Molecular Characterization and Isolation of Extreme ...

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Molecular Characterization and Isolation of Extreme Halophiles from Solar Salterns of Mauritius

QUIRIN Marie Emmanuella Christelle

BSc. (Hons) Biology

University of Mauritius

Department of Biosciences

April 2014

Table of Contents Table of Contents List of Tables List of Figures Acknowledgement

ii iv v vii

Project/dissertation declaration form Abstract

viii ix

List of Abbreviations

x

1. Introduction

1

2. Literature review 2.1. Archaea 2.2. Bacteria 2.3.Extreme halophilic Archaea 2.4.Extreme halophilic Bacteria 2.5.Adaptive mechanisms 2.6.Diversity of extreme halophiles in Solar Salterns 2.7.Application and uses of extreme halophiles 2.7.1. Enzymes 2.7.2. Extracellular polysaccharides 2.7.3. Compatible solutes 2.7.4. Bacteriorhodopsin 2.7.5. Biosurfactant

3 3 3 4 5 6 7 8 8 9 10 10 10

2.7.6. Bioremediation 2.7.7. Food biotechnology 2.7.8. Alternative energy 2.8. Culture-dependent isolating technique 2.9.Culture-independent isolating technique 2.9.1. 16S rRNA phylogeny, primers and bioinformatics 2.10. Pyrosequencing 3. Methodology 3.1.Site of study 3.2.Sampling 3.3.Microscopic observation 3.4.Culture of halophiles 3.4.1. Colony count 3.5. Isolation of microorganisms from environmental samples and isolated colonies 3.6.DNA extraction 3.6.1. Modified Takai and Sako (1999) method

11 11 11 11 12 14 16 18 18 18 19 19 22 23 23 23

3.6.2. Modified Anton et al. (2000) boiling method

24

3.7.Agarose gel electrophoresis 3.8. Amplification of 16S rRNA gene fragments using Polymerase Chain Reaction (PCR) 3.9.Construction of 16S rRNA gene fragment clone libraries 3.9.1. PCR product purification

25 26

ii

28 28

4.

3.9.2. Ligation 3.9.3. Transformation of competent Escherichia coli cells

29 29

3.9.3.1. Method 1 3.9.3.2. Method 2: Using the TransformAid (ThermoScientific) 3.10. Extraction and isolation of plasmids from transformants

29 30

3.11. Bioinformatics

31

Results 4.1.Physical parameters of Tamarin solar saltern

33 33

4.2.Microscopy 4.3.Culture of halophiles

33 34

4.4.Extraction of eDNA 4.5. Extraction of gDNA

36 37

4.6. PCR amplification of 16S rRNA gene fragments

38

4.6.1. Using environmental samples

38

4.6.2. Using isolated colonies

40

4.6.3. PCR product purification and determination of DNA concentration 4.7. Construction of 16S rRNA gene fragment clone libraries

41

4.8. Bioinformatics

43

5. Discussion 5.1.Culture of halophiles 5.2.DNA extraction 5.3.Amplification of 16S rRNA gene fragments

31

42

45 45 46 47

5.4. Construction of 16S rRNA gene fragment clone libraries

48

5.5. Bioinformatics

48 50

6. Conclusions and Recommendations 6.1.Conclusions

50

6.2.Recommendations

50

References Appendix 1: Progress Log Appendix 2: Counted number of colony types per plates Appendix 3: ThermoScientific GeneRuler 1 kb Plus DNA Ladder, ready-to-use. Appendix 4: Sequences used for Multiple Sequence alignment

iii

52 62 65 67 68

List of Tables Table 2.1

2.2 2.3 3.1 3.2 3.3

3.4

4.1

4.2

4.3

Name

Page

Occurrence of extreme halophilic Archaea Enzymes isolated from halophiles and their biotechnological applications Primers used in this study Composition of 30% (w/v) artificial salt water used for MGM Composition of 23% MGM Primers used for 16S rRNA gene fragments of Bacteria and Archaea PCR thermal cycling parameters used The different physical parameters of the water sample collected Different categories of colonies observed Different colony types used for DNA extraction and their sources

iv

5

9 15 20 20 27

27

33

34

36

List of Figures Figure

Name

Page

General trend in the diversity of 2.1

microscopic organisms present in

8

solar salterns 2.2 2.3

Stages of metagenomics Series

of

the

four

13 enzymatic

reactions in pyrosequencing

17

3.1

Site of study

18

3.2

Sampling Area

19

3.3 3.4 3.5

3.6

3.7

3.8 3.9 3.10

4.1

4.2

4.3

Gallenramp cooled incubator and orbital shaker Phytotron POL-EKOAPARATURA Esco Vertical Laminar

21 Flow

cabinet Plate showing the positioning of Q1 and Q2 for colony count Bench top microcentrifuge and Clifton water bath UV transilluminator and connected computer to view and print photographs of gels PCR system used for DNA fragments amplification Different steps involved in the purification of PCR products Microorganisms observed under the microscope Some plates on which different colony types have grown Type 2 colonies grown on plate SP7w

v

21

22

23

25

26 27 29

34

35

35

4.4

4.5

Gel electrophoresis of extracted eDNA Gel

electrophoresis

of

gDNA

extraction

37

38

Gel electrophoresis of amplified 4.6

DNA fragments using 21F/958R

39

primers Gel electrophoresis of amplified 4.7

DNA fragments using 8F/1492R

40

primers Gel electrophoresis of amplified 4.8

DNA

fragments

from

isolated

41

colonies 4.9 4.10 4.11

4.12

Gel electrophoresis of purified PCR products Transformed colonies

42 43

Multiple Sequence Alignment of Arc1 to Arc10 Alignment scores after blasting 21F and 958R against database

vi

44

44

Acknowledgement I thank my supervisors, Dr S Dyall and Dr N Taleb-Hossenkhan for the guidance, help and support they have provided and for the critical review of this thesis. I also thank the Laboratory staff of the Biosciences Department for their help and assistance during the laboratory procedures, especially Mrs Anishta. I show much gratitude to my laboratory partners, Jessica, Sandhya, Aurelie, Ravi, Prishnee and Anushka for their help, support, suggestions, motivation and for making the lab work enjoyable. I would also like to thank Mr J. Langlois of Société Mont Calme Ltd who granted permission for sample collection at the solar saltern of Tamarin. Lastly, I thank my parents and brother for their support, encouragement and comments given on my thesis.

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Abstract Extreme halophiles are organisms that thrive in hypersaline environments with salt concentrations approaching saturation. It was assumed that only halophilic Archaea could survive such conditions while Bacteria and Eukaryotes were automatically excluded. In this study, emphasis is placed on both the halophilic Bacteria and Archaea living in solar salterns due to their biotechnological potential. The objectives were to use culture-independent and culture-dependent methods to identify and isolate extreme halophiles from solar salterns of Mauritius. Successful isolation of halophiles from environmental samples and culture set up on 23% Modified Growth Medium (MGM) was done.

Environmental DNA

(eDNA) and genomic DNA (gDNA) extraction using modified Takai and Sako (1999) and Anton et al. (2000) methods were carried out. PCR amplifications of 16S rDNA fragments using 21F/958R (archaea) and 8F/1492R (bacteria) primer combinations were performed. Bioinformatics tools were used to identify areas of polymorphism and similarity in aligned 16S rDNA genes from halophilic archaea and to determine the reliability of the primers used. Seven morphologically different colonies were observed, showing the effectiveness of the MGM and the possibility of culturing halophiles. A total of ten amplicons were obtained from eDNA, three of archaeal origin and seven of bacterial origin. Three bacterial amplicons were formed from gDNA from the isolated colonies. Multiple Sequence Alignment (performed for haloarchaea only) showed that the 16S rDNA sequence is very conserved but contained several areas of polymorphism which can be used for genetically identifying particular species. The archaeal-specific primers were shown, using BLAST against GenBank database to match uniquely to the archaeal sequences, confirming that the three amplicons were of archaeal origin. This study offers a first insight into culture of halophiles of Mauritius under laboratory conditions. Further work involving sequence and phylogenetic analyses of the amplicons generated in this study will surely contribute to our characterisation of prokaryotes that can survive the harsh conditions of solar salterns and that may thus offer us novel biotechnological tools.

