Neisseria meningitidis - Wiley Online Library

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Sep 21, 2005 - and James W. B. Moir1*. 1Department of Biology (Area ...... Tettelin, H., Saunders, N.J., Heidelberg, J., Jeffries, A.C.,. Nelson, K.E., Eisen, J.A., ...
Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382X© 2005 The Authors; Journal compilation © 2005 Blackwell Publishing Ltd? 2005583800809Original ArticleControl of denitrification in the meningococcusJ. D. Rock et al.

Molecular Microbiology (2005) 58(3), 800–809

doi:10.1111/j.1365-2958.2005.04866.x First published online 21 September 2005

The pathogen Neisseria meningitidis requires oxygen, but supplements growth by denitrification. Nitrite, nitric oxide and oxygen control respiratory flux at genetic and metabolic levels Jonathan D. Rock,1 M. Reda Mahnane,1 Muna F. Anjum,2† Jonathan G. Shaw,3 Robert C. Read3 and James W. B. Moir1* 1 Department of Biology (Area 10), University of York, Heslington, York, YO10 5YW, UK. 2 Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, S10 2TN, UK. 3 Division of Genomic Medicine, University of Sheffield Medical School, Beech Hill Road, Sheffield, S10 2RX, UK. Summary The human pathogen Neisseria meningitidis is the major causative agent of bacterial meningitis. The organism is usually treated as a strict aerobe and is cultured under fully aerobic conditions in the laboratory. We demonstrate here that although N. meningitidis fails to grow under strictly anaerobic conditions, under oxygen limitation the bacterium expresses a denitrification pathway (reduction of nitrite to nitrous oxide via nitric oxide) and that this pathway supplements growth. The expression of the gene aniA, which encodes nitrite reductase, is regulated by oxygen depletion and nitrite availability via transcriptional regulator FNR and two-component sensor-regulator NarQ/NarP respectively. Completion of the two-step denitrification pathway requires nitric oxide (NO) reduction, which proceeds after NO has accumulated during batch growth under oxygen-limited conditions. During periods of NO accumulation both nitrite and NO reduction are observed aerobically, indicating N. meningitidis can act as an aerobic denitrifier. However, under steady-state conditions in which NO is maintained at a low concentration, oxygen respiration is favoured over denitrification. NO inhibits oxidase activity in N. meningitidis with an apparent Ki NO = 380 nM measured in intact cells. The high res-

Accepted 15 August, 2005. *For correspondence. E-mail [email protected]; Tel. (+44) 0 1904 328 677; Fax (+44) 0 1904 328 825. †Present address: Veterinary Laboratories Agency, New Haw, Addlestone, Surrey, KT15 3NB, UK.

© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd

piratory flux to nitrite after microaerobic growth and the finding that accumulation of the denitrification intermediate NO inhibits oxygen respiration support the view that denitrification is a pathway of major importance in N. meningitidis. Introduction The bacterial pathogen Neisseria meningitidis is a major cause of death among infants and young adults. The organism causes two diseases, meningitis and septicaemia, both characterized by a rapid onset and high death rates. The natural habitat of N. meningitidis is the human nasopharynx, where it normally resides asymptomatically, and it has no other known natural reservoir (van Deuren et al., 2000). N. meningitidis is classed as an aerobic bacterium, and is routinely cultured under oxygen rich conditions, in the absence of alternative respiratory electron acceptors. Given that the nasopharynx is an oxygen poor environment, from which both strict aerobes and strict anaerobes are routinely isolated (Brook, 2003), it is worth considering the role of oxygen and other possible respiratory electron acceptors in the lifestyle of N. meningitidis. We have previously shown that the organism is capable of growing in the absence of extensive aeration, and that under these conditions nitrite can be removed from cultures via a denitrification pathway (Anjum et al., 2002). It was not clear from our earlier work the extent to which denitrification was able to supplement growth under oxygen limiting conditions, or whether the denitrification pathway was operating mainly as a detoxification pathway. Nitrite may be an important source of respiratory substrate for N. meningitidis living in an environment where oxygen availability is limited and variable. Nitrite is present in human tissue in vivo due to the oxygenation of nitric oxide (NO), which is produced by various cell types as a signalling molecule and as a toxin produced by the innate immune response in macrophages. Nitrite is also present following dietary intake of nitrate which is reduced to nitrite by facultative nitrate reducers in the mouth and pharynx (Lundberg et al., 2004). Nitrite concentrations in human tissue vary between locations and over time depending on the activation state of the immune response and nutri-

