obtained in this study is highlighted in the context of food preservation and safety. Bacillus cereus growth and sporulation B. cereus and the model spore former Bacillus subtilis thrive in vastly different ecological niches. However, the sporulation mechanism, and the key regulators involved, are highly conserved between the two species (Chapter 2). In our growth-experiments, B. cereus metabolized nearly all of the amino-acids present in the medium during exponential growth, at the same time as glucose. While the amount of amino-acids in the medium decreased, the concentration of NH4+ increased, indicating that B. cereus converted the amino-acids to NH4+ and other compounds. In the presence of D/L-lactate as a carbon and energy source, consumption of amino-acids during exponential growth led to a nearly equimolar increase of NH4+ in the supernatant. In the presence of glucose, however, the increase of NH4+ in the supernatant was approximately 50% of the decrease in aminoacid concentration, indicating that in these conditions B. cereus assimilated approximately half of the nitrogen from the amino-acids into biomass (Chapter 2, 3). Nitrogen assimilation by B. cereus cells was low during exponential growth, but very high during the stationary phase and during sporulation. L-glutamate was an efficient nitrogen source for B. cereus, and preferably taken up during sporulation, while in the absence of L-glutamate, B. cereus assimilated NH4+ during sporulation. We found that in the conditions we employed, significant amounts of carbon and nitrogen sources were still present when the cells started to sporulate (Chapter 3). This implies that sporulation was induced by other triggers than nutrient limitation, presumably via quorum sensing as observed in B. subtilis (Sonenshein, 2000). The alternative sigma factor σB, encoded by the sigB gene, is an important stress response regulator of B. cereus. We observed an increase in sigB transcription upon glucose depletion, coinciding with the transition from exponential growth to the stationary phase (Chapter 2). This increase seems to be specifically associated with the depletion of glucose, and not so much with the growth-phase transition, because in medium lacking glucose, sigB transcription did not peak at the end of exponential growth (Chapter 3). Activity of σB is important for the process of spore formation, because deletion of sigB had a significant impact on the properties of the resulting spores. In particular, these spores showed a slightly reduced heat resistance and a reduced germination response (Chapter 4).

104

Summary, future perspectives and practical applicability of the research described in this thesis

Fig. 1. Summary of the topics of the B. cereus life cycle covered in this thesis, including the most important techniques applied and examples of the results obtained. DGSC; Defined Growth and Sporulation Conditions, described in Chapter 2 and exploited in Chapters 3 and 4. ESR; Electron Spin Resonance, described in Chapter 5. FCM; Flow CytoMetry, described in Chapter 6. The germination of natural isolates was studied in Chapter 7.

The structure of B. subtilis and B. cereus spores Having no metabolic activity, a dormant spore is actually not “alive”, but a mere clump of molecules with specific physical and chemical properties. The physicochemical structure of the spore results in the protection of vital components such as membranes, proteins and DNA. To understand the mechanisms underlying spore resistance, it is of paramount importance to know the physical and chemical properties of the spore. During the more than 100 years of spore research conducted so far, many techniques have been employed to this end, and several important aspects of spore resistance mechanisms have been resolved. Nevertheless, the status of the aqueous

105

Chapter 8

environments in the specific compartments of bacterial spores, a parameter that is believed to be of crucial importance to spore dormancy and resistance, could not be addressed so far. The spin-probe-based Electron Spin Resonance (ESR) concept presented in Chapter 5 deals with this issue, and provides the first direct data on the aqueous environment in the various compartments of B. subtilis and B. cereus spores. From the results obtained, it was concluded that the core cytoplasm is not in a glassy state. Instead, a three-dimensional molecular matrix incorporating free but highly viscous water exists in the core. This is important, because spore wet heat resistance, a parameter of great importance in food processing, has been attributed to a supposed glassy state in the core. Also, stimulation of germination by heat (heat-activation) has been proposed to involve changes in the supposed glassy state in the core. We propose that the wet heat resistance of spores depends on the structuring in the core, and the microviscosity of the core bulk water. Notably, neither heat activation nor partial germination (the excretion of DPA but not full rehydration and enzyme activity) altered the structural properties of the core matrix significantly. Complete germination resulted in the disappearance of the structure in the core, and a decrease of the microviscosity in the core cytoplasm to levels encountered in normal vegetative cells. Spore germination Germination is the process whereby spores change from a metabolically dormant state to a metabolically active state. Germination results in the breakdown of the spore-structure and concomitant loss of resistance properties. Spore germination is essential to the proliferation of spore formers, which may ultimately lead to food spoilage and disease (Moir et al., 2002; Setlow, 2003). For these reasons, germination is important from both fundamental and applied perspectives. The present mechanistic model of the germination response of B. subtilis has a number of gaps, including the mechanism of spore activation, the mechanism of DPA excretion, the exact mode of action of the nutrient receptors, and the signal transduction between these receptors and other components of the germination apparatus (Setlow, 2003). Besides these rather specific gaps, research needs in the field of germination include the behavior of strains other than the B. subtilis 168 model strain, and the heterogeneous distribution of germination capacity in spore populations. These two latter research needs have been addressed in Chapters 6 and 7. In Chapter 6, we have shown that flow cytometry (FCM) is a highly suitable method for the quantitative analysis of the behavior of individual spores in a large, germinating spore-population. By using several different fluorescent dyes, distinct germination parameters such as DNA accessibility and esterase activity were quantified. In Chapter 7, we investigated the germination capacity of spores from strains other than the B. subtilis 168 model strain. Spores from the B. cereus laboratory model strain ATCC14579, which has been cultivated in laboratory conditions for more than a century, germinated rapidly and completely. However, spores from a number of B. cereus strains that were more recently isolated from environmental and industrial settings, had a highly variable germination capacity. Future directions in spore research and food safety Genomics Complete genome sequences from several spore-formers have become available in recent years, and the 2001 anthrax attacks in the US have sparked a tremendous boost in funding for B. anthracis genome sequencing projects (see Enserink et al., 2002). As such, the Bacillus cereus group contains the highest number of closely related fully sequenced genomes, giving the unique opportunity for thorough comparative genomic analyses (Rasko et al., 2005). In a first attempt to extract some hints about how much of the sporulation process and its regulation is conserved

106

Summary, future perspectives and practical applicability of the research described in this thesis

amongst different species, the genomes of several spore-formers and non-spore formers were compared (Stragier, 2002), and it was concluded that the “grand scheme” of sporulation is homologous and inherited from a common ancestor. Nevertheless, differences among less closely related spore-formers were evident, especially between Bacillus and Clostridium spp. Further comparisons between the increasing number of genomes available from sporeformers and non-spore formers will increase our understanding of the evolution of sporulation, and may shed light on the genetic basis of specific spore properties. Interesting candidates for sequencing and comparative analysis would be spore-formers with extra-ordinary properties, such as the spore-forming gram-negative Serratia marcescens (Ajitkumar et al., 2003), and the extremely heat-resistant Bacillus sporothermodurans (Pettersson et al., 1996). Transcriptomics and proteomics An important spin-off of genome sequencing projects is the development of DNA-microarrays, enabling detailed investigations into gene-expression profiles during sporulation and germination. Several systematic investigations into the regulons of specific sigma factors and regulators that are of prime importance during sporulation have been performed (Britton et al., 2002; Eichenberger et al., 2003; 2004; Errington, 2003; Feucht et al., 2003; Hilbert et al., 2004; Molle et al., 2003; Piggot and Hilbert, 2004). Although the outcomes of such studies are subject to variation (e.g. compare Eichenberger et al., 2003 with Feucht et al., 2003), and interpretation is complexed by other factors, such as regulator concentration dependency (Fujita et al., 2005) and functional redundancy (Silvaggi et al., 2004), transcriptomics studies have yielded a wealth of new data and clues. For example, genes involved in the development of heat resistance were identified by transcriptome analysis of a highly heat resistant B. subtilis food isolate (Oomes and Brul, 2004). Furthermore, large-scale transcriptional analysis of C. acetobutylicum sporulation mutants yielded expression patterns that are partly different from what is known in B. subtilis (Tomas et al., 2003). This supports the notion that members of the genus Clostridium may have a sporulation mechanism which is distinct from that of B. subtilis (Bentley et al., 2002; Shimizu et al., 2002). In a pioneering study, Liu et al. (2004) combined transcriptome analysis with high-throughput proteomics. This approach revealed that a large portion of the B. anthracis genome is regulated in a growth phasedependent manner, and this regulation is marked by five distinct waves of gene expression as cells proceed from exponential growth through sporulation. Interestingly, while the genes responsible for assembly and maturation of the spore are tightly regulated in discrete stages, many of the genes encoding proteins found in the eventual spore are expressed throughout and even before sporulation. This novel finding suggests that gene expression during sporulation may be mainly related to the physical construction of the spore, rather than synthesis of eventual spore content. Such studies will continue to significantly increase our understanding of spore formation and structure, in relation to important spore properties, including heat resistance and germination. Detection Tools and marker genes derived from genomics and transcriptomics studies may in future times enable rapid detection, identification, and tracking of specific organisms, for example in food production chains. Furthermore, the physical state of the micro-organisms can be detected through monitoring of specific gene expression, such as stress response genes (see van Schaik, 2005). Early detection of highly resistant spore-formers in the food-process or in raw materials would enable adjustment of processing conditions to the actual needs, avoiding over-processing, and reducing costs and unnecessary loss of organoleptic quality of products. Currently, DNA-based microchips are being developed by food-industries for detection and evaluation of microbial populations in the food process (Oomes and Brul, 2004 and refs therein), while a number of chemical and biological