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List of Abbreviations bp

base pair

dNTP

Deoxynucleotide triphosphate

eDNA

environmental DNA

EDTA

Ethylenediamine tetraacetic acid

gDNA

Isolated colony genomic DNA

LB

Lysogeny Broth

NET

NaCl-EDTA-Tris

PBS

Phosphate buffered saline

rDNA

ribosomal DNA

RNase

Ribonuclease

rRNA

ribosomal RNA

SDS

Sodium dodecyl sulphate

TAE

Tris-Acetate-EDTA

TE

Tris EDTA

x

1. Introduction The Tree of Life is recognised to be made up of three branches, namely the Domains Bacteria, Archaea and Eukarya. This biological classification was put forward by Carl Woese in 1990, based on the analysis of 16S rRNA. It was discovered that even if the Archaea possess similar characteristics and metabolic processes to both the Bacteria and Eukarya, they own unique traits that totally differentiate them from the other two. The Archaea and Bacteria are diverse and have been able to colonise almost every environment on Earth. They are able to thrive in the terrestrial and aquatic environments including the extreme ones. These microorganisms also display peculiar abilities that can help us in better understanding the climate, condition and biodiversity of our early planet. The great Archaeal and Bacterial diversity provides an unexploited source of microorganisms and their products for future biotechnology and medical purposes, for understanding evolutionary processes and for helping scientists in the understanding of biogeochemical cycles and in the search of any extra-terrestrial life. However, it is quite difficult to establish archaeal cultures in the laboratory. Thus, their investigation has been limited compared to the knowledge that scientists have gathered about bacteria over the years (Brochier-Armanet et al., 2011). Both Archaea and Bacteria have been able to colonise a large range of saline habitats that possess different salt concentrations, ranging from sea water (3.5% (w/v) NaCl) to hypersaline (>10% (w/v) NaCl) environments. These saltloving prokaryotes are the halophiles. Those able to live in hypersaline habitats formed mainly by the evaporation of sea water are called the extreme halophiles. Only few extreme halophilic bacteria have been identified up to date, most of them being moderate halophiles (Oren, 2002). Most salt-loving Archaea are found in the order Halobacteriales which require hypersaline conditions (Oren, 2008) to grow and to be physiologically stable. For example, they can be found in salt mines, brine springs, solar salterns, soda lakes, natural salt lakes, deep-sea hypersaline basins and saline soils (Lichfield and Gillevet, 2002). These extreme halophiles have been successfully characterised and identified through the sequencing and analysis of the 16S rDNA gene sequence. Using bioinformatic tools such as Basic Local Alignment Search Tool (BLAST) and Multiple Sequence Alignment (MSA), areas of polymorphism and highly conserved regions were

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identified and compared to establish the relationship between different DNA sequences and species. This project is part of an enterprise to assess archaeal diversity in Mauritius, adding on to previous BSc and MSc research projects (Louis, 2011; Jinerdeb, 2011; Victorien, 2013). The identification of the extreme halophiles will serve a baseline on which future studies can build on. Also, this project will result in the identification and isolation of potentially new strains or species. The aim of the study is to identify and isolate extreme halophiles from solar salterns using cultureindependent and culture-dependent methods. The specific objectives are to: 1. collect water samples from hypersaline environment and measure physical parameters; 2. isolate extreme halophiles present in the brine; 3. extract high quality genomic DNA from isolated microorganisms; 4. use PCR to amplify Archaea-specific and Bacteria-specific 16S rRNA gene fragments; 5. clone fragments into plasmids to produce at least two libraries; 6. sequence at least two amplified cloned fragments from each library; 7. identify halophiles by using bioinformatics tools to analyse sequences; 8. use, in parallel, culture-dependent methods to isolate individual halophiles for long-term analyses and for identification using molecular tools.

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2. Literature Review 2.1. Archaea The development of more advanced molecular techniques has allowed scientists to better understand microbial life. Molecular research on 16S rRNA phylogeny has led to the recognition of the Archaea as the third domain of life (Woese et al., 1990). The domain consists of prokaryotic organisms which have diverse morphology, physiology and niches. They have various shapes and can be either gram positive or gram negative. Some are single cells while others occur as filaments and aggregates. Based on cellular and biochemical analyses, it has been found that these microorganisms possess a combination of both Bacterial and Eukaryal characteristics mixed with unique Archaeal ones (Brown and Doolittle, 1997). Some exclusively Archaeal features include presence of ether-linked lipids in the cell membrane, unusual appendages, unusual resistance to antibiotics, unique composition and structure of ribosomes and modified glycosylation mechanism amongst others (Jarrell et al., 2011). These particularities allow the Archaea to thrive in both mesophilic and extreme environments. Thus, they can be found in lakes, soils, rocks, oceans, animal guts, marshes, hydrothermal vents and mines for example. According to genomic analyses, the Archaea has been currently divided into

Euryarchaeota,

Crenarchaeota,

Korarchaeota,

Nanoarchaeota,

Thaumarchaeota and Aigarchaeota (Eme et al., 2013).

2.2. Bacteria Bacteria are old-prokaryotes that live everywhere on Earth. They can occur as cocci, bacilli, vibrio or spirilli and in different sizes. They lack a well-defined nucleus and most possess a peptidoglycan cell wall that determines whether they are gram-positive or gram-negative. Many of them are motile, using their flagella or pilli to move. The bacteria are able to form spores to resist harsh conditions and move back to their vegetative form under favourable conditions. The domain is divided into 30 phyla containing aerobic and anaerobic members. They are Acidobacteria,

Actinobacteria,

Aquificae,

Armatimonadetes,

Bacteroidetes,

Caldiserica, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres,

Deinococcus,

Thermus,

Dictyoglomi,

Elusimicrobia,

Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospira,

Planctomycetes,

Proteobacteria, 3

Spirochaetes,

Synergistetes,

Tenericutes,

Thermodesulfobacteria,

Thermomicrobia,

Thermotogae,

Verrucomicrobia (LSPN, 2014). One of the most successful groups of microorganisms is considered to be the Proteobacteria (Rappé and Giovannoni, 2003).

2.3. Extreme Halophilic Archaea The extreme halophilic Archaea are salt-loving euryarchaeotes that thrive in hypersaline environments such as natural hypersaline lakes, artificial salterns and deep-sea basins (Table 2.1). They grow at an optimum concentration of 10% (w/v) NaCl or more (Oren, 2008) with pH values ranging from 6 to 11 (Ochsenreiter et al., 2002). They possess a surface-layer (S-layer) made up of proteins held by cations. Unlike the bacterial cell wall, the S-layer does not provide rigidity to the cell. Thus, the halophilic Archaea occur as cocci, rods, discs, triangles and squares. They are represented mainly by the members of the class Halobacteria where “virtually all of them are strictly dependent on high salt concentrations for maintaining growth and cellular integrity” (Andrei et al., 2012). The Halobacteria display an array of different metabolic pathways and nutritional requirements (Andrei et al., 2012). Most are aerobic chemoheterotrophs while some are fermentative or photoheterotrophic anaerobes (Mancinelli, 2005). Even though amino acids are used as energy and carbon source by most of them, some species can metabolise carbohydrates (Andrei et al., 2012). Also, halophilic, anaerobic methanogens feeding on methylated amines and methanol have been identified from the class Methanomicrobia (Ventosa et al., 2012 cited de la Haba et al., 2011a). The members of the genera Methanohalobium, Methanohalophilus, Methanocalculus and Methanosalsum are moderate or extreme halophiles and have been isolated from hypersaline lakes and salterns worldwide (de la Haba et al., 2011a). Narasingarao et al. (2012) have also reported the occurrence of the new class Nanohaloarchaea in hypersaline areas worldwide.

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Table 2.1. Occurrence of extreme halophilic Archaea (Adapted from Chaban et al., 2006). Habitat

Genera

Ancient salt deposits

Halorubrum, Haloarcula, Halococcus

Dead Sea

Halorubrum, Haloferax

Hypersaline

Antarctica

lakes Saline soils

Halorubrum, Haloferax, Haloterrigena

Salt lakes

Natrinema

Salted fish and hides

Halobacterium

Salterns,

crystallizer

ponds Soda lakes

2.4.

Halorubrum

Halorubrum, Haloarcula, Halogeometricum Haloarcula, Natronomonas, Natronococcus

Extreme Halophilic Bacteria

Most halophilic bacteria isolated live in moderate salinities rather than in extreme ones (Oren, 2002) but several poly-extreme ones have been reported in Bowers et al. (2009). They include aerobic and anaerobic phototrophs, chemoheterotrophs and chemolithotrophs (Javor, 2002) which have been identified from the phyla Actinobacteria,

Bacteroidetes,

Cyanobacteria,

Firmicutes,

Proteobacteria,

Spirochaetes, Tenericutes and Thermotogae. (Ventosa et al., 2012). All classes of the Bacteroidetes and the Proteobacteria except the Betaproteobacteria have moderate halophilic members. The largest number of halophilic species is found within the Gammaproteobacteria (de la Haba et al., 2011a). Within the Firmicutes, the classes Bacilli and Clostridia possess halophilic members as well as the order Actinomycetales (Actinobacteria) and genus Spirochaeta (Spirochaetes). The phyla Tenericutes and Thermotogae each possess a sole halophilic member, the Haloplasma contractile and the Petrotoga halophila respectively (Ventosa et al., 2012). Many unicellular and filamentous Cyanobacteria also display halophilic properties and some of them, for example the genus Halospirulina are able to thrive at higher salinities than the other halophilic bacteria (Oren, 2011a). It has been previously reported by Imhoff et al. (1977) and Cayol et al. (1995) that

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Halorhodospira halochloris and Halanaerobium lacusrosei grow at about 3237% (w/v) NaCl (Bowers et al., 2009). Additionally, Salinibacter ruber sp. nov. (Bacteroidetes) displays some characteristics specific to extreme halophilic Archaea and grows at 20-30% (w/v) salt in crystalliser ponds of salterns (Anton et al., 2002).

