Cultivation and characterization of the square haloarchaeon Haloquadratum walsbyi and other novel isolates.
David G. Burns
Submitted in total fulfilment of the requirements of the degree of
Doctor of Philosophy
June 2007
Department of Microbiology and Immunology
The University of Melbourne
This is to certify that
(i)the thesis comprises only my original work towards the PhD except where otherwise indicated
(ii)due acknowledgement has been made in the text to all other material used
(iii)the thesis is less than 100,000 words in length, exclusive of tables, maps, bibliographies and appendices.
David G. Burns B. Sc. (Hons)
Department of Microbiology and Immunology
The University of Melbourne
1
Abstract
The square haloarchaea of Walsby (SHOW group) was first observed in 1980, and dominates hypersaline microbial communities. Despite this,the organism has not been cultured sincetheir discovery 25 years ago. In this work I show that natural water dilution cultures can be used to isolate representatives of this group and, once inpure culture, they can be grown in standard halobacterial media, although optimal growth occurs in a medium optimized for these organisms. Cells display a square morphology and contain gas vesicles andpoly-b-hydroxybutyrate (PHB) granules. The 16S rRNA gene sequence was >99% identical to other SHOW group sequences that have been isolated directly from the environment. The SHOW group prefers high salinities (22 –36 %), and can grow with a doubling time of 1–2 days in optimized media. The ability to culture SHOW grouporganisms makes it possible to study, in a more comprehensive way than before available, the microbial ecology of salt lakes.
Further, many novel isolates from previous studies, spanning a further three genera that had accounted for the balance of uncultured diversity in an Australian solar saltern, are here characterized in preparation for formal taxonomic recognition. It was found that pyruvate, an essential substrate in the isolation and cultivation of the SHOW organism, was an optimal substrate for all of these strains.
There now exists a complete isolate library spanning all of the dominant haloarchaea found in an Australian solar saltern, providing a foundation from which advanced ecological studies may be carried out.
Acknowledgements
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Publicationsarising
Journal articles
* Burns, D.G., Camakaris, H.M., Janssen, P.H., Dyall-Smith, M.L. (2004) Combined use of cultivation-dependent and cultivation-independent methods indicates that members of most haloarchaeal groups in an Australian crystallizer pond are cultivable. Applied and Environmental Microbiology 70(9): 5258-65.
*Experimental work undertaken during Honours year (2002); paper written during PhD
Burns, D.G., Camakaris, H.M., Janssen, P.H., Dyall-Smith, M.L. (2004a) Cultivation of Walsby's square haloarchaeon. FEMS Microbiology Letters 238: 469-473.
Burns, D.G., Janssen, P.H., Itoh, T., Kamekura, M., Li, Z., Jensen, G., Rodrìguez-Valera, F., Bolhuis, H., Dyall-Smith, M.L. (2007) Haloquadratum walsbyi gen. nov., sp. nov., the square haloarchaeaon of Walsby, isolated from saltern crystallizers in Australia and Spain. International Journal of Systematic and Evolutionary Microbiology 57: 387-392.
Book Chapters
Burns, D.G. and Dyall-Smith, M.L. Cultivation of Haloarchaea. In, Extremophiles. (2006) Methods in Microbiology 35:535-552 (ed. A. Oren, F. Rainey). Academic Press / Elsevier ISBN: 0-12-521537-1
Dyall-Smith, M.L., Burns, D.G., Camakaris, H.M., Janssen, P.H., Russ, B.E, Porter, K. (2005) Haloviruses and their hosts. Recent progress in the cultivation of haloarchaea, including square haloarchaea of Walsby, and the isolation of novel haloarchaeal viruses. In, Adaptation To Life at High Salt Concentrations in Archaea, Bacteria, and Eukarya (ed. Nina Gunde-Cimerman, Aharon Oren and Ana Plemenita), Series: Cellular Origin, Life in Extreme Habitats and Astrobiology, volume 9, p555-563, Springer-Dordrecht ISBN 1402036329
Manuscripts in preparation
Burns, D.G., Janssen, P.H., Itoh, T., Kamekura, M., Dyall-Smith, M.L.Halonotius torquis gen. nov., sp. nov., a novel haloarchaeon isolated from a southern Australian salt crystallizer pond.[M1] To be submitted to the International Journal of Systematic and Evolutionary Microbiology.
Burns, D.G., Janssen, P.H., Itoh, T., Kamekura, M., Dyall-Smith, M.L.Characterization of a novel genus of neutrophilic haloarchaea of close phylogenetic relationship to haloalkaliphilic archaea such as Natronomonas pharaonis. To be submitted to the International Journal of Systematic and Evolutionary Microbiology.
Burns, D.G., Janssen, P.H., Itoh, T., Kamekura, M., Dyall-Smith, M.L.Characterization of novel haloarchaeal isolate 2.24.4, a heterotrophic aerobe isolated from an Australian salt crystallizer pond. To be submitted to the International Journal of Systematic and Evolutionary Microbiology.
[M2]The viruses and microbes of Australian salt lakes and salterns. Dyall-Smith, M.L., Burns, D.G., Porter, K., Bath, C.R. & Russ, B. 9th International Society for Salt Lake Research (ISSLR) Conference, Curtin University, Perth, Australia. September 26-30, 2005.