Control of denitrification in the meningococcus 801 tional status. Nitric oxide itself is hard to measure in vivo because it is an unstable gas, but estimates suggest physiological concentrations between 100 nM and 1 μM (see Discussion). Complete denitrification is the reduction of nitrate via nitrite, NO and nitrous oxide to dinitrogen gas (Berks et al., 1995). This predominantly bacterial process is linked to the respiratory chain so that the substrates in denitrification can supplement oxygen as terminal respiratory electron acceptors. N. meningitidis is capable of partial denitrification, having genes required for the reduction of nitrite to nitrous oxide, via nitrite reductase AniA, and NO reductase NorB. We have shown that norB gene is responsible for NO reduction (Anjum et al., 2002), whereas the role of aniA in nitrite reduction was adduced from its similarity to aniA from close relative Neisseria gonorrhoeae (Mellies et al., 1997). The reduction of oxygen to water at the expense of NADH oxidation by organisms generally yields more biochemical energy than the reduction of alternative electron acceptors. Hence alternative respiratory pathways are regulated in such a way that reduction of oxygen is favoured. This regulation operates at a genetic level via the control of transcription. Typically, expression of reductases for denitrification is only activated in the absence of oxygen, a process often controlled by a member of the FNR family of transcriptional regulators (Browning et al., 2002). A second level of regulation is by substrate availability. In Escherichia coli, gene expression in response to the availability of nitrate and nitrite is regulated via a pair of two-component sensor regulators, NarX/L and NarQ/P (Stewart, 1993). N. meningitidis possesses homologues of FNR and NarQ/P (Parkhill et al., 2000; Tettelin et al., 2000). The roles of FNR and NarQ/P in regulation in response to oxygen depletion and nitrite, respectively, have been elucidated for N. gonorrhoeae (Householder et al., 1999; Lissenden et al., 2000). Alternative respiratory pathways are also regulated by oxygen at the metabolic level. The reduction of oxygen to water has a higher standard redox potential than reduction of nitrate to nitrite or nitrite to NO (O2/H2O E°′ = 0.8 V; NO3–/NO2– E°′ = 0.42 V; NO2–/NO E°′ = 0.38 V). Thus, competition for electrons within a respiratory chain is expected to favour oxygen reduction over the reduction of most other respiratory electron acceptors. However, there has been a good deal of controversy concerning the regulation of denitrification by oxygen, and a number of organisms have been reported to be capable of aerobic denitrification (Robertson et al., 1989; Zart and Bock, 1998; Scholten et al., 1999; Chen et al., 2003). The persistence of denitrification aerobically is best understood, from a mechanistic perspective, in Paracoccus pantotrophus which possesses a periplasmic nitrate reductase that catalyses the aerobic reduction of nitrate (Bell et al.,

1990). In this case nitrate reductase competes effectively for electrons with oxygen, as the oxygen reduction pathway is more tightly coupled to proton motive force generation than the nitrate reductase pathway (Richardson and Ferguson, 1992). In this paper we analyse the regulation of the denitrification pathway in N. meningitidis. Although oxygen controls denitrification at both transcriptional and metabolic levels, denitrification can persist under aerobic conditions. The physiology of denitrification in N. meningitidis is discussed in terms of its lifestyle in vivo. Results Neisseria meningitidis does not grow anaerobically We showed previously that N. meningitidis growth in stationary vessels (i.e. with minimum aeration) is supplemented by nitrite but not nitrate. However, this effect was small and there was considerable variation between experiments (Anjum et al., 2002). In order to determine whether N. meningitidis is capable of growth under strictly anaerobic conditions, we sparged 20 ml aliquots of Mueller–Hinton broth (MHB) (supplemented with 10 mM NaHCO3 and 5 mM nitrite) with N2 gas, and then incubated the media in an anaerobic cabinet for 3 days prior to inoculation with N. meningitidis at 37°C. Inocula were derived either from freshly grown aerobic plate cultures or from mid-log phase liquid cultures grown under denitrifying conditions (20 ml MHB + 10 mM NaHCO3 + 5 mM nitrite in 25 ml McCartney bottles, incubated with shaking at 100 rpm). Growth was not observed in these anaerobic cultures, indicating that N. meningitidis requires molecular oxygen. Supplementation of media with herring sperm DNA, chromosomal DNA from N. meningitidis or deoxyribonucleoside triphosphates did not allow growth to be supported anaerobically. Nitrite supplements growth of N. meningitidis under oxygen limitation To achieve growth under conditions of oxygen limitation N. meningitidis was cultured in 20 ml MHB + 10 mM NaHCO3 in 25 ml McCartney bottles, which were incubated with shaking at 100 rpm. Supplementing cultures with nitrite altered the growth kinetics from linear growth to exponential growth, consistent with a change from oxygen limitation to unrestricted growth (Fig. 1A). The increase in growth rate in the presence of nitrite was coincident with expression of nitrite reductase (aniA) and the disappearance of nitrite (Fig. 1B). A strain deficient in aniA displays growth characteristics identical to the wildtype grown in the absence of nitrite, whereas growth of a norB mutant strain is poor compared with the aniA mutant (Fig. 1C), presumably due to the accumulation of toxic NO in that strain.

© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 58, 800–809

802 J. D. Rock et al. sured value of oxygen uptake of 20 nM min−1 per mg protein. The flux of electrons through the respiratory chain to the denitrifying reductases is similar to the flux to oxygen (noting, of course, that oxygen reduction to water is a fourelectron reduction reaction whereas nitrite reduction to nitrous oxide is a two-electron reduction). This highlights the importance of this mode of respiratory growth to the meningococcus under microaerobic conditions. After aerobic growth of N. meningitidis nitrite reduction was negligible, whereas the rate of oxygen uptake was similar to the rate of oxygen uptake from denitrifying cultures, indicating that oxygen respiration is constitutive. Nitrite reductase is controlled by oxygen availability and nitrite via FNR and NarQP To assess the environmental cues affecting aniA expression we used an N. meningitidis strain containing an aniA promoter lacZ fusion. aniA expression was very low during aerobic growth, and the supplementation of aerobic cultures with nitrite had little effect on expression levels. A fivefold increase in aniA-lacZ expression occurred on switching from aerobic to oxygen-limited growth conditions. Inclusion of nitrite in the growth media increased expression levels by a further 10-fold (Fig. 2). Mutant strains were constructed with lesions in fnr (which encodes a homologue of the anaerobic transcriptional activator from E. coli and other bacteria) and narQP (which encodes a two-component regulatory system that may regulate gene expression in response to nitrate and/ or nitrite). In each case the mutant strains were generated by insertion of antibiotic resistance genes into the genes encoding the regulators. Polar effects are considered unlikely due to neighbouring genes being transcribed from the opposite DNA strand. To ensure against second site Fig. 1. Growth of N. meningitidis under oxygen-limited conditions. A. The effect of nitrite on the growth of N. meningitidis MC58. Growth is plotted as OD600 versus time, with OD600 plotted on a logarithmic scale, and on a linear scale (inset). Growth in the absence of nitrite shown as open diamonds, plus 5 mM nitrite as filled diamonds. Linear growth rate minus nitrite is 0.025 OD units per hour. Exponential growth rate plus nitrite is 0.15 per hour. B. Disappearance of nitrite (closed squares) and induction of aniA expression (measured using a promoter aniA-lacZ translational fusion) (open squares). C. Growth of N. meningitidis MC58 plus nitrite (filled diamonds) compared with N. meningitidis aniA plus nitrite (filled circles) and N. meningitidis norB plus nitrite (open circles).

The rate of nitrite reduction was calculated, from the nitrite data in Fig. 1B, to be 45 nmol min−1 per mg protein in late exponential phase in a representative microaerobic denitrifying culture of N. meningitidis. Once the nitrite had disappeared, cultures were assayed for nitrite reductase activity under anaerobic conditions in an oxygen electrode chamber. Rates of nitrite reduction of 40 nmol min−1 per mg protein were recorded. This compares with a mea-

Fig. 2. Expression of aniA in response to oxygen availability and nitrite. The expression of aniA was monitored using a promoter aniAlacZ translational fusion carried by N. meningitidis MC58. Aerobic growth no nitrite (open circles), aerobic growth plus 5 mM nitrite (filled circles), microaerobic growth no nitrite (open diamonds), microaerobic growth plus 5 mM nitrite (filled diamonds).

© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 58, 800–809

Control of denitrification in the meningococcus 803 with measurements of growth, nitrite and aniA expression. Thirty millilitres of MHB supplemented with 5 mM nitrite in a sterile glass chamber fitted with NO and O2 electrodes and was inoculated with aerobically grown N. meningitidis and agitated with a magnetic stirrer at 37°C. Stirring of the vessel caused little perturbation of the surface of the medium, and oxygen became depleted over 1–2 h depending on the rate of oxygen supply to the medium (controlled by altering stirrer speed). Experimental data for the transition of N. meningitidis from aerobic to denitrifying growth are shown in Fig. 4. Following anaerobiosis, NO began to accumulate. This only occurred when nitrite was included in the medium, and did not occur when fnr or aniA strains were used (data not shown). NO accumulation continued and when the [NO] exceeded 100–200 nM oxygen began to accumulate. NO accumulation was unaffected by the increase in oxygen availability in the medium. NO accumulation continued until it reaches a plateau at 1–5 μM. During this period of high [NO] growth ceased. Approximately 1 h after NO accumulation began [NO] declined at an accelerating rate, indicating that NO reduc-

Fig. 3. Growth of regulatory mutants deficient in fnr and/or narQP. Data shown are the means of three independent growth experiments and error bars show the standard errors. Cultures of N. meningitidis MC58 (open diamonds), fnr (closed diamonds)and narQP (open circles) were grown under oxygen-limited conditions in the presence of 5 mM nitrite. (A) growth measured as OD600 (B) nitrite disappearance, and (C) aniA-lacZ β-galactosidase activity.

mutations we analysed three independently isolated mutant strains for each gene knockout and found them, in each case, to have essentially identical properties. Growth of strains deficient in fnr and/or narQP relative to the wildtype N. meningitidis MC58 under microaerobic conditions in the presence of nitrite are shown in Fig. 3. Both mutant strains grew poorly under oxygen-limited conditions and the removal of nitrite was slow (narQP) or non-existent (fnr). Transition from oxygen utilization to denitrification is not a simple aerobic to anaerobic switch To understand the relationship between oxygen availability and denitrification, cultures were established in a vessel in which media aeration could be manipulated and the concentrations of oxygen and NO monitored coincident

Fig. 4. Transition from oxygen respiration to denitrification. Aerobically grown N. meningitidis promoter aniA-lacZ strain was inoculated into 30 ml MHB + 5 mM nitrite + 10 mM NaHCO3 in a glass vessel maintained at 37°C. Oxygen (grey line) and NO (black line) were monitored continuously with electrodes. Growth was followed as OD600 (filled diamonds), the expression of aniA was followed by measuring β-galactosidase activity (open circles) and nitrite was measured periodically colourimetrically (open squares).

© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 58, 800–809

804 J. D. Rock et al. nitrite stimulated NO accumulation, inhibition of oxygen respiration and ultimately active NO removal, as seen in Fig. 4. Nitrite disappeared from the system during the aerobic period of NO accumulation and disappearance and transient inhibition of growth of the bacterium occurred during this period. Nitric oxide inhibits oxidase activity in N. meningitidis

Fig. 5. A low concentration of nitrite leads to accumulation of NO and aerobic denitrification. Experimental set-up as in Fig. 4, except that cultures were supplemented with 100 μM nitrite. NO (black line), O2 (grey line), nitrite (open squares), OD600 (filled diamonds).

tase is being expressed in response to NO accumulation. This was supported by the observation that rapid NO removal did not occur in a norB mutant strain (data not shown). After NO became depleted, oxygen respiration resumed and the [O2] fell. This was accompanied by resumption in growth of cultures. Oxygen concentration tended to zero, aniA expression resumed, growth continued and eventually nitrite and NO became completely depleted. (A steady-state [NO] of 30–50 nM was observed until nitrite became completely depleted, after which [NO] drops towards zero). The series of phases during which the bacteria adapt to oxygen-limited growth supplemented by denitrification does not take the form of a simple aerobic to anaerobic switch. Rather, the transient production of the denitrification intermediate NO, which is an inhibitor of oxygen respiration, gives rise to a period during the adaptation in which oxygen is available yet denitrification continues, i.e. N. meningitidis behaves as an aerobic denitrifier.