2.5. Adaptive mechanisms The development of various mechanisms to respond and adapt to osmotic stress have allowed the haloarchaea to thrive in the harsh saline environments. Actually, most of them undergo lysis in freshwater and are least likely to grow there (Ventosa et al., 2012). So, to be able to survive high salinity, they have to maintain their cytoplasm isotonic with respect to their surroundings. Two different strategies have been employed in order to prevent desiccation and both involve the building up of osmotic pressure inside the cells (Oren, 2002). The first strategy is adopted by the Halobacteria, the Halanerobiales (Firmicutes) and Salinibacter ruber sp. nov. (Bacteroidetes). They maintain an osmotic balance with the environment by accumulating potassium ions into their cytoplasm and spend little amount of energy (Oren, 2011b). The second strategy is more energy demanding and is performed by halotolerant and halophilic Bacteria and Eukaryotes as well as halophilic

methanogenic

Archaea

(Kerkar,

2004).

They accumulate

or

biosynthesize compatible organic solutes to create the osmotic equilibrium. These compounds are neutral and do not affect any enzymatic activities. They include amino acids, simple sugars, glycerol, glycine betaine and ectocine amongst others (Oren, 2002; Oren, 2011b; Kerkar, 2004). The organisms that survive at a wide range of salt concentrations use the second strategy to do so (Oren, 2011b). Membrane proteins are highly affected by salt which can disrupt hydrophobic and electrostatic interaction in them (Reed et al., 2013). Folding and stability can be altered and cause aggregation and precipitation of the proteins. Extreme halophiles require their proteins to adapt and maintain their three-dimensional structures to remain stable and functional at high salinity. They do so by increasing the number of acidic residues on the protein surfaces, decreasing the number of large hydrophobic residues, using salt to fold and increasing halophilic peptide insertion (Reed et al., 2013). Furthermore, extreme halophilic Archaea are able to maintain membrane stability and prevent leakiness by having special membrane lipids. 6

These lipids have ether bonds and branched isoprenoid chains that confer much more stability. Tenchov et al. (2006) have reported that the diacidic phospholipid, archaetidylglycerol methylphosphate (PGP-Me) made up 50-80% of total polar lipids of the membrane and confers stability at high salt concentrations (approx. 17.5-30% (w/v) NaCl). PGP-Me has a double charge and a big head group, preventing approach and aggregation by steric repulsion (Tenchov et al., 2006). The lipid creates a poorly interacting bilayer which accounts for the membrane stability.

2.6.

Diversity of extreme Halophiles in Solar Salterns

Solar salterns are artificial, hypersaline ponds found in tropical and sub-tropical areas where salt is produced by the evaporation of sea water. The increasing gradient in salinity from pond to pond causes a decrease in the microbial diversity (Fig. 2.1). This easily accessible saline environment with different ranges of salinity has allowed scientists to study the life that thrive there. It is to be noted that organisms from all three domains have been identified in solar salterns (Sabet et al., 2009). The main constituent of the microbial mats, which form at the bottom of evaporation pans, are the cellular and filamentous cyanobacteria (Oren, 2006). Depending on the salinity of the ponds, the primary producers can either be the cyanobacteria or the eukaryotic microalgae Dunaliella (Ochsenreiter et al., 2002). In certain salterns, it has been reported that photosynthetic purple bacteria belonging to the genera Halochromatium, Thiohalococcus and Halorhodospira have also been identified in the benthic mats (Oren, 2006). Due to their ease to grow in laboratories rather than their abundance in worldwide solar salterns (Bardavid et al., 2008), the haloarchaea that have been mostly identified are from the genera Halobacterium, Haloferax, Haloarcula and Halorubrum (Ma et al., 2010). But, the most abundant (>0.5% of cell number) halophilic Archaea are those that contain gas vesicles and are flat and square or rectangular in shape (Bardavid et al., 2008). Among the eukaryotes, members of certain genera of diatoms, protozoa and fungi are halophilic and have been identified in saltern water (Mancinelli, 2005). Additionally, Litchfield and Gillevet (2002) stated that “frequently heavy populations of the brine shrimp Artemia” can also be observed in solar salterns.

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Figure 2.1. General trend in the diversity of microscopic organisms present in solar salterns (Oren, 2006).

2.7. Application and uses of Extreme Halophiles Their adaptation to harsh environments and characteristics make extreme halophiles interesting organisms. Their special properties allow us to use them and their products in biotechnology.

2.7.1.

Enzymes

A variety of enzymes from halophilic microorganisms has been isolated and used in biotechnology (de la Haba et al., 2011b) due to their thermostability and performance at high salt concentrations (Abrevaya, 2013). Proteases are used in the detergent, dairy, baking and leather industries. For example, an active ingredient of detergents is a protease from Bacillus species and antifouling coatings contain a protease isolated from the Halobacterium species (Karan et al., 2012). Lipases and esterases are commonly produced by the haloarchaea and are used in detergents to help in stain removal (de Lourdes Moreno et al., 2013). Amylases have been isolated from bacterial species of the various genera such as Halobacillus, Streptomyces, Chromohalobacter and Halothermothrix. The amylase and protease from Chromohalobacter has been reported to maintain their stability in presence or

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absence of NaCl (Setati, 2010). Table 2.2 shows some more enzymes isolated from halophiles, their properties and biotechnological uses.

Table 2.2. Enzymes isolated from halophiles and their biotechnological applications (Adapted from Karan et al., 2012). Enzymes

Source

Properties/Applications

Saccharopolyspora sp. Amylases

A9 Chromohalobacter sp. TVSP 101

Chitinases

Salinivibrio sp. Marinobacter

Cellulases

Esterases

Starch hydrolysis Salt stable

sp.

MSI032

Alkaline, pH stable

Halobacillus sp.

Salt stable

Haloarcula sp.

Alkaline and salt stable

β-galactosidase Haloferax alicantei Lipases

Detergent formulation

Salt stable

Salicola strain IC10

Alkaline and salt stable

Natronococcus sp.

Salt and thermal stable Alkaline, salt and solvent stable. Fish

Proteases

Halobacterium sp.

Nucleases

Bacillus sp.

Alkaline, salt and thermal stable

Xylanases

Thalassobacillus sp.

Salt stable

2.7.2.

sauce preparation, antifouling coating

Extracellular polysaccharides

Exopolysaccharides (EPS) are produced by halophiles to protect themselves from environmental stresses and to prevent viral attacks (Abrevaya, 2013). They occur as a capsule or slime and their compositions vary greatly (Llmas et al., 2012). In various industrial fields, they are used as chelating, emulsifying and viscosifying agents (de la Haba et al., 2011b). They have been isolated from various organisms of the genus Halomonas, Halobacterium, Haloferax, Haloarcula and Halomonas. (Abrevaya, 2013; Margesin and Schinner, 2001). For example, the EPS produced

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by Halomonas eurithalina has a gel texture and has good emulsifying properties (Llmas et al., 2012).

2.7.3.

Compatible solutes

The previously mentioned compatible solutes (Section 2.5) are low molecular weight and water-soluble compounds. They prevent any heat or osmotic stress to denature, inactivate and inhibit proteins, membranes and whole cells (de la Haba et al., 2011b). Thus, they are being used as salt antagonists, stress-protective agents and stabilizers of biomolecules and whole cells (Margesin and Schinner, 2001). Ectoines are the mostly used compatible solutes. Halomonas elongate and other Halomonas species are being used for its production in biotechnology. When exposed to an osmotic shock at 3% (w/v) NaCl, the halophilic bacteria release the accumulated compound which is then harvested. Ectoines are used in the field of medicine, pharmacy and agriculture (de la Haba et al., 2011b).

2.7.4.

Bacteriorhodopsin

Bacteriorhodopsin (BR) is a transmembrane protein which pumps protons across the cell membrane driven by light energy. The protons are pumped outwardly across the membrane and allowed to flow back inside through ATP synthase to produce ATP (Margesin and Schinner, 2001). BR has been isolated from Halobacterium salinarum and its photochemical stability gives it various applications in biotechnology. “Such applications include protein films, used in artificial retinal implants, light modulators, three-dimensional optical memories, colour photochromic sensors, photochromic and electrochromic papers and ink, biological camouflage and photo detectors for biodefense and non-defense purposes” (Saeedi et al., 2012).

2.7.5.

Biosurfactant

Biosurfactant are complex, amphiphilic molecules produced by microbial cells to facilitate diffusion of insoluble substrates (Karanth et al., 1999). They reduce surface tension and increase mobility and solubility of hydrocarbons (Margesin and Schinner, 2001). Thus, small amounts of biosurfactants increase the degradation of hydrocarbon pollutants and play an important role in oil recovery and soil and water remediation. Biosurfactants are also used in food, cosmetic, 10

textile and metal industries amongst others (Abrevaya, 2013). Members of the genera Haloarcula and Halovivax were identified by Kebouche-Gana et al. (2009) to be optimal producers of biosurfactants (Abrevaya, 2013).

2.7.6.