107

Chapter 8

sensor systems have been described that could serve as early detectors (Sadik et al., 2004 and references therein). Integration of such sensors in lab-on-chip devices, combined with innovative self-learning software tools, is under development and holds great promise for the future. For rapid evaluation of microbial cells and spores, innovative growth methods are available. An example is the use of anopore micro-chips in combination with fluorescence microscopy (Ingham et al., 2005). With this method, detection and parameters such as growth-rate and heterogeneity can be established much faster than with traditional agar-based growth methods. Furthermore, by using fluorescent in situ hybridizing probes, species can be rapidly identified onchip. In a preliminary study, we have designed a convenient anopore-based method for the analysis of micro colony formation by individual spores. This method was ideal for the investigation into population heterogeneity and the early stages of colony formation, and time-saving, as micro colonies were clearly visible after only two hours of incubation (Y. de Vries and C. Ingham, 2005 unpublished results). Two major challenges for the food-industry are to cope with interference from the complex food-matrix, which is detrimental to the functioning of most sensors (Sadik et al., 2004), and the detection of very low numbers of micro-organisms. Future technologies lowering detection limits and reducing the interference from the food-matrix may include innovative enrichment techniques, such as immuno-magnetic separation (Pyle et al., 1999; Sun et al., 2002). In the well-funded defense and medical research fields, considerable progress has been made in the development of techniques for detection and identification of low levels of chemicals and micro-organisms in a variety of environments. Developments from these fields may have great potential for application in the food-sector. Decontamination and inactivation Increased understanding of spore killing mechanisms may lead to improvement of sporicidal treatments. With this in mind, traditional spore-inactivation methods are nowadays being evaluated with state-of-the-art technology, and the killing mechanisms of a variety of sporicidal agents on B. subtilis have been systematically investigated (Genest et al., 2002; Loshon et al., 2001; Melly et al., 2002; Setlow et al., 1997; Setlow et al., 2002; Setlow, 2004; Shapiro et al., 2004; Tennen et al., 2000; Young and Setlow, 2004; 2004b). In a study on the heat-inactivation mechanisms of B. cereus spores, we have studied the kinetics of DPA excretion during a heat-treatment. It is known that spores release their DPA upon a lethal heat treatment (Kort et al., 2005), and that DPA excretion is generally accompanied by core rehydration, loss of spore stability and heat resistance. Therefore, it was expected that DPA excretion coincides with spore death in a heat resistance assay. However, we found that B. cereus spores heated in a nutrient-less buffer at 95 ºC and 100 ºC released all of their DPA in the first 30 seconds of the treatment, whereas a decrease of colony counts was evident only after 1 minute of treatment (Y. de Vries, unpublished data). This suggested that spores were able to survive the heat treatment for a significant amount of time after release of their DPA, a new insight into spore heatresistance. Future experiments will shed more light on this important issue. Practically, in spore heat resistance assays, survivors are counted usually after overnight incubation, while the number of colonies may increase after additional incubation. The colonies that come up after prolonged incubation are smaller in size than those that are visible already after overnight incubation, and originate from spores that are damaged by the heat treatment. Investigating the nature of this damage, we found that the growth-rate of the bacteria in both colony types is identical, which indicated that the retarded growth of colonies from heat damaged spores results from a defective germination or outgrowth mechanism (Y. de Vries, unpublished data). Incubation of heat treated spores in the presence of Ca-DPA, which circumvents the nutrient receptors and induces germination directly by activation of the cortex lytic system, resulted in more

108

Summary, future perspectives and practical applicability of the research described in this thesis

colonies and a negligible increase in colony count upon prolonged incubation, which suggested that the nutrient receptors were damaged during the heat treatment. Inclusion of B. subtilis mutants that lack all nutrient receptors in these experiments indicated that the initial decrease (0.5-1 log reduction) of the number of colonies that is observed in a spore inactivation curve, is due to damage to the nutrient receptors, while the further decrease (1-4 log reduction) is reduced in the mutant, suggesting that the absence of nutrient receptors reduces heat-inactivation. In the final part of the inactivation curve (4-6 log reduction), the inactivation rates of the mutant and the wild-type were identical. Future experiments in which more different treatment temperatures and conditions are included may provide additional clues to the mechanism whereby heat kills spores. Another important challenge ahead is the question whether the results obtained for the laboratory model strains apply for other strains and other species of spore formers, especially Clostridium spp. Inclusion of a broad range of spore formers in future investigations is of prime importance to the practical applicability of the results obtained in such studies. Next to the investigations into the mechanisms of spore resistance to traditional spore inactivation methods, new sporicidal agents are being developed and tested, such as nano-particles, bacteriophages, plasma, and nano-photo-sensitizers (Hartley and Axtell, 2004; Koper et al., 2002; Lee et al., 2005; Purevdorj et al., 2003 and references therein; Schuch et al., 2002). The use of cold plasma is currently under investigation as a novel surface decontamination method (Mastwijk, 2004), while the space industry has developed plasma-based air-decontamination systems that are already operable and commercially available (Anonymous, 2004). A decontamination treatment suitable for food and highly effective against spores is ionizing radiation. Irradiation has been approved by the FDA for several food-products, and also by the EC for a smaller selection of products (Scott Smith and Pillai, 2004; Steele, 2001). In contrast to wet heat resistance, which may be several orders of magnitude higher, the resistance of spores to ionizing radiation is only 5-15fold greater than that of their vegetative counterparts (Gould, 1977; Farkas, 1994). Furthermore, while the heat-resistance of spores from various Bacillus species may range over 3 orders of magnitude, their radiation resistance varies only 4-fold (Farkas, 1994). Therefore, the need for considerable over-processing to eliminate specific extremely resistant species is far less with irradiation treatments than with heat. In addition, radiation treatments greatly sensitize spores to heat, and are suitable for complementary use with traditional food-processing treatments in a hurdle-based approach (Farkas, 1994; Leistner, 2000). Currently, the major hurdle thwarting the application of irradiation as a food-preservation attribute is its low consumer acceptance. Finally, a wide range of natural antimicrobial systems has evolved in animals, plants and micro-organisms. A number of natural antimicrobials are already employed as food preservatives, and several other are in development (Gould, 2001). Such antimicrobials do not kill spores, but rather impede germination and/or outgrowth, thus preventing food spoilage and poisoning. Natural antimicrobials are expected to have an increasing role in the future of food preservation, particularly in combination with other mild preservation techniques (Gould, 2001). Practical application of the research described in this thesis B. cereus spores occur ubiquitously in the environment, and contamination of raw materials used in food production is practically unavoidable. The attachment of spores to processing equipment is an accompanying problem, resulting in a source of continuous contamination. Because of their high resistance, spores can only be inactivated with fairly harsh treatments such as heat sterilization, which are not only costly and time-consuming, but also damaging to processing equipment and detrimental to the organoleptic properties of the food products. As consumers increasingly demand for foods that have a naturally fresh taste and texture, producers are looking for new approaches to cope with the presence of spores. Therefore, research on critical spore

109

Chapter 8

properties such as resistance to heat and other stresses, adherence properties, and germination mechanisms, is of paramount importance. The methodology developed in the course of this project provides a firm basis for further research into sporulation and spore properties of the important food-borne pathogen Bacillus cereus and other species. Important achievements are the efficient protocol for convenient production and harvesting of large amounts of B. cereus spores, while the defined sporulation conditions developed in Chapter 2 are essential for detailed investigations into the sporulation process of B. cereus. One of the initial goals was the identification of links between sporulation conditions, gene expression and spore properties, so as to find a molecular basis for critical spore properties such as heat resistance and germination capacity. To this aim, sporulation in different conditions was achieved which yielded spores with varying resistance and germination properties. Follow-up projects employing B. cereus micro-arrays for the analysis of the total transciptomes in these different conditions will greatly benefit from the availability of this setup. The germination studies performed with isolates of industrial importance, described in Chapter 7, indicate that germinability is highly variable among different strains, and that many naturally occurring strains have a relatively poor germination response compared to the model laboratory strain. However, we have shown that with the proper activation and germination conditions all strains eventually germinated. This may pave the way for application of in situ spore germination as an attribute to spore inactivation treatments. The use of FCM for analysis of spore-populations, described in Chapter 6, has great potential. FCM provides large amounts of quantitative data on the properties or behavior of individual spores, and can be used to quantify the heterogeneity of natural spore populations. Furthermore, FCM can be applied for quantitative analysis of the germination response of heatdamaged spores, and combined with fluorescent labeling of specific damage indicator genes, FCM allows monitoring of the effectiveness of spore inactivation treatments. The obtained data will provide a sound basis for computer modeling studies, which in the future may enable precise adjustment of food processing conditions, resulting in a reduction of costs and a reduced loss of organoleptic properties. Finally, the spin-probe-based ESR approach for the study of the inner structure of spores, described in Chapter 5, is of special interest. This approach provides a new set of specific structural parameters to be linked with spore properties. Notably, these parameters can be used to identify the structural basis of the super heat-resistance of certain isolates of industrial importance. Specific knowledge of the structural basis for wet heat resistance, which is still lacking at present, will aid in the identification of weak points in spore resistance. This may open the door to new preservation methods exploiting the Achilles heel of bacterial spores. Final remarks Spore biology is a very active and highly exciting research field, in which fundamental scientific interest, practical applicability and industrial relevance go hand in hand. The research described in this thesis has yielded new fundamental data on spore formation and spore structure, and has resulted in a number of novel tools with great practical applicability. These are expected to bring us closer to solving some of the myriad of problems caused by the omni- and ever-present bacterial spores.