The genome of N. meningitidis encodes a single oxidase that is a homologue of the high oxygen affinity cytochrome cbb3 type oxidases found in other bacteria capable of microaerobic growth. This oxidase has been reported to possess NO reductase activity in Pseudomonas stutzeri (Forte et al. 2001). In order to determine the effect of NO on the oxidase activity of N. meningitidis, we measured the rate of oxygen respiration in a norB mutant in response to boluses of NO. Figure 6 shows that oxidase activity is inhibited by NO, and an apparent Ki for NO of 380 nM was calculated. This is in keeping with the results of other experiments in this paper in which oxygen accumulates coincidentally with the accumulation of NO. Competition between oxygen and nitrite respiration From the data in Figs 4 and 5 it is clear that, in the presence of accumulated NO, nitrite reduction persists in the presence of molecular oxygen. After growth of N. meningitidis under oxygen-limited conditions with nitrite, the bacteria have active reductases for oxygen, nitrite and

Low concentrations of nitrite bring about a transitional increase in NO following oxygen depletion The concentration of nitrite typically available in human tissue is likely to be significantly lower than the 5 mM used in the experiments described heretofore. Nitrite is likely to vary widely in space and time within tissues, but a concentration of 100 μM is physiologically relevant (Al-Waili and Boni, 2004). Experiments were carried out as described in the previous section but supplementing cultures with 100 μM nitrite (Fig. 5). The low concentration of

Fig. 6. Inhibition of N. meningitidis oxidase by NO. N. meningitidis norB was grown under aerobic conditions and oxygen respiration was followed using a Clark-type oxygen electrode. Simultaneous measurements of NO were obtained using an electrode. NO was added to the oxygen respiring cell suspension as a bolus from an NO saturated aqueous solution. The inhibition of oxidase activity was measured as a percentage of the uninhibited oxidase rate and plotted against log10[NO]. The concentration of NO sufficient to inhibit oxygen respiration by 50% is marked with an arrow.

© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 58, 800–809

Control of denitrification in the meningococcus 805

Fig. 7. Competition between oxygen and nitrite during respiration. The reduction of oxygen by a suspension of wild-type N. meningitidis grown under denitrifying conditions was followed using a Clark-type oxygen electrode. The addition of nitrite is marked by an arrow and the rate of oxygen uptake is shown by a dotted line. NO accumulation was measured using an NO electrode and is displayed as a solid line.

NO. Nitrite and oxygen respiration by N. meningitidis grown under these conditions were followed simultaneously in order to assess the impact of nitrite respiration on oxygen reduction, and vice versa (Fig. 7). During the course of oxygen respiration a small increase in NO concentration was observed in response to the addition of nitrite, which had a minor inhibitory effect on the rate of oxygen uptake. Nitrite disappearance under these conditions was slow. NO accumulated to a higher steady-state concentration once oxygen became completely depleted, and the overall rate of nitrite reduction increased. Nitrite reduction aerobically was typically less than 10% of the anaerobic rate. Clearly, in the absence of sufficient NO, oxygen is favoured as respiratory electron acceptor over nitrite. Discussion Denitrifying bacteria typically use nitrate and/or nitrite as alternative electron acceptors to oxygen under anaerobic conditions. Paradoxically, N. meningitidis is strictly dependent on the presence of oxygen for growth, and yet we show here that the bacterium supplements growth by denitrification. Presumably the requirement for oxygen is therefore not as a respiratory electron acceptor, but rather molecular oxygen is required for a metabolic reaction in the cell. Analysis of the genome of N. meningitidis reveals that the organism possesses a single ribonucleotide reductase, and that this belongs to a family of enzymes that require molecular oxygen for their activation. This should be sufficient to prevent the prolonged growth of N. meningitidis under strictly anaerobic conditions. In recent work with Bacillus species (Folmsbee et al., 2004), it was demonstrated that B. subtilis (which only possesses an oxygen-dependent ribonucleotide reductase) and B. mojavensis can be made to grow anaerobically if provided