Bioremediation

The use of halophiles for bioremediation and biodegradation of contaminated environments and toxic effluents and wastes has been widely explored (Abrevaya, 2013). Halophilic Archaea from the genera Haloferax, Halobacterium, Halococcus and Haloarcula (Bonfa et al., 2011) and halophilic bacteria of the genus Halomonas, Chromohalobacter, Salinococcus and Halobacillus (Le Borgne et al., 2008) are able to metabolise aromatic compounds, ammonia, arsenite, mercury (Abrevaya, 2013).

2.7.7.

Food biotechnology

Halofermentation has been used for the production of various salted, fermented or preserved food products and sauces. The halophilic fermentative bacteria and Archaea used include Halobacteria, Halococci, Natronococci, Pediococci and Tetragenococci (Kivistö and Karp, 2011).

2.7.8. Alternative energy Due to harmful effects of using fossil fuels and other environmental issues, sources of alternative energy has intensively been studied during the past years. For example, hydrogen is a clean, environmental friendly and renewable energy source. A fermentative by-product of halophilic bacteria is hydrogen (Kivistö and Karp, 2011). Kivistö et al. (2010) were able to show that Halanaerocium saccharolyticum subspecies senegalense and saccharolyticum can produce hydrogen by using glycerol as substrate.

2.8. Culture-dependent isolating technique To culture microorganisms in laboratory, we need to re-create the environment in which they grow. Many times this has proven to be quite challenging because one should know the specific growth requirement of the target microorganism. Then prepare a liquid or solid medium containing the appropriate amount and combination of nutrients and exposed to the correct physical conditions. Even 11

though all requirements are provided, in some experiments sufficient time is not given to the microorganism to adapt to the laboratory setting and to establish colonies. Burns et al. (2004) observed that long incubation period (12 weeks) increased the number of viable colonies. Hypersaline culture media commonly contain yeast extract, peptone, tryptone, NaCl and water but can also contain magnesium, calcium, potassium and vitamins (Schneegurt, 2012). The medium is inoculated with the halophiles and incubated at suitable temperature until colonies appear or turbidity changes (Hugenholtz, 2002). Since extreme halophiles thrive at temperatures above 35°C and salt concentration ranging from 15-25% (w/v) (The Halohandbook, chapter 1), small changes in salinity and temperature can alter the growth rate and appearance of the colonies and also the nutritional needs (Schneegurt, 2012). The Modified Growth Medium (MGM) used in this study was formulated by Holmes and Dyall-Smith (1990) based on that previously developed by RodriguezValera et al. (Holmes and Dyall-Smith, 1990 cited Rodriguez-Valera et al., 1980). MGM of different concentrations can be prepared by varying the concentration of artificial salt water used (The Halohandbook, chapter 2.2, 2.3). This medium was primarily designed for the culture of haloarchaea but it was observed that halophilic bacteria could also grow on it. For example, Halomonas spp. and Salinibacter ruber sp. nov. were able to grow well on the MGM (Burns et al., 2004). The mostly used 23% MGM was used in this study because it allowed the growth of a variety of halophiles.

2.9. Culture-independent isolating technique (Metagenomics) The traditional culture techniques used to identify the microbial diversity of certain environment are limited to those that are able to grow under laboratory setting, thus, giving a false picture of the actual microbial community. However, we are now able to better understand the genetic and phenotypic diversity of previously cultivated as well as uncultivated microorganisms through a culture-independent technique. It also allows us to understand the relationships between coexisting populations and the community structure and function (Hugenholtz and Tyson, 2008). This technique is referred to as metagenomics, which is the study and analysis of the collection of genomes of the total microorganisms obtained directly from the environment they inhabit (Hugenholtz and Tyson, 2008; Rondon et al., 12

2000). It is a sequence-based approach and is divided into various stage of analysis (Fig. 2.2). The first step involves the isolation of the microorganism(s) of interest from the environmental sample. Collective genomic DNA is then extracted and the 16S rRNA gene fragment is then amplified by Polymerase Chain Reaction (PCR). The amplification is done by using universal primers which bind to the conserved regions of the gene fragment. The collection of sequences obtained is the inserted into vectors which are replicated by a bacterial host (Escherichia coli). Eventually, gene fragment libraries are produced by the host which are then used for sequencing and identification of the sampled species. The use of PCR amplification of bacterial and archaeal 16S rRNA genes and metagenomics studies, confirmed the dominance of some species in hypersaline environments worldwide (Narasingarao et al., 2012).

Figure 2.2. Stages of metagenomics. A. Sampling from the environment; B. Isolation of desired organism; C. Cell lysis and DNA extraction; D. Cloning of

13

desired DNA fragment and library formation; E. Sequencing; F. Annotation and assembly of sequenced data. (Wooley et al., 2010).

2.9.1.

16S rRNA phylogeny, primers and bioinformatics

Ribosomes form an essential component of the protein synthesis machinery of cells. The 16S rRNA together with 21 proteins forms the 30S small subunit of prokaryotic ribosomes. Thus, the structure and function of the 16S rRNA gene sequence has not changed over time. It is present in all prokaryotes and is long enough (approx. 1500 bp) to provide information to researchers (Rastogi and Sani, 2011). The 16S rRNA gene has highly conserved regions alternating with very variable ones. This is due to the different folding, looping and bonding of rRNA molecule. The degree of variability is sufficient to determine the relationship between closely related groups of organisms. These properties made this gene an important tool in the study of prokaryotic phylogeny and taxonomy, to understand microbial life, interaction and evolution. Primers are short oligonucleotides that are required by DNA polymerases to start DNA replication. When using two different primers (forward and reverse), it is possible to define the region to be amplified by PCR. A number of primers have been designed to selectively amplify the conserved 16S rDNA regions (archaeal or bacterial) because binding to unknown microbial DNA would also occur. Therefore, primers have allowed the identification and classification of prokaryotes that are unculturable under laboratory conditions. Some primers are specific to certain taxa whilst others are said to be universal. However, some Archaea-specific primers have Euryarchaeota and Crenarchaeota bias because they were designed before the discovery of the other four phyla (Baker et al., 2003). The primers 8F, 21F, 958R and 1492R were used in this study (Table 2.3). 21F/958R are archaealspecific primers, 8F is a bacterial-specific primer and 1492R is a universal primer. The combinations 21F/958R and 8F/1492R yield PCR products of approximately 950 bp (DeLong, 1992) and 1400 bp (Bockelmann et al., 2000) respectively.

14

Table 2.3. Primers used in this study. Priming sites indicated by E. coli 16S rRNA numbering. Y indicate C or T; M indicate A or C. (van der Lelie et al., 2011; Radax et al., 2001). Primer used

Orientation

Priming site

Sequence (5´- 3´)

8F

Forward

8-27

AGAGTTTGATCCTGGCTCAG

21F

Forward

2-21

TTCCGGTTGATCCYGCCGGA

958R

Reverse

958-976

YCCGGCGTTGAMTCCAATT

1492R

Reverse

1492-1513

GGTTACCTTGTTACGACTT

Since genome data is increasingly produced given new and high throughput technologies, researchers are encouraged to submit their new sequences to an international nucleotide sequence database such as GenBank, DDBJ and EMBL to make them available to the public. Daily exchange of sequences between the different databases provides the most up to date data to the scientific community (Mount, 2001). GenBank is an open access sequence database managed by the National Center for Biotechnology Information (NCBI). It allows free access and submission of entries and is involved in data curation. BLAST is an algorithm used to compare biological sequences stored on GenBank. It provides homology searches against all publicly available sequences. This has several purposes including the identification of species and their homologues, the location of domains, DNA mapping, genetic comparison and the generation of phylogenetic trees (Madden, 2002). Multiple Sequence Alignment has been developed to align three or more sequences at the same time based on the sequential alignment of most alike sequences. It is a method used to distinguish regions of similarity between the sequences. Once the alignment has been done, highly conserved regions and areas of polymorphism can be identified. This allows the demonstration of homology between sequences, the design of primers for related gene identification and the set-up of evolutionary and phylogeny models (Mount, 2001).

15

2.10. Pyrosequencing Pyrosequencing is a de novo, chemiluminometric DNA sequencing technique involved

in

sequencing

and

synthesising

complementary

DNA

strand

enzymatically (Fakruddin et al., 2012). It depends on the recognition of the release of pyrophosphate (PPi) during DNA synthesis. A single-stranded DNA is hybridised to a primer and incubated with adenosine 5´ phosphosulfate (APS), luciferin, DNA polymerase, ATP sulfurylase, luciferase and apyrase. In a series of four enzymatic reactions, light is produced when a known nucleotide adds up to the sequence (Fig. 2.3). Therefore, the chemiluminescent signals produced upon addition of various nucleotides, permit the determination of the DNA sequence (Fakruddin et al., 2012). At room temperature, the series of reactions proceed within 3-4 seconds and the amount of light is perceived by a photon detection device (Ronaghi, 2001). For pyrosequencing, any source DNA can be used but only 100-200 bp DNA strand can be sequenced (Petrosino et al., 2009). It is useful in sequencing samples containing different species (minor and dominant) (Fakruddin et al., 2012) by making use of the multiple sequencing primer approach (Gharizadeh, 2003). Thus, the sequence signal of less abundant species can be detected. Pyrosequencing provides us with more information about the diversity of halophiles present in solar salterns for example. Genomes sequencing can be performed more rapidly by using this method, allowing identification of biotechnologically useful genes and enzymes. In addition, pyrosequencing is a simple method used to analyse polymorphisms, aiding in the identification and classification of closely related species (Gharizadeh, 2003). Specifically one of the most highly variable regions on the 16S rDNA gene can be sequenced to distinguish between two different strains of the same species.