110

Summary, future perspectives and practical applicability of the research described in this thesis

References: 1. 2. 3. 4. 5.

6.

7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Ajithkumar, B., V. P. Ajithkumar, R. Iriye, Y. Doi, and T. Sakai. 2003. Spore-forming Serratia marcescens subsp. sakuensis subsp. nov., isolated from a domestic wastewater treatment tank. Int J Syst Evol Microbiol 53:253-258. Anonymous 2004, posting date. Space tech captures toxic micro-organisms. European Space Agency news. [Online: http://www.esa.int/export/esaCP/SEMKAWL26WD_index_0.html] Bentley, S., M. Holden, N. Thomson, and J. Parkhill. 2002. Armed to the teeth. Trends Microbiol 10:163-164. Britton, R. A., P. Eichenberger, J. E. Gonzalez-Pastor, P. Fawcett, R. Monson, R. Losick, and A. D. Grossman. 2002. Genome-wide analysis of the stationary-phase sigma factor (sigma-H) regulon of Bacillus subtilis. J Bacteriol 184:4881-4890. Eichenberger, P., M. Fujita, S. T. Jensen, E. M. Conlon, D. Z. Rudner, S. T. Wang, C. Ferguson, K. Haga, T. Sato, J. S. Liu, and R. Losick. 2004. The program of gene transcription for a single differentiating cell type during sporulation in Bacillus subtilis. PLoS Biol 2:e328. Eichenberger, P., S. T. Jensen, E. M. Conlon, C. van Ooij, J. Silvaggi, J.-E. GonzálezPastor, M. Fujita, S. Ben-Yehuda, P. Stragier, J. S. Liu, and R. Losick. 2003. The sigE regulon and the identification of additional sporulation genes in Bacillus subtilis. Journal of Molecular Biology 327:945-972. Enserink, M. 2002. Microbial genomics. TIGR begins assault on the anthrax genome. Science 295:1442-1443. Farkas, J. 1994. Tolerance of spores to ionizing radiation: mechanisms of inactivation, injury and repair. Soc Appl Bacteriol Symp Ser 76:81S-90S. Feucht, A., L. Evans, and J. Errington. 2003. Identification of sporulation genes by genome-wide analysis of the sigma(E) regulon of Bacillus subtilis. Microbiology 149:30233034. Fujita, M., J. E. Gonzalez-Pastor, and R. Losick. 2005. High- and low-threshold genes in the Spo0A regulon of Bacillus subtilis. J Bacteriol 187:1357-1368. Genest, P. C., B. Setlow, E. Melly, and P. Setlow. 2002. Killing of spores of Bacillus subtilis by peroxynitrite appears to be caused by membrane damage. Microbiology 148:307314. Gould, G. W. 1977. Recent advances in the understanding of resistance and dormancy in bacterial spores. J Appl Bacteriol 42:297-309. Gould, G. W. 2001. Symposium on 'nutritional effects of new processing technologies'. New processing technologies: an overview. Proc Nutr Soc 60:463-474. Hartley, S. M., and H. Axtell. 2004. Presented at the 24th Army Science Conference, Orlando, Florida, Nov 29 - Dec 2. Hilbert, D. W., and P. J. Piggot. 2004. Compartmentalization of gene expression during Bacillus subtilis spore formation. Microbiol Mol Biol Rev 68:234-262. Ingham, C. J., M. van den Ende, D. Pijnenburg, P. C. Wever, and P. M. Schneeberger. 2005. Growth of Microorganisms on a Highly Porous Inorganic Membrane (Anopore). SUBMITTED. Koper, O. B., J. S. Klabunde, G. L. Marchin, K. J. Klabunde, P. Stoimenov, and L. Bohra. 2002. Nanoscale powders and formulations with biocidal activity toward spores and vegetative cells of Bacillus species, viruses, and toxins. Curr Microbiol 44:49-55. Kort, R., A. C. O'Brien, I. H. van Stokkum, S. J. Oomes, W. Crielaard, K. J. Hellingwerf, and S. Brul. 2005. Assessment of heat resistance of bacterial spores from

111

Chapter 8

19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.

112

food product isolates by fluorescence monitoring of dipicolinic acid release. Appl Environ Microbiol 71:3556-3564. Lee, S. H., S. Pumprueg, B. Moudgil, and W. Sigmund. 2005. Inactivation of bacterial endospores by photocatalytic nanocomposites. Colloids Surf B Biointerfaces 40:93-98. Leistner, L. 2000. Basic aspects of food preservation by hurdle technology. Int J Food Microbiol 55:181-186. Liu, H., N. H. Bergman, B. Thomason, S. Shallom, A. Hazen, J. Crossno, D. A. Rasko, J. Ravel, T. D. Read, S. N. Peterson, J. Yates, 3rd, and P. C. Hanna. 2004. Formation and composition of the Bacillus anthracis endospore. J Bacteriol 186:164-178. Loshon, C. A., E. Melly, B. Setlow, and P. Setlow. 2001. Analysis of the killing of spores of Bacillus subtilis by a new disinfectant, Sterilox. J Appl Microbiol 91:1051-1058. Mastwijk, H. C. 2004. Presented at the 2nd Innovative Foods Centre Conference, Sydney, 14-15 sept. Melly, E., A. E. Cowan, and P. Setlow. 2002. Studies on the mechanism of killing of Bacillus subtilis spores by hydrogen peroxide. J Appl Microbiol 93:316-325. Moir, A., B. M. Corfe, and J. Behravan. 2002. Spore germination. Cell Mol Life Sci 59:403-409. Molle, V., M. Fujita, S. T. Jensen, P. Eichenberger, J. E. Gonzalez-Pastor, J. S. Liu, and R. Losick. 2003. The Spo0A regulon of Bacillus subtilis. Mol Microbiol 50:1683-1701. Oomes, S. J. C. M., and S. Brul. 2004. The effect of metal ions commonly present in food on gene expression of sporulating Bacillus subtilis cells in relation to spore wet heat resistance. Innov Food Sci Emerg Technol 5:307-316. Pettersson, B., F. Lembke, P. Hammer, E. Stackebrandt, and F. G. Priest. 1996. Bacillus sporothermodurans, a new species producing highly heat-resistant endospores. Int J Syst Bacteriol 46:759-764. Purevdorj, D., N. Igura, O. Ariyada, and I. Hayakawa. 2003. Effect of feed gas composition of gas discharge plasmas on Bacillus pumilus spore mortality. Lett Appl Microbiol 37:31-34. Pyle, B. H., S. C. Broadaway, and G. A. McFeters. 1999. Sensitive detection of Escherichia coli O157:H7 in food and water by immunomagnetic separation and solid-phase laser cytometry. Appl Environ Microbiol 65:1966-1972. Rasko, D. A., M. R. Altherr, C. S. Han, and J. Ravel. 2005. Genomics of the Bacillus cereus group of organisms. FEMS Microbiol Rev 29:303-329. Sadik, O. A., A. K. Wanekaya, and S. Andreescu. 2004. Advances in analytical technologies for environmental protection and public safety. J Environ Monit 6:513-522. Schuch, R., D. Nelson, and V. A. Fischetti. 2002. A bacteriolytic agent that detects and kills Bacillus anthracis. Nature 418:884-889. Scott Smith, J., and S. Pillai. 2004. Irradiation and food safety. Food Tech 58:48-55. Setlow, B., C. A. Loshon, P. C. Genest, A. E. Cowan, C. Setlow, and P. Setlow. 2002. Mechanisms of killing spores of Bacillus subtilis by acid, alkali and ethanol. J Appl Microbiol 92:362-375. Setlow, B., C. A. Setlow, and P. Setlow. 1997. Killing bacterial spores by organic hydroperoxides. Journal of Industrial Microbiology & Biotechnology 18:384-388. Setlow, P. 2003. Spore germination. Curr Opin Microbiol 6:1-7. Setlow, P. 2004. Presented at the 24th Army Science Conference, Orlando, Florida, Nov 29 - Dec 2. Shapiro, M. P., B. Setlow, and P. Setlow. 2004. Killing of Bacillus subtilis spores by a modified fenton reagent containing CuCl2 and ascorbic acid. Appl Environ Microbiol 70:2535-2539.