with deoxyribonucleosides. That work was critical of previous claims that Bacillus species, including B. subtilis, are facultative anaerobes, arguing that the earlier work lacked the necessary stringency to ensure oxygen-free conditions. The inability of the microaerophilic pathogen Campylobacter jejuni to grow anaerobically is likely due to the absence of an anaerobically active ribonucleotide reductase (Sellars et al., 2002). Supplementation with deoxyribonucleosides or DNA was unable to support anaerobic growth of N. meningitidis, but this is probably due to the inability of this organism to transport or metabolize the deoxyribonucleosides. It is notable that the close relative of N. meningitidis, N. gonorrhoeae, which is often stated to be a facultative anaerobe, also appears to lack an anaerobically functional ribonucleotide reductase in its genome, suggesting that the status of this bacterium as a facultative anaerobe needs to be re-evaluated. Neisseria meningitidis possesses a single set of oxidase genes, and these are predicted to encode a cytochrome cbb3 type oxidase. In other bacteria containing this oxidase, its function is thought to be a high O2 affinity enzyme operating under microaerobic conditions. Nitrogen fixation in Bradyrhizobium japonicum requires the presence of cytochrome cbb3 oxidase, which is capable of scavenging oxygen so that its intracellular concentration is kept sufficiently low to protect the nitrogenase from oxygen damage (Preisig et al., 1996). Like N. meningitidis, the cytochrome cbb3 oxidase is the only oxidase in the strict microaerophile Helicobacter pylori (Tomb et al., 1997). In facultative anaerobes Paracoccus denitrificans and Rhodobacter species cytochrome cbb3 is inducible under microaerobic conditions (Van Spanning et al., 1997). The role of cytochrome cbb3 in other bacteria supports the notion that N. meningitidis is adapted for a microaerobic lifestyle, in which the limited supply of oxygen as a respiratory electron acceptor is supplemented by denitrification of nitrite to nitrous oxide. In this paper we have shown that in the presence of accumulated NO, denitrification occurs regardless of the presence of oxygen when nitrite reductase and NO reductases are expressed. This is argued to be primarily due to the inhibition of oxidase activity by NO, which favours utilization of nitrite and NO as respiratory electron acceptors. Although denitrification is generally thought of as an anaerobic process, there have been many reports of aerobic denitrification (Robertson et al., 1989; Bell et al., 1990; Zart and Bock, 1998; Scholten et al., 1999; Chen et al., 2003), the mechanistic basis of this has only been extensively analysed for the first step of denitrification, the reduction of nitrate to nitrite. The periplasmic nitrate reductase of P. pantotrophus is expressed under aerobic conditions, particularly when the bacteria are cultured on a highly reduced carbon substrate (Richardson and Ferguson, 1992). The enzyme is active under these condi-

© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 58, 800–809

806 J. D. Rock et al. tions, and its physiological role has been argued to be in the dissipation of excess reducing equivalents (NADH) via a pathway that is poorly coupled to the generation of a proton motive force. Here we show that nitrite and NO reductase activities in N. meningitidis are insensitive to the presence of oxygen when NO has accumulated to concentrations sufficient to render oxygen respiration inactive. Thus, denitrification is favoured as the only route available for respiratory electron transport under these conditions. The sensitivity of oxidase activity to inhibition by NO (apparent Ki = 380 nM at 50 μM O2) is similar to that observed for other oxidases (Borisov et al., 2004; Brown and Cooper, 1994). Oxidase activity is inhibited by NO at concentrations lower than those required to inhibit other cellular processes. In experiments reported here, when NO accumulates to concentrations ∼1 μM the growth of N. meningitidis ceases, indicating that other cellular processes are becoming affected by NO. Nitric oxide is a relatively short-lived species in aqueous systems, and therefore it is difficult to obtain good estimates of [NO] within the tissues colonized by the meningococcus. However, we do know that nasal airways are a significant source of exhaled NO gas compared with the mouth (Kimberly et al., 1996), supporting the view that occupation of this niche is facilitated for bacteria possessing active NO detoxification systems. Whether NO accumulates sufficiently to inhibit oxygen respiration in the nasopharynx is an open question, but recent work has shown that NO concentrations sufficient to inhibit the N. meningitidis oxidase are required for some NO-regulated physiological processes (Thomas et al., 2004) and NO concentrations in the range 100 nM−1 μM have been measured with probes in animal tissues [e.g. brain (Kirkeby et al., 2000), venous endothelium (Gerova et al., 1998) vitreous humour of the eye (Hoshi et al., 2003)]. In addition to NO generation from arginine via NO synthase activity, NO may be synthesized from dietary nitrate which is reduced to nitrite by oral nitrate reducing bacteria leading to salivary nitrite concentrations ranging from 10 μM to >1 mM (Lundberg et al., 2004). Nitrite increases dramatically following a nitrate-rich meal and acidification or bacterial metabolism may lead to NO production (Palmerini et al., 2003). Oxygen availability within nasal and pharyngeal mucosa is also likely to vary widely. [O2] varies in response to the distance from airways or perfusion by oxygen-rich blood, and the extent of microbial colonization at a site. The variable availability of oxygen is highlighted by the isolation of bacteria which are strict anaerobes and strict aerobes from the same nasal and nasopharyngeal tissues (Brook, 2003). Neisseria meningitidis is shown here to adapt to the changing availability of oxygen, nitrite and NO by regulating expression of the various reductases involved in res-