16

Figure 2.3. Series of the four enzymatic reactions in pyrosequencing. X indicates one of the four nucleotides (Ronaghi, 2001).

This review clearly shows that the approaches used during previous studies were successful in the isolation and characterisation of extreme halophiles in solar salterns water and sediment. In this context, similar approaches were used in the present study: metagenomics and 23% MGM were used as culture-independent and culture-dependent isolation methods, with the support of bioinformatics analyses.

17

3. Methodology 3.1. Site of study Samples were collected from Les Salines de Tamarin found in Tamarin village, on the West coast of Mauritius (Fig. 3.1). Water is pumped directly from the sea in the main reservoir from which it is allowed to flow into the shallow basins. The basins are interconnected by a network of channels which permits the continual flow of water.

The salt pans can be divided into the intermediate reservoirs where

evaporation occurs and the crystallizing pans where salt is harvested. Thus, as we move from the main reservoir to the last crystallizing pans, the salinity of water keeps on increasing.

Figure 3.1. Site of study. (Source: Google Maps)

3.2. Sampling Water and sediment samples were collected from 11 different intermediate reservoirs (Fig. 3.2A, B, C) and 2 crystallizing pans (Fig. 3.2D) using sterile 50 ml polypropylene tubes. The tubes were used as corer to take the upper layers of the sediments from the area where water was collected. The pans were labelled SP1(less concentrated sea water) to 13(most concentrated brine), water samples were labelled SPxw (x: salt pan number) and the sediment samples were labelled SPxsed. The samples were carried to the laboratory at ambient temperature and placed in the sunlight until processing. Temperature of the water was taken in situ 18

while

the

pH

and

salinity

were

measured

in

the

laboratory.

Figure 3.2. Sampling Area. A: Salt Pan 1. B: Salt Pan 4. C: Salt Pan 7. D: Crystalizing pan (SP12).

3.3. Microscopic observation The samples were observed under both the light microscope (Olympus BH-2) at 100X-400X magnification and the inverted microscope (Leica) at a magnification of 400X. A drop of the water samples was spotted on normal glass slide and covered with a 24 x 24 mm cover slip. To observe the sediment samples, a solution was first produced by mixing 1 cm of sediment with 5 ml of 1X PBS (137 mM NaCl; 2.7 mM KCl; 10 mM Na2HPO4; 2 mM KH2PO4) and shaking vigorously. The next step was performed in the same way as the water samples.

3.4. Culture of halophiles 23% Modified Growth Medium (MGM) was used as culture medium (liquid and solid) and is prepared as described in table 3.2. Technical agar (Oxoid) was used as gelling agent.

The agar solution was heated to dissolve it prior to mixing.

Alternatively, other MGM formulation using natural salt pan fine and coarse salts 19

(instead of laboratory NaCl) were also used. Prior to the preparation of the MGM, a 30% (w/v) artificial salt water stock solution was prepared, autoclaved and stored (table 3.1)

Table 3.1. Composition of 30% (w/v) artificial salt water used for MGM (The Halohandbook, Chapter 2.2). Salt

g for 1 litre

NaCl (Alpha Chemika or Natural)

240

MgCl2.6H2O (AnalaR)

30

MgSO4.7H2O(Alpha Chemika)

35

KCl (AnalaR)

37

CaCl2 (Sigma-Aldrich)

0.5

Add distilled water to final volume, warm a little to dissolve salts and mix with a glass rod.

Table 3.2. Composition of 23% MGM (The Halohandbook, chapter 2.3). Ingredients

For 1 litre

Salt water (stock)

767 ml

Technical agar (Agar no.3) (Oxoid)

15 g

Tryptone (Fluka analytical)

5g

Yeast extract (Sigma-Aldrich)

1g

Distilled water

Top up to 1000 ml

Use 1 M Tris.Cl, pH 7.5 to adjust pH to 7.5. Agar was not added to the liquid medium during preparation.

The medium was poured in Duran bottles and autoclaved at 101kPa for 30 minutes. The autoclaved solid medium was allowed to cool and was mixed before being poured into sterile plastic petri dishes. When cool and well-set, 100 μl of water samples from each salt pan was spread onto the medium using a „hockey stick‟ under sterile conditions. A sediment suspension was prepared as per section 3.3 and plated. The plates were sealed with Parafilm „M‟ to prevent loss of moisture and were incubated in the incubator (Gallenramp) (Fig. 3.3A) at 37°C for 4 days.

20

Figure 3.3. A: Gallenramp cooled incubator. B: Gallenramp orbital shaker.

Isolated colonies that grew on the solid medium were picked with sterile loops or toothpicks and grown in 10 ml of liquid medium placed into covered, sterile 50 ml conical flasks. They were shaken overnight at 37°C at 220 rpm in the orbital shaker (Gallenramp) (Fig. 3.3B) under light. 15 μl of the medium incubated overnight was pipetted and streaked onto the solid medium using a loop and incubated into a Phytotron (POL-EKO-APARATURA) (Fig. 3.4) at 37°C under 12 hours of light and darkness.

Figure 3.4. Phytotron POL-EKO-APARATURA.

21

The culture of colonies onto alternatively solid and liquid medium was repeated until a culture pure enough was obtained. During the final transfer of colonies from liquid to solid medium, 1.5 ml of the solution (colonies and medium) was pipetted into different microcentrifuge tubes and stored at -20°C for future DNA extraction. All the manipulation of the samples and plates was done in sterile conditions in the Esco Vertical laminar flow cabinet (Fig. 3.6).

Figure 3.5. Esco Vertical Laminar Flow cabinet.

3.4.1. Colony count The first set of plates containing different colonies were used for the colony count. Two opposite segments each covering one/eighth (Q1 and Q2) that best represent the colony number and distribution of the whole plate, were identified (Fig. 3.6). The different colony types were counted and their shapes, sizes and colours were described. The approximate number of individual colonies of different types in a plate was estimated by multiplying the sum of Q1 and Q2 by four.

22

Figure 3.6. Plate showing the positioning of Q1 and Q2 for colony count. (This plate is not relevant to this project, only used to demonstrate position of Q1 and Q2).

3.5. Isolation of microorganisms from environmental samples and isolated colonies 1.5 ml of water and resuspended sediment samples were pipetted into microcentrifuge tubes and centrifuged at 10,000 X g for 2 minutes at 4°C. The supernatant was discarded. The microcentrifuge tube was re-filled with the sample and centrifuged under same conditions. This step was repeated for 3 times until a pellet of satisfactory size was obtained. It was resuspended in 100 µl of distilled water and stored at -20°C for DNA extraction afterwards. Frozen liquid cultures of isolated colonies were thawed and centrifuged at 10,000 X g for 5 minutes at 4°C. The supernatant was discarded and the pellet formed was resuspended in 100 µl of distilled water and stored at -20°C.

3.6. DNA extraction 3.6.1. Modified Takai and Sako (1999) method This method was used to extract eDNA and gDNA. Samples were thawed, resuspended in 240 µl of NET buffer (50 mM Tris.Cl, pH 8.0; 150 mM NaCl; 100 mM EDTA) containing 0.2% (w/v) lysozyme, to digest the bacterial peptidoglycan and incubated at 37°C for 15 minutes. To lyse the cells (both bacterial and archaeal) and denature „harmful‟ enzymes, 20 µl of 2% proteinase K (Sigma) and 30 µl of 10% (w/v) sodium dodecylsulfate (SDS) were added to the tubes before incubating them at 65°C for 30 minutes. Afterwards, the tubes were centrifuged at 15,000 X g for 10 minutes at 4°C using a bench-top microcentrifuge (Fig 3.7A).

23

The supernatants were extracted with equal volumes of phenol saturated with 100 mM of Tris.Cl (pH 8), vortexed for 10 seconds and centrifuged at 12,500 X g for 10 minutes at 4°C. Subsequently, extraction was performed on the aqueous layers using

phenol/chloroform/isoamyl

alcohol,

25:24:1

(v/v)

followed

by

chloroform/isoamyl alcohol, 24:1 (v/v). The tubes were vortexed and centrifuged under same conditions. Phenol dissolved the proteins and lipids, chloroform ensured the formation of two well-demarcated layers and isoamyl alcohol caused the inactivation of RNase. DNA was precipitated from solution by using 0.1 volume of 3 M sodium acetate, pH 5.2 and 2 volumes of absolute ethanol and centrifuging at 15,000 X g for 15 minutes. Following removal of the supernatant, the DNA pellet was rinsed by vortexing with 500 µl of 70% (v/v) ethanol and centrifuged at 15,000 X g for 10 minutes. Ethanol was decanted and the pellet was allowed to air dry for 15 minutes before being resuspended in 200 µl TE buffer (10 mM Tris.Cl, pH 8; 1 mM EDTA, pH8). After standing for 5 minutes, the tubes were vortexed, centrifuged at 10,000 X g for 2 minutes and stored at -20°C.