Summary, future perspectives and practical applicability of the research described in this thesis

40.

41. 42. 43. 44. 45.

46. 47.

48. 49.

Shimizu, T., K. Ohtani, H. Hirakawa, K. Ohshima, A. Yamashita, T. Shiba, N. Ogasawara, M. Hattori, S. Kuhara, and H. Hayashi. 2002. Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater. Proc Natl Acad Sci U S A 99:9961001. Silvaggi, J. M., D. L. Popham, A. Driks, P. Eichenberger, and R. Losick. 2004. Unmasking novel sporulation genes in Bacillus subtilis. J Bacteriol 186:8089-8095. Sonenshein, A. L. 2000. Control of sporulation initiation in Bacillus subtilis. Curr Opin Microbiol 3:561-566. Steele, J. H. 2001. Food irradiation: a public health challenge for the 21st century. Clin Infect Dis 33:376-377. Stragier, P. 2002. A gene odyssey: exploring the genomes of endospore-forming bacteria, p. 519-527. In A. L. Sonenshein, J. A. Hoch, and R. Losick (ed.), Bacillus subtilis and its closest relatives: from genes to cells. ASM Press, Washington D. C. Sun, W., F. Khosravi, H. Albrechtsen, L. Y. Brovko, and M. W. Griffiths. 2002. Comparison of ATP and in vivo bioluminescence for assessing the efficiency of immunomagnetic sorbents for live Escherichia coli O157:H7 cells. J Appl Microbiol 92:1021-1027. Tennen, R., B. Setlow, K. L. Davis, C. A. Loshon, and P. Setlow. 2000. Mechanisms of killing of spores of Bacillus subtilis by iodine, glutaraldehyde and nitrous acid. J Appl Microbiol 89:330-338. Tomas, C. A., K. V. Alsaker, H. P. Bonarius, W. T. Hendriksen, H. Yang, J. A. Beamish, C. J. Paredes, and E. T. Papoutsakis. 2003. DNA array-based transcriptional analysis of asporogenous, nonsolventogenic Clostridium acetobutylicum strains SKO1 and M5. J Bacteriol 185:4539-4547. Young, S. B., and P. Setlow. 2004. Mechanisms of Bacillus subtilis spore resistance to and killing by aqueous ozone. J Appl Microbiol 96:1133-1142. Young, S. B., and P. Setlow. 2004b. Mechanisms of killing of Bacillus subtilis spores by Decon and Oxone, two general decontaminants for biological agents. J Appl Microbiol 96:289-301.

113

Chapter 8

114

Gearfetting Ynlieding Baktearje spoaren binne spesjale kapsules, dy’t bepaalde soarten fan baktearjen meitsje om ûngeunstige omstannichheden te oerlibjen. Dizze baktearjen foarmje de spoaren binnen yn harren sellen, wêrby’t eltse sel ien inkelde spoare produsearje kin. It proses fan spoarefoarming wurdt spoarulaasje neamd. Tidens de spoarulaasje wurde alle komponinten fan de spoare produsearre, en ynelkoar setten oant de spoare ryp is, wêrnei’t de sel dêr’t de spoare yn foarme is ôfstjert en út elkoar falt. Hjirby komt de spoare frij yn it miljeu. Spoaren hawwe in unike struktuer, dy’t sterk ferskillend is fan dy fan gewoane baktearjesellen. Dizze struktuer ferlient de spoare bjusterbaarlike eigenskippen, lykas in grutte bestindichheid tsjin bygelyks hite, straling, soer, mechanyske skea, antibiotika, gemyske stoffen, en útdroegjen. Fierders hawwe spoaren gjin stofwikseling, en dus gjin itensboarne nedich om te oerlibjen. Hjirtroch kinne spoaren ekstreme omstannichheden en tige lange tiden oerlibje. Sa binne der bygelyks artikels ferskynd oer de isolaasje fan libbensfetbere spoaren út fossilen en ûnderierdske sâltlagen, dy’t 40 oant 250 miljoen jier âld wiene! Fanwegen harren metaboalyske slieptastân kinne spoaren net groeie en harren ek net fermannichfâldigje. Spoaren hawwe lykwols in soarte fan ôftaastsystemen yn harren,dêr’t se de hiele tiid har omjouwing mei ôftaaste op de oanwêzichheid fan iten. Sadree’t se fernimme dat der genôch iten is foar de groei fan de baktearjen, reageare de spoaren troch út te spruten. Yn it proses fan útspruten feroaret de spoare yn in gewoane baktearjesel, wêrby’t de unike struktuer en de bjusterbaarlike eigenskippen fan de spoare ferlern geane. De útspruten spoaren feroarje yn baktearjesellen, dy’t groeie en harren fermannichfâldigje, oant it momint dat de omjouwing wer ûngeunstich wurdt om fierder te groeien. Dan foarmje de sellen wer nije spoaren. Yn de praktyk stjoere spoaren faak de boel yn ‘e hobbels at der omstannichheden fan hygiëne nedich binne, bygelyks by it tarieden fan iten yn ‘e yndustry. Spoaren sitte oeral, dus ek op masjines, yn grûnstoffen, en op ferpakkingsmaterialen dy’t yn de itensyndustry brûkt wurde. Dêrom is it praktysk net mooglik om foar te kommen dat spoaren yn it iten bedarje. Mei harren bjusterbaarlike eigenskippen oerlibje spoaren de tariedingsprosessen fan iten, wêrnei’t se yn it tarette iten ûntkime en groeie kinne, wêrtroch de brut bedjert en sels fergiftich wurde kin. Spoaren kinne allinnich mar deamakke wurde troch bûtengewoane maatregels lykas tige hege ferhiting (sterilisaasje), dat sawat altyd ten koste giet fan ‘e smaak en de tekstuer fan it iten. De hjoeddeiske konsumint mei graach farsk, natuerlik en lekker iten keapje. Sadwaande stiet de itensyndustry no foar de útdaging om iten te meitsjen dat farsk, natuerlik en lekker is, mar dochs ek mikrobiologysk feilich, dus frij fan spoaren. Dêrom siket men nei nije metoades om spoaren dea te meitsjen sûnder sterilisaasje. Ekstra kennis oer baktearjespoaren kin hjirby helpe, en dêrom is der binnen it Wageningen Sintrum foar Itenswittenskip (WCFS) in projekt op gong brocht om mear kennis oer baktearjespoaren te garjen. It ûndersyk dat yn dit proefskrift beskreaun is, is in part fan dat projekt. As modelbaktearje hawwe wy Bacillus cereus útkeazen, in baktearje dy’t spoaren makket, yn de praktyk faak molke bedjert, en fergiftigingen feroarsaakje kin. B. cereus heart by in nau besibbe groepke baktearjen, dêr’t ek Bacillus anthracis, de feroarsaker fan myltfjoer, by heart. Op it genoom fan B. cereus lizze dan ek in grut tal firulinsje en toksine genen. B. cereus is spesialisearre yn it metaboalisme fan aaiwiten, en groeit tige bêst yn molke. Hjirtroch, en trochdat de spoaren makkelik pasteurisaasje behannelingen oerlibje kinne, is B. cereus ien fan de wichtichste bedjerders fan pasteurisearre suvelprodukten. Boppedat kin B. cereus, troch toksinefoarming, yn oare itensprodukten ferskate foarmen fan fergiftiging feroarsaakje. Oer’t algemien is dêrby sprake fan frij swakke symptomen, lykas spuie en skiterij. Der binne lykwols in pear seldsume gefallen bekend wêrby’t fergiftiging fan iten troch B. cereus ta de dea laat hat.