piratory metabolism. One of the consequences of this regulatory process is that NO can accumulate to cytotoxic concentrations rapidly, and then become rapidly depleted, depending on the prevailing environmental conditions and due to the finite response time of the bacterium to changing environmental conditions. NO accumulation and disappearance occur consecutively in response to nitrite when oxygen availability is limited. The capacity of the meningococcus to manipulate the NO concentration in its local environment in this way may have consequences for the lifestyle of the bacterium in vivo. In response to the changing availability of nitrite (e.g. after a meal) the meningococcus may cause the production of a burst of NO which (i) is toxic to microorganisms competing for colonization of the nasopharyngeal tissue, (ii) impacts upon host signalling systems that are dependent upon NO, and (iii) inhibits the meningococcal oxidase activity thus favouring denitrification by the bacterium. The regulation of gene expression in response to oxygen and nitrite in N. meningitidis is reminiscent of that observed in the close relative of the meningococcus, N. gonorrhoeae. In that organism nitrite reductase expression is controlled by FNR and NarQP (Lissenden et al., 2000) as found here for the meningococcus, and the NO reductase is controlled in response to NO by an unknown regulator (Householder et al., 2000). It has recently been reported that nitrite reductase and NO reductase expression in N. meningitidis may be controlled by the iron responsive repressor Fur (Delany et al., 2004), although it is unclear whether this regulator is responsible for control in response to NO under conditions relevant for denitrification. In this paper we have shown that denitrification is a major sink for respiratory reductant in the strictly aerobic bacterium N. meningitidis. Under microaerobic conditions, which are likely to be encountered by the bacterium in its natural habitat in the human nasopharynx, denitrification contributes approximately equally with oxygen respiration as a route for electron transfer in respiration. Furthermore, under conditions where NO has accumulated to a few hundred nanomolar, denitrification is favoured over oxygen respiration, thus providing an alternative means of respiratory activity under conditions relevant to the natural environment of this important pathogen.

Experimental procedures Bacterial strains, plasmids and growth conditions All N. meningitidis strains used in this study were derived from N. meningitidis MC58. E. coli strains were routinely cultured at 37°C using Luria–Bertani (LB) medium (Roth, 1970). N. meningitidis strains were routinely cultured at 37°C on Columbia-horse blood agar plates, or during liquid culture in MHB supplemented with 10 mM NaHCO3. Aerobic culture

© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 58, 800–809

Control of denitrification in the meningococcus 807 was performed using 5 ml of broth in a 25 ml McCartney shaking at 180 rpm. (New Brunswick Scientific, C25K incubator shaker). Microaerobic culture was carried out using 20 ml of broth in a 25 ml McCartney shaking at 100 rpm.