3.6.2. Modified Anton et al. (2000) boiling method This method was performed on the environmental samples only. The pellets of microorganims were thawed and 100 µl of sterilised water was added before heating them in a water bath (Fig 3.7B) at 65°C for 10 minutes. They were then centrifuged at 13,000 X g for 10 minutes at 4°C. The supernatant and pellet formed were stored in separate microcentrifuge tubes at -20°C. The tubes containing the supernatant were labelled as SPxw super or SPxsed super depending on the origin of the colonies. The stored supernatants were further used for direct PCR amplification.

24

Figure 3.7. A: Bench top microcentrifuge (Micro 200R, Hettich Zentrifugen) used for microbial isolation and DNA extraction. B: Clifton water bath used for boiling method of DNA extraction

3.7. Agarose Gel electrophoresis Agarose gel electrophoresis was performed to observe any DNA species in this work. TAE buffer (40 mM Tris base; 20 mM glacial acetic acid; 1 mM Na2EDTA.H2O; pH 8.3) was added to agarose powder (HiMedia) and heated in a microwave until the 0.8% (w/v) agarose melted completely. The volume of gel prepared is dependent on the volume of the casting tray being used. 10 µl of 0.5 µg/ml ethidium bromide was added to the gel when it was warm to touch. The gel was poured in the casting tray with the appropriate comb in place. 10 µl of samples containing DNA was mixed with 4 µl of 6X gel loading buffer (10 mM Tris.Cl, pH 7.6; 0.03% bromophenol blue; 0.03% xylene cyanol FF; 60% glycerol; 60 mM EDTA) and 10 µl of distilled water. The mixture was loaded in the corresponding lane and 10 µl of 1 kb DNA ladder (GeneRuler 1 kb plus DNA ladder, ready-touse, ThermoScientific) was loaded in one lane. After electrophoresis for 30-45 minutes (depending on the volume of gel) at 120V, the gel was viewed on a UV transilluminator (Fig. 3.8) and imaged with Advanced American Biotechnology software (Valencia, USA).

25

Figure 3.8. UV transilluminator and connected computer to view and print photographs of gels.

3.8. Amplification of 16S rRNA gene fragments using Polymerase Chain Reaction (PCR) The amplification was performed using the ProFlex PCR system (Fig. 3.9) and Maxima Hot Start PCR Master Mix (2X) (Fermentas) containing Maxima Hot Start Taq DNA Polymerase, optimized Hot Start PCR buffer, Mg2+ and dNTPs. Each reaction comprised of 25 µl of the PCR Master Mix, 5 µl of 1 µM forward primer (21F or 8F) and 5 µl of 1 µM reverse primer (958R or 1492R) (Table 3.3). 2 µl of template DNA was added and the total volume was made up to 50 µl by adding 13 µl of nuclease-free water. For the positive and negative controls, instead of adding template DNA, a stored PCR product of archaeal 16S rRNA (Sornum, 2012) or E. coli and nuclease-free water were added respectively. When preparing the PCR mixtures, primer solutions and template DNA were placed on ice and the PCR Master Mix was vortexed after thawing. The PCR thermal cycling parameters used are shown in table 3.4. The PCR products formed were stored at -20°C.

26

Figure 3.9. PCR system used for DNA fragments amplification.

Table 3.3. Primers used for 16S rRNA gene fragments of Bacteria and Archaea. Y: C or T, M: A or C. Type of 16S rRNA

Primer used

Sequence (5´- 3´)

gene 21F

Archaeal

958R

YCCGGCGTTGAMTCCAATT AGAGTTTGATCCTGGCTCAG

8F

Bacterial

TTCCGGTTGATCCYGCCGGA

1492R

GGTTACCTTGTTACGACTT

Table 3.4. PCR thermal cycling parameters used. Archaeal primers Step

Temperature (°C)

Bacterial primers

Number Time

of Cycles

Temperature (°C)

Number Time of Cycles

Initial denaturation/

95

4 mins

Denaturation

95

30s

Annealing

48

45s

enzyme

1

95

4 mins

1

activation 35

27

95

30s

48

45s

35

Extension Final extension

72

1min16

72

5 mins

72

1

72

2 mins 10 mins

1

3.9. Construction of 16S rRNA gene fragment clone libraries 3.9.1. PCR product purification Purification was done using GeneJET PCR Purification kit (ThermoScientific). 40 µl of PCR product was diluted to a total volume of 200 µl by adding 160 µl of distilled water. An equal volume of binding buffer was added, causing the mixture to turn yellow which indicated optimal binding pH. The tube was vortexed before transferring all the solution to the GeneJET purification column preassembled with the collection tube. The tube was then centrifuged at 12,500 X g for 2 minutes and the flow-through was discarded. The empty collection tube was again centrifuged under the same conditions. 700 µl of wash buffer was added to the purification column which was centrifuged under same conditions. The flow-through was discarded, the empty purification column was placed back into the collection tube and was centrifuged. The purification column was then transferred into a clean, uncapped microcentrifuge tube and 50 µl of elution buffer (10 mM Tris.Cl, pH 8.5) was added. After incubation at room temperature for 2 minutes, it was centrifuged under same conditions as before. The collected mixture was transferred into a capped microcentrifuge tube and stored at -20°C. The purification column, collection tube and uncapped microcentrifuge tube were discarded. The various steps are summarised in fig. 3.10. The concentrations (ng/µl) of the purified PCR products were determined by running an agarose gel electrophoresis and comparing the intensity of the DNA bands obtained with those of the ladder. 10 µl of purified PCR products were mixed with 4 µl of 1X gel loading buffer and 10 µl of distilled water. A diluted 1 kb DNA ladder (0.05 µg/µl) was also loaded. The concentration of the purified product was then calculated by dividing the amount (ng) of the corresponding ladder band by 10.

28

Figure 3.10. Different steps involved in the purification of PCR products.

3.9.2. Ligation Ligation of PCR amplicons obtained from the environmental samples was performed using the CloneJET PCR Cloning kit (ThermoScientific). Since the DNA fragments produced had sticky ends, a blunting reaction was first set up on ice in two series (A and B). For series A, 10 µl of 2X reaction buffer, 7 µl of purified PCR product and 1 µl of DNA blunting enzyme were added to a fresh microcentrifuge tube. The mixture was vortexed briefly and incubated at 70°C for 5 minutes. Afterwards, the ligation reaction was also set up on ice. 1 µl of pJET1.2/blunt Cloning vector and 1 µl of T4 DNA ligase were added to the blunting reaction mixture. The mixture was then incubated at room temperature for 10 minutes. For series B, the same steps were followed but 20 µl of 2X reaction buffer, 15 µl of purified PCR product, 1 µl of DNA blunting enzyme, 0.5 µl of pJET1.2/blunt Cloning vector and 1 µl of T4 DNA ligase were used instead. After ligation, the mixtures were stored at -20°C. Ligation of PCR amplicons obtained from the isolated colonies was performed using the InsTAclone PCR Cloning kit (Fermentas). 2 µl of vector pTZ57R/T, 8 µl of purified PCR product, 4 µl of 5X ligation buffer, 5 µl of water and 1 µl of T4 DNA were added to an empty microcentrifuge tube. After gently vortexing, the mixture was incubated at room temperature for a few hours and stored at -20°C.

3.9.3. Transformation of competent Escherichia coli cells 3.9.3.1. Method 1 Frozen competent E. coli cells were placed on ice until just thawed and the following steps were carried out under sterile conditions. 5 µl of ligation reaction mixture was added to the cells and incubated on ice for at least 30 minutes. The cells were heat-shocked at 42°C for 1 minute and then placed onto ice for 2

29

minutes. 900 µl of LB (1% tryptone; 0.5% yeast extract; 0.5% NaCl) was added to the transformed cells and incubated at 37°C for 1 hour in the orbital shaker. After incubation, the microcentrifuge tubes were centrifuged at 10,000 X g for 2 minutes. 800 µl of the supernatant was discarded and the cells were gently resuspended in the remaining volume. Using a sterile hockey stick, 100 µl of the culture was spread onto LB plates supplemented ampicillin (100 µg/ml) and incubated overnight at 37°C without sealing the plates. For the blue-white selection, when using the vector pTZ57R/T, prior to plating, 40 µl of 0.1M IPTG (isopropyl-β-D-thiogalactopyranoside) and 16 µl of 20 mg/ml X-Gal (5-bromo-4chloro-3-indolyl-β-D- galactopyranoside) were spread onto the plates and allowed to dry. As positive and negative controls, 5 µl of supercoiled plasmid and TE buffer were used respectively for transformation.