115

Gearfetting

B. cereus groei en spoarulaasje Oan it begjin fan dit ûndersyk wie de earste útdaging om standert protokollen te ûntwikkeljen foar bygelyks it kweken fan B. cereus, it produsearjen , it rispjen en it bewarjen fan de spoaren. Dêrfoar hawwe wy standerdisearre en krekt definiearre kondysjes tapast, om de reprodusearberheid fan ús eksperiminten te garandearjen. Mei dizze definearde opset koe dêrnei it proses fan spoarefoarming yn detail bestudearre wurde. Hjirby die bliken dat, hoewol’t B. cereus yn hiele oare omstannichheden libbet as de modelspoarefoarmer Bacillus subtilis, it mechanisme fan spoarulaasje foar it grutste part itselde is yn beide soarten (Haadstik 2). B. cereus metabolisearret aminosoeren tidens de groei tagelyk mei glukoaze, wêrby’t de aminosoeren as koalstofboarne en as brânstof fungearje. Yn de oanwêzichheid fan glukoaze set B. cereus ûngefear de helte fan de aminosoeren om yn ammoania, wat oanjout dat dizze aminosoeren net as boustof foar aaiwiten brûkt wurde. At der laktaat ynstee fan glukoaze as koalstofboarne oanwêzich is, wurde sawat alle aminosoeren omsetten yn ammonia. It grutste part fan de stikstofopname bart ûnder de stasjonaire groeifase en ûnder spoarulaasje. L-glutamaat is in prima stikstofboarne foar B. cereus, en wurdt by foarkar ûnder de spoarulaasje opnommen (Haadstik 3). Oer’t algemien wurdt oannommen dat in gebrek oan iten it wichtichste teken is foar de sellen om spoaren te foarmjen. Yn ús eksperiminten, lykwols, begûnen de sellen al te spoarulearjen lang foardat harren iten op wie, wat derop wiist dat de spoarulaasje yn dit gefal oanstjoerd waard troch oare faktoaren, lykas seltichtheid. Yn ús eksperiminten waard it dúdlik dat de transkripsje fan it gen fan in wichtige regulator fan stress sinjalen, de sigmafaktor σB, omheech gie op itselde stuit as guon spoarulaasje sigmafaktoaren. Dit hat fan dwaan mei de oergong fan glukoaze nei oare koalstofboarnen wêrby’t de sellen blykber oanset wurde ta spoarulaasje. It die bliken dat in mutant wêryn’t it gen foar σB útskeakele is, abnormaal rûne spoaren makke. Dizze rûne spoaren fan de σB mutant hienen in legere bestindigens tsjin hitens, en in sterk oantaaste útsprutingsrespons. Dit wiist derop dat σB belutsen is by it spoarulaasjeproses fan B. cereus (Haadstik 4). De struktuer fan B. cereus en B. subtilis spoaren Fanwege syn metaboalyske slieptastân is in spoare eigentlik net “yn libben”, mar in samling fan biologyske molekulen, mei in spesifike fysyske en gemyske struktuer. Dizze struktuer beskermet de belangrike ûnderdielen fan de spoare, lykas membranen, wichtige aaiwiten en it DNA. Om it mechanisme fan wjerstân te begripen, is it fan it heechste belang om de fysyske en gemyske eigenskippen fan de spoare krekt yn kaart te bringen. Ien fan de wichtichste parameters hjirby is de fysyske steat fan it spoaresytoplasma, en hoewol’t der al mear as hûndert jier ûndersyk nei dien is, is it noch hieltyd net dúdlik hoe’t dy derút sjocht. Wy hawwe in elektron spin resonânsje metoade betocht (Haadstik 5), en mei dizze metoade is it foar it earst slagge om spesifyk yn de kearn fan B. cereus en B. subtilis spoaren te sjen, en ferskate wichtige saken lykas molekulaire beweeglikheid en fiskeusiteit te mjitten. Hjirby die it bliken dat it sytoplasma fan baktearjespoaren net yn in glêseftige tastân is, wêr’t wol ferskillende kearen yn ‘e literatuer oer skreaun is. Ynstee fan in glêseftige tastân wize ús mjittingen derop dat der yn de kearn fan de spoare in soarte fan spûnseftige struktuer oanwêzich is, wêryn’t de aaiwytkomponinten fan it sytoplasma foar in grut part fêstlein binne, wylst it tige fiskeuze sytoplasmatyske wetter wól frijwat beweeglik is.Troch waarmte-aktivaasje en it foar in part útspruten, wurdt dizze spûnsstruktuer net sa bot feroare. At de spoare alhielendal útsprút, lykwols, ferdwynt de struktuer yn syn gehiel, en giet de sytoplasmatyske fiskeusiteit danich nei ûnderen ta. It útspruten fan B. cereus spoaren Utsprute is it proses dêr’t spoaren troch feroarje fan in ynerte, resistinte tastân nei in metaboalysk aktive tastân. Sa’t hjirboppe al sein is, soarget it útspruten derfoar dat de struktuer fan de spoare ôfbrutsen wurdt. Sa liedt it útspruten ta it ferlies fan de bjusterbaarlike wjerstân

116

Gearfetting

eigenskippen fan de spoare. Utsprute is wêzentlik foar de prolyferaasje fan spoarefoarmers, en dus ek foar it úteinliks ûntstean fan itensbedjer en –fergiftigingen. Om dizze redenen is it proses fan útsprute net allinnich wittenskiplik besjoen ynteressant, mar ek fanút it praktyske tapassingsperspektyf wei nijsgjirrich. It mechanistyske model foar útspruten is basearre op eksperiminten mei B. subtilis, en is net hielendal folslein. Neist spesifike mechanistyske fragen, lizze der foaral fragen oer it útspruten fan oare soarten as de modelstammen, foaral de soarten dêr’t men yn de praktyk lêst fan hat. Boppedat is foar de praktyk ynsjoch yn de heterogenisiteit fan de útsprútreaksje yn spoarepopulaasjes fan grut belang. Dizze twa kwestjes hawwe wy ûnder hannen naam yn de haadstikken 6 en 7. Yn haadstik 6 hawwe wy in nije technyk tapast om it gedrach fan yndividuele spoaren yn útsprutende spoarepopulaasjes te bestudearjen. Troch ferskate fluoresinte kleurmetoades te brûken, wie it mooglik om ferskillende stadia fan útspruten, bygelyks DNA tagonkelikheid en esterase aktiviteit, spesifyk yn de populaasje te kwantifisearjen. Yn Haadstik 7 waard it útsprútgedrach fan spoaren fan in oantal yndustriële isolaten ûnder de lûp nommen. Wylst spoaren fan de B. cereus modelstam ATCC14579 fluch en folslein útspruten, die it bliken dat de yndustriële isolaten in tige grutte fariaasje hienen yn harren manier en mate fan útspruten. De tapasberheid fan de risseltaten De metoades en techniken dy’t ûntwikkele en tapast binne yn dit ûndersyk foarmje in poerbêste basis foar fierder ûndersyk oan spoaren fan B. cereus en oare spoarefoarmers. De definiearde omstannichheden foar spoarulaasje, sa’t dy beskreaun binne yn haadstik 2, binne essinsjeel foar it krijen fan in detaillearre ynsjoch yn it spoarulaasje proses fan B. cereus. Yn haadstik 3 waard spoarulaasje bewurkstellige yn ferskate omstannichheden, wêrby’t spoaren mei ferskillende eigenskippen rispe wurde koenen. Takomstige projekten dy’t de beskikking hawwe oer B. cereus mikro-arrays om in ferbân te finen tusken gen-ekspresje en spoare-eigenskippen, binne tige holpen mei dizze opset. Oare belangrike ûntjouwings binne it effisjinte protokol foar it maklik produsearjen en rispjen fan in grut tal B. cereus spoaren, de real-time qPCR metoade foar it kwantifisearjen fan genekspresje, en de ferskate metoades om spesifike eigenskippen fan spoaren te mjitten, lykas hite-bestindigens, kymkrêft, DPA ynhâld, en soartlik gewicht. De kymstúdzjes mei isolaten út de yndustry, beskreaun yn haadstik 7, litte sjen dat de útsprútreaksje fan spoaren dy’t yn de praktyk foarkomme tige fariabel is en ferskilt fan dy fan de model stam. Hoewol’t guon stammen relatyf min útsprute woene yn ferliking mei it laboratorium model, hawwe wy sjen litten dat mei de goeie stimulaasje ek dizze stammen ta útspruten brocht wurde kinne. Dit makket it paad frij foar de tapassing fan útspruten yn de praktyk om de ynaktivaasje fan spoaren makliker te meitsjen, bygelyks by it skjinmeitsjen en desinfektearjen fan masjines en proses liedingen yn de itens-yndustry. De Flow-Sytometryske metoade (FCM), beskreaun yn haadstik 6, is tige geskikt foar de kwantitative analyse fan spoarepopulaasjes. Mei FCM kinne in hiele protte mjittingen dien wurde, wêrby’t de heterogenisiteit fan natuerlike spoarepopulaasjes kwantifisearre wurde kin. Dit kin tapast wurde op spoaren dy’t skansearre binne, bygelyks yn de praktyk by de tariedingsprosessen fan iten. Sa kin mei fluoresinte kleurmetoades en merkergenen de effektiviteit fan spoaren ynaktivaasje metoades ûndersocht wurde. De grutte mannichten kwantitatieve data dy’t FCM dêrby opleveret binne boppedat tige brûkber foar ferwurking yn kompjûter modellen, dy’t brûkt wurde kinne om proses parameters krekt ôf te stellen. Hjirtroch kinne kosten besparre en produkten ferbettere wurde. Ta einbeslút foarsjocht de ESR metoade, beskreaun yn haadstik 5, yn in nije set fan parameters dy’t fuortendaliks weromslaan op de fysyske struktuer fan spoaren. Dizze parameters kinne fuort ferbûn wurde oan spesifike spoare-eigenskippen. Benammen it geheim fan de ekstreme wjerstân fan bepaalde soarten tsjin hitens kin mei dizze metoade yn takomstige eksperiminten opheldere wurde. De kennis dy’t dit opleveret sil helpe by it finen fan swakke plakken yn de

117

Gearfetting

struktuer fan spoaren. Dit iepenet de doar ta nije konservearingsmetoades, dy’t gebrûk meitsje fan de Achilleshakke fan baktearje spoaren. Slotwurd De biology fan spoaren is in ynternasjonaal, tige dynamysk en spannend ûndersyksfjild, wêryn’t fundamenteel wittenskiplike ynteresse, praktyske tapasberheid en yndustriële relevânsje mei inoar mjukse wurde. It ûndersyk dat yn dit proefskrift beskreaun is hat nije fundamentele kennis oplevere oer de foarming en struktuer fan spoaren, hat nije metoades oplevere mei praktyske tapasberheid, en hat sa foarsjoen yn in stevige basis foar fierder spoare-ûndersyk.