Growth supplements and measurement of culture parameters Nitrite was used at final concentrations of 5 or 0.1 mM as described for each experiment. Antibiotics were used routinely during culture at the following concentrations: kanamycin 50 μg ml−1, spectinomycin 50 μg ml−1, erythromycin 100 μg ml−1 and chloramphenicol 25 μg ml−1 (2 μg ml−1 for N. meningitidis). DNA (herring sperm or N. meningitidis chromosomal) and dNTPs were included at 1 g l−1 when used as growth supplements. Amount of protein in 1 ml of bacterial cells was measured by both BCA assay (Pierce, Rockford IL, USA) and Bradford assay (Bio-Rad GmbH, München, Germany). Saturated NO solution was made by sparging NO gas (Sigma-Aldrich, UK) through 1 M NaOH and into 10 ml of 1 M Tris pH 8 and a sample was analysed to check that the pH had not become acidic. The presence of oxygen in culture was measured by digital oxygen system 10 electrode (Rank Bros, Bottisham, UK) and NO concentration was measured by iso-NO markII electrode (World Precision Instruments, Stevenage, UK). Nitrite concentration in culture was measured by colourimetric assay (Nicholas and Nason, 1957).

and MRM2 to clone aniA and ligation of the Ω cassette into a naturally occurring SspI site. Following transformation of this plasmid into N. meningitidis, transformants were selected by plating onto Columbia-horse blood agar containing spectinomycin, and correct chromosomal rearrangement verified by PCR using primers MRM1 and MRM2. The N. meningitidis FNR mutant strain was created using the gonococcal fnr gene that had been previously disrupted by the insertion of erythromycin cassette and was donated by Prof Jeff Cole, University of Birmingham. The disrupted gonococcal fnr gene was cloned into pGIT5 (Dr Tom Baldwin, University of Nottingham) and the resulting plasmid was linearized by restriction with XbaI and used to transform N. meningitidis MC58. Transformants were selected as above using erythromycin and the correct chromosomal orientation was confirmed by Southern hybridization. The norB mutant was constructed as described previously (Anjum et al., 2002). A mono-allelic promoter-lacZ fusion of the aniA promoter was inserted into the proAB region of N. meningitidis MC58. Primers MFA7 and MFA8 were used to amplify the aniA promoter, which was inserted upstream of lacZ in pLES94 (Silver and Clark, 1995). Linearized plasmid was then transformed into N. meningitidis MC58 and translational fusions selected for by plating onto Columbia-horse blood agar plates containing chloramphenicol and Xgal. Constructs were confirmed by PCR. Double mutants and promoter fusion/ regulator mutant strains were constructed by transformation of existing mutant or promoter fusion strains with chromosomal DNA from mutant strains. All strains were verified for correct chromosomal rearrangement by PCR.

Construction of strains Genetic mutations were produced by allelic replacement with an insertionally inactivated cloned N. meningitidis MC58 gene. The narQP mutant was created by blunt end TOPO cloning (Invitrogen Life Technologies, Carlsbad CA, USA) of the narQ and P genes using primers NarPF1 and NarQR1 (Table 1). The resultant plasmid (pJR101) was digested with XmnI and the Ω cassette, encoding spectinomycin resistance from pHP45Ω (Prentki and Krisch, 1984), ligated within the reading frame of narP. This plasmid (pJR105) was then transformed into N. meningitidis MC58 (Seifert et al., 1990). Transformants were selected for by plating on Columbia-horse blood agar containing spectinomycin, and correct chromosomal rearrangement verified by polymerase chain reaction (PCR) using primers NarPF1 and NarQR1. The aniA mutant was constructed in an identical manner using primers MRM1

Table 1. Oligonucleotide primer sequences. Primer name

Sequence

Function

NarPF1 NarQR1 MRM1 MRM2 MFA7

gaagcggtgcggattaagttt gggcaaacggaaaacactaa cgtcggccaggcgtgttttc ccgcatcgataacgggcagt gggatcccgtttcataatgttttccttttgt

MFA8

gggatcccgtcccattttgagagctcctttt

Cloning of narQP gene Cloning of narQP gene Cloning of aniA gene Cloning of aniA gene Cloning of aniA promoter Cloning of aniA promoter

Acknowledgements We are grateful to J. A. Cole and T. W. Overton, University of Birmingham, UK for supplying N. gonorrhoeae fnr::eryR construct used in this work. We are grateful to V. L. Clark, University of Rochester Medical School, USA for providing vector pLES94. This work was funded by Wellcome Trust Grant 070268/Z/03/Z awarded to JWBM and RCR.

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