3.9.3.2. Method 2: Using the TransformAid (ThermoScientific) Frozen stock E. coli cells were inoculated in 5 ml of C-medium overnight at 37°C, shaking at 220 rpm. A 50 ml sterile conical flask containing 22.5 ml of TransformAid C-medium was pre-warmed at 37oC. 2.5 ml of the overnight grown culture was inoculated in the pre-warmed C-medium and grown for 20 minutes at 37oC, shaking at 220 rpm. All plates to be used were pre-warmed at 37°C. But, for blue-white selection, 40 µl of 0.1 M IPTG and 40 µl of 20 mg/ml X-Gal were spread onto the plates prior to pre-warming. 3.75 ml of TransformAid T-Solution (A) and 3.75 ml of TransformAid T-Solution (B) were mixed and kept on ice. After 20 minutes, 750 µl of culture was pipetted into fresh microcentrifuged tubes and centrifuged at 10,000 X g for 1 minute at 4oC. After discarding the supernatant, the pellet was resuspended in 150 µl of TransformAid T-Solution and incubated on ice for 5 minutes. The tubes were then centrifuged under same conditions and the supernatant was discarded. Before incubating on ice for 5 minutes, 60 µl of TransformAid T-Solution was added to resuspend the pellet. 5 µl of ligation reaction mixture was added to a fresh microcentrifuge tube and was incubated on ice for 2 minutes. Afterwards, 50 µl of the resuspended cells was pipetted to each tube and incubated on ice for 5 minutes. Using a sterile hockey stick, the cells were spread onto the pre-warmed plates which were incubated overnight at 37°C.

30

3.10. Extraction and isolation of plasmids from transformants The GeneJET Plasmid Miniprep Kit was used to carry out the plasmid extraction. Single colonies were picked from plate and inoculated in 10 ml of LB medium containing 100 µg/ml of ampicillin. The colonies were allowed to grow overnight in the orbital shaker set at 37°C, 220 rpm. 1.5 ml of the bacterial culture was transferred to a microcentrifuge tube and was centrifuged at 8,000 X g for 2 minutes. The supernatant was decanted and bacterial culture was added to the same microcentrifuge tube which was centrifuged again. This was repeated 3 times to generate a pellet from 4.5 ml of culture. The pellet was completely resuspended in 250 µl of resuspension solution and 250 µl of lysis solution was added. A viscous and slightly clear solution was formed when the tube was inverted for mixing. 350 µl of neutralizing solution was added and the tube was inverted gently to mix the solution thoroughly. The tube was centrifuged at 12,000 X g for 5 minutes and the supernatant formed was pipetted carefully to a GeneJET spin column. It was centrifuged at 12,000 X g for 1 minute and the flow-through was discarded afterwards. The column was placed back into the same collection tube and 500 µl of wash solution was added. After centrifugation under same conditions, the flow-through was discarded and 500 µl of wash solution was added again. The centrifugation and flow-through discard steps were repeated. The empty spin column was centrifuged for another 1 minute under same conditions to remove any residual of wash solution. The spin column was then transferred into a fresh, uncapped microcentrifuge tube. 50 µl of elution buffer was pipetted to the centre of the column. After incubation at room temperature for 2 minutes, the tube was centrifuged at 12,000 X g for 2 minutes. The column was discarded, the eluate was transferred to a fresh microcentrifuge tube and stored at -20°C.

3.11. Bioinformatics The NCBI Nucleotide database was searched for 16S rRNA gene that had been sequenced from isolated haloarchaea of environmental origin. Data on the members of the class Halobacteria only were retrieved by using the keyword „halobacteria‟. Ten partial 16S rRNA gene sequences ranging from 791 bp to 919 bp were used for MSA which was generated by MultAlin (Corpet, 1988). The 31

sequences were named Arc1 to Arc10 (Appendix 4). After pasting all sequences, in FASTA format, in the rectangle area, the results were calculated by the server and displayed. BLAST was used to input the primer sequences (21F and 958R) as queries against all public sequence databases to identify if the primers used were annealing and amplifying Archaeal 16S rDNA sequences only. After pasting the primer sequence in the query sequence box and blasting, the results are posted.

32

4. Results 4.1. Physical parameters of Tamarin solar saltern The samples were collected on the 26th September 2013 at about 10 a.m. on a hot, sunny day. The physical parameters measured are recorded in table 4.1. There was a lot fluctuation in the three parameters measured due to heavy rainfall on the previous day. However, the pH remained more or less constant and an overall increase in salinity from SP1 (intermediate pan) to SP13 (crystallizing pan) was noticed. The average temperature was 37.6°C, average pH was 6.79 and average salinity was 30.8%.

Table 4.1. The different physical parameters of the water sample collected Salt Pan

Temperature/°C

pH

Salinity/%

1

36

8.15

19

2

35

7.74

13

3

35

6.94

9

4

36

6.69

10

5

37

6.74

11

6

38

6.11

18

7

38

7.20

45

8

39

5.96

45

9

43

6.75

30

10

38

5.82

41

11

Not measured

6.32

46

12

35

7.02

50

13

41

6.88

63

4.2. Microscopy It was difficult to clearly identify the microorganisms present on the slides. Mainly Dunaliella cells and possibly Halospirulina filaments were observed under the microscope (Fig. 4.1) due to their size.

33

Figure 4.1. Microorganisms observed under the microscope (x400). A: Dunaliella spp. B: Halospirulina

4.3. Culture of halophiles Colonies were observed on all plates (Fig. 4.2) except from plates SP9, SP12 and SP13. All of them were more or less round in shape and were categorised as 7 types (Table 4.2). Type 2 (Fig. 4.3) was the most abundant (approx. 896 in plate SP11w) and was present in all plates.

Table 4.2. Different categories of colonies observed. Type

Description

Size (mm)

1

Large orange

0.5-2.0

2

Glossy pale cream

6.0-0.5

Small yellow with

1.0

3

translucent ring

4

Translucent cream

< 0.5

5

Glossy yellow

6

Translucent

< 1.0

7

Tiny black

< 0.2

2.0

34

Figure 4.2. Some plates on which different colony types have grown. A: SP1sed with type 2 and 3 colonies. B: SP4sed with type 2 and 4 colonies. C: SP10w with type 2, 4 and 7 colonies. D: SP11w with type 1, 2 and 6 colonies.

Figure 4.3. Type 2 colonies grown on plate SP7w.

Different colony types were picked at random from all plates (Table 4.3) to form pure cultures for DNA extraction afterwards. But not all colony types present from individual plates were picked.

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Table 4.3. Different colony types used for DNA extraction and their sources. Pure culture name

Colony type

Taken from plate

a1

2

SP1w

a2

2

SP1sed

b

3

SP2sed

c1

5

SP3Aw

c2

2

SP3w

d

2

SP4w

e1

1

SP5w

E1

2

SP6w

e2

4

SP5sed

E2

1

SP6w

f

2

SP6Bsed

F

4

SP4sed

g1

2

SP7w

g2

3

SP7sed

h1

4

SP8w

h2

6

SP8w

h3

3

SP8w

i1

7

SP10w

i2

1

SP10Ased

i3

3

SP10Ased

j

6

SP11w

4.4. Extraction of eDNA Genomic DNA was extracted directly from the halophiles isolated from the water and sediment samples using Takai and Sako (1999) method. High molecular weight DNA has been isolated from SP7w, SP8Aw (Fig. 4.4A, lane 15, 19), SP11w, SP10Ased and SP10Bsed (Fig. 4.4B, lane 4, 5, 6) when extraction was performed. However, the DNA bands appeared very pale on the gel electrophoresis photograph. DNA for SP7w stayed in the well, the band for SP8Aw was of approximately 18000 bp and the bands for SP11w, SP10Ased and SP10Bsed were

36

at 11000 bp. From the graphs drawn, the size of DNA for SP8Aw was 17205 bp and for SP11w, SP10Ased and SP10Bsed were 10794 bp.

Figure 4.4. Gel electrophoresis of extracted eDNA. A: Lane 1: O‟GeneRuler 1 kb ladder, Lane 2: SP1w, Lane 3: SP1sed, Lane 4: SP2sed, Lane 5: SP3w, Lane 6: SP3Ased, Lane 7: SP3Bsed, Lane 8: SP4w, Lane 9: SP4sed, Lane 10: SP5w, Lane 11: SP5sed, Lane 12: SP6w, Lane 13: SP6Ased, Lane 14: SP6Bsed, Lane 15: SP7w, Lane 16: SP7sed, Lane 17: SP8w, Lane 18: SP8(i)w, Lane 19: SP8Aw. B: Lane 1: O‟GeneRuler 1 kb ladder, Lane 2: SP9sed, Lane 3: SP10w, Lane 4: SP10Ased, Lane 5: SP10Bsed, Lane 6: SP11w, Lane 7: SP12w.

4.5. Extraction of gDNA Extraction performed on the isolated colonies resulted in mostly degraded DNA. DNA was obtained from a1, a2, d, e1, f and F (refer to table 4.3 for colony type and source). DNA for a1, a2 and d remained in the wells (Fig. 4.5A, lane 2, 3, 4). The DNA band for e1 (Fig. 4.5A, lane 5) was approximately at 21000 bp. For f (Fig. 4.5B, lane 5) and for F (Fig. 4.5C, lane 6), highly degraded DNA was obtained and was found to be approximately 200 bp and 600 bp respectively. From the graphs drawn, e1 was 20980 bp, f was 214 bp and F was 678 bp.