118

Samenvatting Inleiding Bacteriële sporen zijn speciale overlevingscapsules, die worden gemaakt door specifieke soorten bacteriën om ongunstige omstandigheden te overleven. Deze bacteriën vormen de sporen binnenin hun cellen, waarbij elke cel één enkele spore kan produceren. Het proces van sporenvorming wordt sporulatie genoemd. Tijdens de sporulatie worden alle componenten van de spore geproduceerd, en geassembleerd totdat de spore rijp is, waarna de cel waarin de spore werd gevormd afsterft en lyseert. Hierbij komt de spore vrij in het milieu. Sporen hebben een unieke structuur die sterk verschilt van de structuur van normale bacteriecellen. Deze unieke structuur maakt sporen zeer resistent tegen hitte, straling, zuur, mechanische beschadigingen, antibiotica, chemische middelen, uitdroging, en ga zo maar door. Bovendien hebben sporen geen metabolisme, en dus geen voedsel nodig om te overleven. Hierdoor kunnen sporen extreme omstandigheden en zeer lange tijd overleven. Zo zijn er publicaties verschenen over de isolatie van levensvatbare sporen uit fossielen en onderaardse zoutlagen (40 en 250 miljoen jaar oud!). Daarnaast is het bekend dat sporen ruimtereizen kunnen overleven, hetgeen theorieën over de verspreiding van het leven door het heelal (“panspermia”) middels sporen voedt. Door hun metabolische slaaptoestand kunnen sporen niet groeien of zich vermenigvuldigen. Echter, sporen beschikken over monitoringsystemen waarmee ze continue hun omgeving screenen op de aanwezigheid van nutriënten. Zodra bepaalde nutriënten aanwezig zijn, die duiden op gunstige omstandigheden voor groei, reageert de spore door te kiemen. Tijdens het kiemingsproces verandert de spore in een vegetatieve bacteriecel, waarbij zijn unieke structuur verloren gaat. De onstane bacteriecel kan vervolgens gaan groeien en zich vermenigvuldigen. Als de omstandigheden ongunstig worden, vormen de cellen weer nieuwe sporen. In de praktijk veroorzaken sporen vaak problemen in omstandigheden waar hygiëne gewenst is, bijvoorbeeld bij het bereiden van voedsel. Sporen zijn overal aanwezig, dus ook op machines, in grondstoffen, en op verpakkingsmaterialen, die in de levensmiddelen industrie gebruikt worden. Het is daarom praktisch onmogelijk om te voorkomen dat sporen in voedselproducten terecht komen. Door hun hoge resistentie kunnen sporen de processen die in de industrie gebruikt worden voor de bereiding van levensmiddelen overleven, waarna ze in de eindproducten kunnen gaan ontkiemen en groeien, waardoor uiteindelijk bederf en voedselvergiftigingen ontstaan. Afdoding van sporen in de eindproducten kan alleen bereikt worden met een zeer forse hittebehandeling (sterilisatie), wat leidt tot ernstig smaak- en textuurverlies. Dit is voor de hedendaagse consument, die steeds meer eist dat levensmiddelen vers en natuurlijk eruit zien en smaken, niet langer acceptabel. Aldus ziet de levensmiddelenindustrie zich geconfronteerd met de uitdaging om producten op de markt te brengen die minimaal behandeld zijn en daardoor vers en natuurlijk smaken, maar toch microbiologisch veilig zijn, dus vrij van sporen. Daarom is men op zoek naar nieuwe methodes om sporen te inactiveren zonder sterilisatie. Extra kennis van bacteriële sporen kan helpen bij deze zoektocht, en daarom is binnen het Wageningen Centre for Food Sciences (WCFS) een project gestart om meer kennis over bacteriële sporen te ontwikkelen. Het onderzoek beschreven in dit proefschrift maakt deel uit van dat project. Als modelorganisme is gekozen voor Bacillus cereus, een sporenvormende bacterie die in de praktijk bederf en voedselvergiftiging veroorzaakt. B. cereus hoort bij een groep van nauw verwante bacteriën, waar ook Bacillus anthracis, de veroorzaker van miltvuur (anthrax), deel van uitmaakt. Het B. cereus genoom bevat talrijke virulentie en toxinegenen. B. cereus is gespecialiseerd in het metabolisme van eiwitten, en gedijt uitstekend in melk. Hierdoor, en doordat de sporen gemakkelijk pasteurisatie behandelingen overleven, is B. cereus één van de belangrijkste bederforganismen in gepasteuriseerde zuivelproducten. Bovendien kan B. cereus, door

119

Samenvatting

toxinevorming, in andere voedselproducten verschillende vormen van voedselvergiftiging veroorzaken. Er is daarbij over het algemeen sprake van vrij milde symptomen, waaronder diarree, braken en verkoudheid. Echter, er zijn enkele zeldzame gevallen bekend waarbij voedselvergiftiging door B. cereus tot de dood heeft geleid. Bacillus cereus groei en sporulatie Bij de aanvang van het in dit proefschrift beschreven onderzoek was de eerste uitdaging om standaard protocollen te ontwikkelen voor bijvoorbeeld het groeien van B. cereus, sporenproductie, sporen oogst, opslag, enzovoort. Hierbij werden gestandaardiseerde en precies gedefinieerde condities toegepast, om de reproduceerbaarheid van experimenten te garanderen. Met deze gedefinieerde opzet kon vervolgens het proces van sporenvorming in detail worden bestudeerd. Hierbij bleek dat, hoewel B. cereus in een heel andere ecologische niche leeft dan de modelsporenvormer, Bacillus subtilis, het mechanisme van sporulatie zeer geconserveerd is tussen deze twee soorten (Hoofdstuk 2). B. cereus metaboliseerde aminozuren tijdens zijn groei tegelijkertijd met glucose, waarbij de aminozuren als koolstofbron en brandstof voor de groei fungeerden. In de aanwezigheid van glucose zette B. cereus ongeveer de helft van de aminozuren om in ammonia, wat erop duidt dat deze aminozuren als brandstof en niet als bouwstof gebruikt worden. Als er lactaat in plaats van glucose als koolstofbron aanwezig was, werden bijna alle aminozuren omgezet in ammonia (Hoofdstuk 2 en 3). Het leeuwendeel van de stikstofopname geschiedde tijdens de stationaire groeifase en tijdens de sporulatie. L-glutamaat bleek een uitstekende stikstofbron voor B. cereus, en werd bij voorkeur opgenomen tijdens de sporulatie. B. cereus kon ook ammonia als stikstofbron gebruiken. Over het algemeen wordt aangenomen dat voedseltekort het belangrijkste signaal is voor de cellen om te gaan sporuleren. Echter, in onze experimenten begonnen de cellen al te sporuleren lang vóórdat er sprake was van nutriënten depletie, wat erop duidt dat sporulatie werd gestart onder invloed van andere factoren zoals celdichtheid (Hoofdstuk 3). Tijdens onze experimenten werd duidelijk dat de transcriptie van het gen voor een belangrijke regulator van de B. cereus stressresponse, de alternatieve sigma factor σB, werd opgereguleerd op hetzelfde moment als enkele sporulatie regulatoren. Deze opregulatie had te maken met de overgang van glucose naar andere koolstofbronnen. Bij nader onderzoek bleek dat een mutant waarin het gen voor σB is geïnactiveerd abnormaal ronde sporen vormde. De ronde sporen van de σB mutant hadden een lagere hitteresistentie en ernstig aangetaste kiemingsrespons, wat erop wijst dat σB is betrokken bij het sporulatie mechanisme van B. cereus (Hoofdstuk 4). De structuur van B. subtilis en B. cereus sporen Vanwege zijn metabolische slaaptoestand is een spore eigenlijk niet “in leven”, maar een verzameling van biologische moleculen, met een specifieke fysische en chemische structuur. Deze fysisch-chemische structuur bewerkstelligt de bescherming van de vitale onderdelen van de spore, zoals membranen, essentiële eiwitten en het DNA. Om het mechanisme van resistentie te begrijpen, is het van het grootste belang om de fysische en chemische eigenschappen van de spore nauwkeurig in kaart te brengen. Eén van de belangrijkste parameters hierbij is de fysische staat van het spore cytoplasma, en ondanks meer dan 100 jaar onderzoek, is het nog steeds niet duidelijk hoe die eruit ziet. De Elektron Spin Resonantie (ESR) methode, gepresenteerd in Hoofdstuk 5, gaat in op deze kwestie. Met deze nieuwe methode is het voor het eerst gelukt om specifiek in de kern van B. subtilis en B. cereus sporen te kijken, en diverse parameters zoals moleculaire beweeglijkheid en viscositeit te meten. Hieruit bleek dat het cytoplasma van deze sporen niet in een glasachtige toestand verkeert, hetgeen herhaalde malen in de literatuur is beweerd. In plaats daarvan suggeren onze metingen dat er een driedimensionale moleculaire matrix in de kern aanwezig is, waarin de eiwitcomponenten van het cytoplasma grotendeels zijn geïmmobiliseerd, maar waarin het zeer viskeuze cytoplasmatische water wél vrij beweeglijk is. Hitte-activatie en gedeeltelijke ontkieming

120

Samenvatting

leidden niet tot een grote verandering in deze matrix. Echter, volledige kieming resulteerde in een complete afbraak van deze matrix, en leidde bovendien tot een grote afname van de cytoplasmatische viscositeit. B. cereus sporen kieming Kieming is het proces waarbij sporen veranderen van een metabole slaaptoestand naar een metabolisch actieve toestand. Zoals eerder werd vermeld, gaat kieming gepaard met de afbraak van de specifieke structuur van de spore, wat leidt tot het verlies van resistentie eigenschappen. Bovendien is kieming essentieel voor de proliferatie van sporenvormers, en dus voor het uiteindelijk ontstaan van voedselbederf en -vergiftiging. Om deze redenen is het proces van kieming niet alleen interessant om fundamenteel wetenschappelijke redenen, maar ook vanuit een toepassingsgericht perspectief. Het mechanistische model voor kieming is geënt op experimenten met B. subtilis, en is niet helemaal compleet. Naast specifieke mechanistische vragen, liggen er veel vragen over de kieming van andere soorten, met name industrieel relevante stammen. Bovendien is voor de praktijk inzicht in de heterogeniciteit van de kiemingsrespons in sporenpopulaties van groot belang. Deze twee kwesties zijn behandeld in Hoofdstuk 6 en 7. In Hoofdstuk 6 werd een nieuwe techniek toegepast om het gedrag van individuele sporen in kiemende sporenpopulaties te bekijken. Door verschillende fluorescente kleurmethodes te gebruiken, was het mogelijk om diverse stadia van kieming, zoals DNA toegankelijkheid en esterase activiteit, specifiek in de kiemende populaties te kwantificeren. In Hoofdstuk 7 werd het kiemingsgedrag van sporen van een aantal industriële isolaten onder de loep genomen. Terwijl sporen van de B. cereus model stam ATCC14579 snel en volledig kiemden, bleken de industriële isolaten een grote variabiliteit in hun kiemingsrespons te laten zien. Toepasbaarheid van de verkregen resultaten De methodes en technieken die zijn ontwikkeld en geïmplementeerd tijdens dit onderzoek vormen een uitstekende basis voor verder onderzoek aan sporen van B. cereus en andere sporenvormers. De gedefinieerde condities voor sporulatie, beschreven in Hoofdstuk 2, zijn essentieel voor het verkrijgen van gedetailleerd inzicht in het sporulatie proces van B. cereus. In Hoofdstuk 3 werd sporulatie gerealiseerd in verschillende condities, waarbij sporen met verschillende eigenschappen werden verkregen. Vervolgprojecten die gebruik maken van B. cereus micro-arrays om een verband te vinden tussen sporeneigenschappen en genexpressie tijdens de sporulatie, zijn zeer gebaat bij deze opzet. Andere belangrijke ontwikkelingen zijn het efficiënte protocol voor het gemakkelijk produceren en oogsten van grote hoeveelheden B. cereus sporen, de real-time qPCR methode voor het kwantificeren van genexpressie, en de verschillende methodes om specifieke sporeneigenschappen te meten, waaronder hitte resistentie, ontkiembaarheid, DPA inhoud, hydrophobiciteit, en dichtheid. De kiemingsstudies met industriële isolaten, beschreven in Hoofdstuk 7, laten zien dat de kiemingsrespons van sporen die in de industriële praktijk voorkomen zeer variabel is. Hoewel enkele stammen relatief slecht kiemen in vergelijking met het laboratorium model, hebben we aangetoond dat met de juiste stimulatie ook deze stammen tot kieming kunnen worden gebracht. Dit maakt de weg vrij voor toepassing van kieming in de praktijk om de inactivatie van sporen te vergemakkelijken, bijvoorbeeld bij het reinigen en desinfecteren van machines en procesleidingen in de levensmiddelen industrie. De Flow-cytometrische methode (FCM) gepresenteerd in Hoofdstuk 6 is zeer geschikt voor de kwantitatieve analyse van sporen populaties. Met FCM kunnen grote hoeveelheden data worden gegenereerd, waarbij de heterogeniciteit van natuurlijke sporenpopulaties kan worden gekwantificeerd. Dit kan worden toegepast op sporen die beschadigd zijn, bijvoorbeeld in de praktijk door procescondities. Zo kan met fluorescente kleurmethodes en merkergenen de effectiviteit van sporen-inactivatiemethodes worden onderzocht. De grote hoeveelheden

121

Samenvatting

kwantitatieve gegevens die FCM daarbij oplevert zijn bovendien uiterst geschikt voor verwerking in computer modellen, die kunnen worden gebruikt om de procescondities nauwkeuriger af te stellen, wat kan leiden tot kostenreducties en verbetering van de producten. Tenslotte voorziet de ESR methode, beschreven in Hoofdstuk 5, in een nieuwe set van parameters die direct betrekking hebben op de fysieke structuur van sporen, en kunnen worden verbonden aan specifieke sporeneigenschappen. Met name het geheim van de extreme hitte resistentie van bepaalde industriële isolaten kan met deze methode in toekomstige experimenten worden ontrafeld. De kennis die hierbij gegenereerd wordt zal helpen bij het vinden van zwakke plekken in de resistentie mechanismen van sporen. Dit opent de deur naar nieuwe conserveringsmethodes die gebruik maken van de Achilleshiel van bacteriële sporen. Slotwoord De biologie van sporen is een internationaal, zeer actief en spannend onderzoeksveld, waarin fundamenteel wetenschappelijke interesse, praktische toepasbaarheid en industriële relevantie hand in hand gaan. Het onderzoek dat is beschreven in dit proefschrift heeft nieuwe fundamentele data opgeleverd over sporenvorming en -structuur, heeft geresulteerd in nieuwe methodes met praktische toepasbaarheid, en heeft voorzien in een stevige basis voor verder sporenonderzoek. Deze zullen helpen in de voortdurende zoektocht naar oplossingen voor de vele problemen die worden veroorzaakt door de immer en alom aanwezige bacteriële sporen.

122

List of Publications International Congresses Ynte P. de Vries, Y. Takahara, Y. Ikunaga, Y. Ushiba, M. Hasegawa, Y. Kasahara, H. Shimomura, S. Hayashi, Y. Hirai, and H. Ohta. Degradation of branched nonylphenol by Sphingomonas sp. Presented at the Annual Meeting of the Japanese Society for Micriobial Ecology, November 2000, Tsukuba, Japan. Ynte P. de Vries, Luc M. Hornstra, and Marjon Bennik. Germination properties of spores of foodborne Bacillus cereus. Presented at the 9th International Symposium on Microbial Ecology, August 2001, Amsterdam, the Netherlands. Luc. M. Hornstra, Ynte P. de Vries, Marjon H. Bennik, Willem M. de Vos, and Tjakko Abee. GerR, a novel ger operon involved in L-alanine and inosine germination in B. cereus ATCC 14579. Presented at Functional Genomics of Gram-Positive Microorganisms, 12th International Conference on Bacilli, June 2003, Baveno, Italy. Ynte P de Vries, Luc M. Hornstra, Willem M. de Vos, and Tjakko Abee. Sporulation of B. cereus in defined conditions: Carbon source utilization, gene expression and the effect on spore properties. Presented at the European Spores Conference, June 2004, Smolenice Castle, Slowakia. Luc M. Hornstra, Ynte P. de Vries, Willem M. de Vos, and Tjakko Abee. Germination operons in Bacillus cereus ATCC 14579; Characterization and function. Presented at the European Spores Conference, June 2004, Smolenice Castle, Slowakia. Elena A. Golovina, Susanne M.C. Claessens, Jan Dijksterhuis, Ynte P. de Vries and Folkert A. Hoekstra. ESR study of cytoplasmic viscosity in hydrated, dormant spores and seeds. Presented at the International Symposium on the Environmental Physiology of Ectotherms and Plants, Juli 2005, Roskilde, Denmark. Peer reviewed publications Ynte P. de Vries, Y. Takahara, Y. Ikunaga, Y. Ushiba, M. Hasegawa, Y. Kasahara, H. Shimomura, S. Hayashi, Y. Hirai, and H. Ohta. Organic nutrient-dependent degradation of branched nonylphenol by Sphingomonas sp. YT isolated from a river sediment sample. Microbes and Environments, 2001, Volume 16, issue 4, pages 240-249. Yasuhiro Oda, Ynte P. de Vries, Larry J. Forney and Jan C. Gottschal. Acquisition of the ability for Rhodopseudomonas palustris to degrade chlorinated benzoic acids as the sole carbon source. FEMS Microbiology Ecology, Volume 38, Issues 2-3, 26 December 2001, Pages 133-139. Ynte P. de Vries, Luc M. Hornstra, Willem M de Vos and Tjakko Abee. Growth and sporulation of Bacillus cereus ATCC 14579 under defined conditions: temporal expression of genes for key sigma factors. Applied and Environmental Microbiology, 2004, Volume 70, issue 4, pages 2514-2519.