37

Figure 4.5. Gel electrophoresis of gDNA extraction. A: Lane 1: O‟GeneRuler 1 kb, Lane 2: a1, Lane 3: a2, Lane 4: d, Lane 5: e1. B: Lane 1: O‟GeneRuler 1 kb, Lane 2: not relevant, Lane 3: c2, Lane 4: i2, Lane 5: f, Lane 6: j, Lane 7: not relevant. C: Lane 1: O‟GeneRuler 1 kb, Lane 2: b, Lane 3: c1, Lane 4: E1, Lane 5: c2, Lane 6: F, Lane 7: g1, Lane 8: g2.

4.6. PCR amplification of 16S rRNA gene fragments 4.6.1. Using environmental samples Using the Archaea specific primers 21F and 958R, the positive control (Fig. 4.6A, lane 12) was smeary but appeared to have a diffuse band estimated at approximately 900 bp. As expected no band was observed for the negative control (Fig. 4.6A, lane 13). An amplicon (PCR 24) of approximately 900 bp and two (PCR 27, 28) of approximately 850 bp were observed (Fig. 4.5A, lanes 5, 8, 9). These amplicons came from the amplification of the DNA obtained from Salt pan 10 sediment (SP10sed). Two of them (Fig 4.6A, lane 8, 9) resulted from DNA extraction using the Anton et al. (2000) boiling method and the other one (Fig 4.6A, lane 5) from Takai and Sako (1999) extraction method. From the graph (Fig 4.6B), the size of DNA in lanes 5 and 12 were found to be 947 bp and in lanes 8 and 9 were 868 bp.

38

Figure 4.6. A: Gel electrophoresis of amplified DNA fragments using 21F/958R primers. Lane 1: O‟GeneRuler 1 kb ladder, Lane 2: SP8Aw (PCR 21), Lane 3: SP11w (PCR 22), Lane 4: SP10Ased (PCR 23), Lane 5: SP10Bsed (PCR 24), Lane 6: SP8Aw super (PCR 25), Lane 7: SP11w super (PCR 26), Lane 8: SP10Ased super (PCR 27), Lane 9: SP10Bsed super (PCR 28), Lane 10: d(PCR 28), Lane 11: e1 (PCR 30), Lane 12: positive control (PCR 31) and Lane 13: negative control (PCR 32). Genomic DNA extraction using Anton et al. boiling method (lane 6-9) and Takai et al. method (lane 2-5, 10,11). For lane 10 and 11, DNA extraction was performed using isolated colonies. B: semi-log graph representing size of DNA (log10 bp) plotted against migration distance (mm).

Using Bacterial primers 8F and 1492R, positive control of approximately 1500 bp and four amplicons (PCR 4, 6, 7, 8) of approximately the same size (Fig. 4.7A, lane 5, 7-9,13) were observed. As expected no band was observed for the negative control (Fig. 4.7A, lane 12). These amplicons came from the amplification of the DNA obtained from Salt pan 10 sediment (SP10sed) and salt pan 11 water (SP11w). Three of them (Fig 4.7A, lane 7, 8, 9) resulted from the DNA extraction using Anton et al. (2000) boiling method and the other one (Fig 4.7A, lane 5) from Takai and Sako (1999) extraction method. From the graph (Fig 4.7B), the size of DNA in lanes 5, 7, 8, 9 and 13 were all found to be 1488 bp.

39

Figure 4.7. A: Gel electrophoresis of amplified DNA fragments using 8F/1492R primers. Lane 1: O‟GeneRuler 1 kb ladder, Lane 2: SP8Aw (PCR 1), Lane 3: SP11w(PCR 2), Lane 4: SP10Ased(PCR 3), Lane 5: SP10Bsed(PCR 4), Lane 6: SP8Aw super(PCR 5), Lane 7: SP11w super(PCR 6), Lane 8: SP10Ased super(PCR 7), Lane 9: SP10Bsed super(PCR 8), Lane 10: d(PCR 9), Lane 11: e1(PCR 10), Lane 12: negative control(PCR 11) and Lane 13: positive control(PCR 12). Genomic DNA extraction using Anton et al. boiling method (lane 6-9) and Takai et al. method (lane 2-5, 10,11). For lane 10 and 11, DNA extraction was performed using isolated colonies. B: semi-log graph representing size of DNA (log10 bp) plotted against migration distance (mm).

4.6.2. Using isolated colonies For the isolated colonies, both Archaeal (21F/958R) and Bacterial (8F/1492R) primers combinations were used. No bands were observed for the positive controls (PCR products of Archaeal 16S rDNA fragment or E. coli) (Fig. 4.8A, lane 6, 11). No DNA bands were observed with the Archaeal primers (Fig. 4.8A, lane 2-5). However, two amplicons (PCR 5, 7) were observed at approximately 1400 bp (Fig. 4.7A, lane 7, 9) and one (PCR 6) at 1300 bp (Fig. 4.8A, lane 8) when the Bacterial specific primers were used. a1(PCR 5) was a type 2 colony isolated from Salt Pan 1 water sample (SP1w). f (PCR 6) was also a type 2 colony isolated from Salt Pan 6 sediment (SP6Bsed). F (PCR 7) was a type 4 colony isolated from Salt Pan 4 sediment sample (SP4sed). From the graph, it was found that a1 and F were 1412 bp and f was 1307 bp (Fig. 4.8B).

40

Figure 4.8. A: Gel electrophoresis of amplified DNA fragments from isolated colonies. Lane 1: O‟GeneRuler 1 kb ladder, Lane 2: a1 (PCR 1), Lane 3: f(PCR 2), Lane 4: F(PCR 3), Lane 5: SP7w(PCR 4), Lane 6: positive control, PCR product of Archaeal 16S rDNA fragment (PCR 9), Lane 7: a1(PCR 5), Lane 8: f(PCR 6), Lane 9: F(PCR 7), Lane 10: SP7w(PCR 8), Lane 11: positive control, E. coli (PCR 10). Amplification using Archaeal primers (lane 2-6) and Bacterial primers (lane 711). B: semi-log graph representing size of DNA (log10 bp) plotted against migration distance (mm).

4.6.3.

PCR product purification and determination of DNA concentration.

After purification and running a gel electrophoresis, DNA bands were observed for all products except for SP10Ased super (Archaeal primers). The concentration of the purified PCR product for SP11w super, SP10Ased super, a1 (Bacterial primers) and SP10Bsed super (Archaeal primers) were found to be 2.5 ng/µl (Fig. 4.9A, lane 2, 3, 6; Fig. 4.9B, lane 6). The concentration of SP10Bsed super, f and F (Bacterial primers) was 8 ng/µl (Fig. 4.9A, lane 4; Fig. 4.9B, lane 7, 8).

41

Figure 4.9. Gel electrophoresis of purified PCR products. A: Bacterial primers (lane 2-4), Archaeal primers (lane 5,6), Lane 1: diluted O‟GeneRuler 1 kb ladder, Lane 2: SP11w super, Lane 3: SP10Ased super, Lane 4: SP10Bsed super, Lane 5: SP10Ased super, Lane 6: SP10Bsed super. B: Lane 1-4: not relevant, Lane 5: diluted O‟GeneRuler 1 kb ladder, Lane 6: a1, Lane 7: f, Lane 8: F.

4.7. Construction of 16S rRNA gene fragment clone libraries For ligation of amplicons from environmental samples, the purified PCR products from SP10Bsed super bacterial and archaeal were used for series A ligation (8a, 28a). Only the purified PCR products from SP10Bsed super archaeal was used for series B (28b). For ligation using the vector pTZ57R/T, a1, f and F (5, 6, 7) were used.

All six ligation reaction mixtures were used for both methods of

transformation. Ligation 5, 6 and 7 were used for the blue-white selection. After overnight incubation, some colonies grew on plates 8a, 28a and 28b (Fig. 4.10) but there were signs of contamination. For the other transformation done, the plates were contaminated and no colonies had grown. Colonies (28b.1, 28b.2, 28b.3) from plate 28b were picked for plasmid extraction. When a gel electrophoresis was run after the extraction, no DNA bands were observed.

42

Figure 4.10. Transformed colonies. A: 8a. B: 28a. C: 28b.

4.8.

Bioinformatics

When the GenBank database was searched using the „halobacteria‟ keyword, 32,158 nucleotide sequences were found. The 16S rRNA partial sequences retrieved were seven uncultured halobacteria archaeon, Halorubrum sp. M5, Halococcus thailandensis and Halococcus sp. IARI-SGAB1. Following Multiple Sequence Alignment (MSA), it was found that sequence heterogeneity lies approximately in the first 530 to 820 sequence bases of the 5‟ end (Fig. 4.11). Greatest heterogeneity was observed between positions 529-531, 550-554, 562-566 and 583-587. Also, most of the sequences were conserved in all 10 entries.

43

Figure 4.11. Multiple Sequence Alignment of Arc1 to Arc10.

After the two archaeal-specific primers were searched against the database, BLAST resulted in an alignment score of 0.5 because of the primers are degenerate ones. The presence of another alphabet other than A, T, C and G affected the results. For primer 21F, when Y was replaced by T or C and blasted again, the E value decreased to 0.091 48

in both cases. Values