123

Ynte P. de Vries. The role of calcium in bacterial spore germination. Environments, 2004, Volume 19, pages 199-202.

Microbes and

Ynte P. de Vries, Menno van der Voort, Janneke Wijman, Willem van Schaik, Luc M. Hornstra, Willem M. de Vos and Tjakko Abee. Progress in food-related research focussing on B. cereus. Microbes and Environments, 2004, Volume 19, pages 265-269. Luc M. Hornstra, Ynte P. de Vries, Willem M. de Vos, Tjakko Abee, and Marjon H. Wells-Bennik. gerR, a novel ger operon involved in L-alanine- and inosine-initiated germination of Bacillus cereus ATCC 14579. Applied and Environmental Microbiology, 2005, Volume 71, issue 2, pages 774-781. Ynte P. de Vries, Ratna D. Atmadja, Luc M. Hornstra, Willem M. de Vos, and Tjakko Abee. Influence of glutamate on growth, sporulation and spore properties of Bacillus cereus ATCC 14579 in a defined medium. Applied and Environmental Microbiology, 2005, Volume 71, issue 6, pages 3248-3254. Ynte P. de Vries, Luc M. Hornstra, Ratna D. Atmadja, Willem van Schaik, Willem M. de Vos, and Tjakko Abee. Deletion of sigB in Bacillus cereus affects spore properties. FEMS Microbiology Letters, 2005, Volume 252, pages 169-173. Luc M. Hornstra, Ynte P. de Vries, Marjon H. J. Wells-Bennik, Willem M. de Vos, and Tjakko Abee. Characterization of germination receptors of Bacillus cereus ATCC 14579. Applied and Environmental Microbiology, 2006, Volume 72, issue 1, in press. Luc M. Hornstra, Ynte P. de Vries, Willem M. de Vos, and Tjakko Abee. The influence of sporulation media composition on the transcription of ger operons and the germination response of Bacillus cereus ATCC 14579 spores. Submitted for publication. Ynte P. de Vries, Elena A. Golovina, Luc M. Hornstra, Marjon Wells-Bennik, Willem M. de Vos, Tjakko Abee and Folkert A. Hoekstra. Water in the core of Bacillus spores is not in a glassy state: a spin-probe study. Manuscript in preparation. Ynte P. de Vries, Kaouther Ben Amor, Luc M. Hornstra, Willem M. de Vos, Marcel Zwietering and Tjakko Abee. Bacillus cereus spore germination quantified by flow-cytometry. Manuscript in preparation. Ynte P. de Vries, George Aboagye, Luc M. Hornstra, Willem M. de Vos, Tjakko Abee and Marcel Zwietering. Germination capacity and heat resistance of spores from naturally occurring Bacillus cereus strains. Manuscript in preparation.

124

Dankwoord Toen ik begin 2001 naar Wageningen kwam om mijn aio-schap te doen, viel het me aanvankelijk niet mee om als niet-in-Wageningen-gestudeerd-hebbende te integreren in “het Wageningse”. Gelukkig mocht ik met een aantal heel fijne collega’s werken, op het ATO, bij het WCFS, bij de Leerstoelgroep Levensmiddelenmicrobiologie en bij de onderzoekschool VLAG. Zitting in de VLAG aio-raad, in het WCFS promovendi forum, en de daarbij behorende organisatie van vele leuke uitjes, feesten en teambuildings: dit alles heeft heel veel contacten opgeleverd en de tijd doen vliegen. Ik wil hiervoor de raads- en forumleden van harte bedanken. Een mooie plek in mijn herinnering hebben de beide VLAG aio-reizen naar de VS en naar Japan, die een uitstekende gelegenheid hebben geboden om een groot aantal collega’s beter te leren kennen. Ook in sportief opzicht is er veel gebeurd tijdens mijn aio-schap; dankzij het enthousiasme en organisatorische talent van een paar mensen heb ik een aantal jaren heerlijk mogen zaalvoetballen met een leuke groep. Bovendien hebben enkele WCFS en WUR collega’s me in de fietssport meegetrokken, en dat is echt een verrijking van mijn sportieve carrière geworden. Enorm bedankt voor dit alles! Met betrekking tot het aio-onderzoek zelf is de steun van een aantal mensen cruciaal geweest. Van groot belang was natuurlijk de inzet van mijn promotoren: Tjakko, Willem, en Marcel, ik wil jullie bij deze hartelijk danken voor alle tijd die jullie met mij in deze studie hebben willen steken. Ook de initiator van het B. cereus project binnen WCFS verdient een vermelding: Marjon, hoewel we je al vroeg moesten missen, is je bijdrage zeer belangrijk geweest. Important contributions to this thesis were made by my dear stagiairs Ratna and George. Without your efforts several of the chapters in this thesis would be rather empty: thank you very much for your contributions! Ook Folkert en Elena van Plantenfysiologie wil ik van harte bedanken: onze samenwerking was niet alleen essentieel voor de totstandkoming van hoofdstuk 5, maar ook uiterst prettig. Terugkijkend kan ik zeggen dat mijn detachering op het ATO (later A&F), met de vele ups en downs, een onvervangbare leerervaring is geweest. Met name mijn naaste collega’s Andreas, Rob, Katia, Patrick, Irene, Roy, Jannie en Ineke, maar natuurlijk ook de overige ATO-collega’s: hartstikke bedankt! Een speciale collega op het ATO was kantoor- en lotgenoot Luc Hornstra, die van alle collega’s toch wel de meeste van mijn hersenspinsels heeft aangehoord op de vroege ochtend, en mijn vele al of niet relevante monologen altijd weer glansrijk heeft doorstaan. Luc kerel, hartstikke bedankt voor de samenwerking, het was me daadwerkelijk een waar genoegen! Ook de collega’s bij de leerstoelgroep Levensmiddelenmicrobiologie wil ik bedanken voor de samenwerking, met name de andere B. cereus freaks, Willem, Janneke, en Menno. Aan René wil ik hier een woord van speciale dank richten: ik heb erg genoten van het gezamenlijk vissen, bierdrinken, en (veel te hard) fietsen van en naar het werk, en ik hoop dat we in de toekomst kans zullen zien om deze activiteiten voort te zetten. Aan het thuisfront, ten slotte, ben ik wel de meeste dank verschuldigd. Juul, zonder jouw steun en opofferingen was deze studie waarschijnlijk ergens halverwege gestrand. Zoals Roy in het voorwoord al zei, ging de “jungle-tocht” niet altijd over rozen. In dezelfde periode is er thuis ook ontzettend veel veranderd, en toch is het gelukt om er iets moois van te maken. Juul, hartstikke bedankt; ik weet zeker dat wij, met onze kleine Lola, een geweldige toekomst tegemoet gaan!

125

126

Curriculum vitae Ynte Piet de Vries werd geboren op 14 oktober 1976 te Sneek. Al spoedig verhuisde het gezin de Vries naar Nijega, een plattelandsdorpje in Friesland waar Ynte opgroeide temidden van boerderijen, weidevogels en water. Aldus werd de basis gelegd voor Ynte’s passie voor de biologie. Via de basisschool in de Tike kwam Ynte terecht op het VWO (Andreas College, Drachten) en vervolgens op het Gymnasium (Ichthus College, Drachten), waar hij in 1995 voor 9 keuzevakken examen deed en slaagde. Ynte’s wens om zich verder te ontwikkelen in de biologie bracht hem naar de Rijksuniversiteit Groningen, waar hij in 1995 begon en in 2000 Cum Laude afstudeerde in de Microbiële Ecologie. Tijdens zijn studie deed Ynte onderzoek aan de evolutie van het vermogen tot afbraak van 3-chloorbenzoaat in de anoxygene fototroof Rhodopseudomonas palustris, bij de vakgroep Microbiële Ecologie, onder begeleiding van Prof. Larry J Forney, Dr. Jan C. Gottschal en MSc.Yasuhiro Oda. Vervolgens bracht Ynte een half jaar in Japan door, waar hij werkte aan de afbraak van nonylphenol door Sphingomonas sp. bij de vakgroep Microbial Ecology aan de Ibaraki University, onder begeleiding van Prof. Hiroyuki Ohta. In januari 2001 trok Ynte naar Wageningen, om te beginnen aan zijn promotieonderzoek aan de leerstoelgroep Levensmiddelenmicrobiologie. Dit onderzoek viel binnen het onderzoeksprogramma “Microbial functionality and safety” van het Wageningen Centre for Food Sciences, vond plaats onder de paraplu van onderzoekschool VLAG, en werd grotendeels uitgevoerd op werklocatie ATO. Het promotieonderzoek werd uitgevoerd onder begeleiding van Prof. Dr. Tjakko Abee, Prof. Dr. Willem de Vos, en Prof. Dr. Ir. Marcel Zwietering, en heeft geresulteerd in dit proefschrift. Vanaf oktober 2005 is Ynte werkzaam als Researcher Food Microbiology voor Friesland Foods, te Leeuwarden.

127