Salt Lake Microbiology: The Weird World of Haloarchaea and Their Viruses
Mike Dyall-Smith, David Burns, Kate Porter, Brendan Russ
Aust. Soc. Micro. (ASM) annual scientific congress, Canberra, 2005. SY38/PP38.02, Thur 29th Sept, 2005.
Haloviruses and their Hosts
Dyall-Smith M., Bath C., Burns D., Porter K., Russ B., and Tang S.L.
HALOPHILES 2004, International Congress on Halophilic Microorganisms
Ljubljana, 4 - 9 September 2004
Abbreviations
aaamino acid/s
ATCCAmerican Type Culture Collection
BLASTBasic Local Alignment Search Tool
bpbase pair(s)
BSABovine Serum Albumin
CCelsius
DNAdeoxyribonucleic acid
dNTPdeoxynucleotide triphosphate
DSM Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
(German Collection of Microorganisms and Cell Cultures)
EDTAethylene diamine tetra-acetic acid
EMelectron microscope
EtBrethidium bromide
G+Cguanosine plus cytosine
JCM Japan Collection of Microorganisms
hrhour
kbkilobase pairs
MGMModified Growth Medium
minminute
NCBINational Center for Biotechnology Information
nt/snucleotide/s
ODoptical density
PCRpolymerase chain reaction
PHApolyhydroxyalkanoate
PHB polyhydroxybutyrate
RNAribonucleic acid
rpmrevolutions per minute
rRNAribosomal RNA
secsecond
spp.species
SWsalt water
TE Tris-EDTA
volvolume
wtweight
X-gal5-bromo-4-chloro-3-indolyl--galactopyroanoside
Abstract
Acknowledgements
Publicationsarising
Abbreviations
Chapter 1
Introduction
Archaea
Archaea: phylogeny
History
Halobacteria
Hypersaline Lakes
Adaptation mechanisms
The Square Haloarchaea of Walsby (SHOW)
Cultivation of “uncultivables”.
Isolation
Chapter 2
Materials and Methods
Sample Collection
Microscopic Observation
Medium Formulations
DBCM1
DBCM2
MGM
Incubation Conditions
Liquid Medium
Plate Incubation Conditions
Extinction cultures
Standard Polymerase Chain Reaction (PCR) Protocols
PCR Amplification of Isolate 16S rDNA
PCR Protocol DB194
DB195
Multiplex PCR Protocol DB239
PCR Cleanup
Sequencing PCR
DNA visualisation:
Clone library generation
RFLP
16S rRNA gene identification and phylogenetic tree reconstructions.
Phenotypic tests
Media and growth conditions
Anaerobic respiration
Antibiotic sensitivity
Beta-galactosidase activity
Casein hydrolysis
Catalase Test
Microscopy
GC content and DNA-DNA hybridisation
Indole test
Lipids
Magnesium Optima
Motility
Oxidase Test
Salinity Optima
Substrate utilisation and acid production
Temperature Optima
Chapter 3
Characterisation of Natronomonas-like, and Halogeometricum-like isolates, and isolates related to the Deep-10 phylotype (Antarctic Deep Lake)
Introduction
Part 1: Isolates 1.15.5, 2.27.5, 5.24.4 and 6.14.5
Putative ADL Group
Introduction
Results and Discussion
Motility
Temperature Optimum
Salinity and Magnesium Optimum
pH Optimum and Antibiotic Sensitivity
16S rRNA gene phylogeny
GENUS and SPECIES description
Part 2: Isolates 4.03.5 and 8.8.11
Natronomonas-like group
Results and Discussion
Temperature Optimum
Salinity and Magnesium Optimum
pH Optimum and Antibiotic Sensitivity
16S rRNA gene phylogeny
GENUS and SPECIES description
Part 3: Isolate 2.24.4
Halogeometricum-like group
Results and Discussion
Temperature Optimum
Salinity and Magnesium Optimum
pH Optimum and Antibiotic Sensitivity
16S rRNA gene phylogeny
GENUS and SPECIES description
Summary Tables
Chapter 4
Isolation of the Square Haloarchaea of Walsby
Overview
Solid medium cultivation
Liquid isolation
Low-substrate liquid isolation
Screening techniques
SHOW cultivation using natural water extinction culturing
Passaging of mixed SHOW culture
Improving SHOW isolation medium
Low substrate medium supplementation – amino acids and acetate
Low substrate medium supplementation – pyruvate
SHOW isolation
Passaging of SHOW cultures
SHOW medium variation and optimization
Isolate Purity
Microscopic examination
Restriction Fragment Length Polymorphism (RFLP)
Sequencing
Isolate Selection
Chapter 5
Characterisation of Haloquadratum walsbyi
Preface
Microscopy
Macroscopic appearance
Further characteristics
GENUS and SPECIES description
Chapter 6
Final Discussion
Bibliography
1
Chapter 1
Introduction
1
Archaea
The advent of molecular techniques applied to natural ecologies heralded a dramatic expansion in our understanding of the diversity of microbial populations. Early molecular studies began with protein sequencing, and later moved to nucleotide sequencing and then specifically the 5S universally-conserved ribosomal RNA gene, and later again to the larger and more complex (and current benchmark) 16S rRNA gene. This last gene codes for the 16S small subunit rRNA ribosome component, an essential part of all living cells. The genetic sequence for the 16S rRNA gene is highly conserved, but with sufficient slow changes that its many variants may be compared to determine phylogenetic relationships between organisms.
Molecular techniques have provided a radically different picture of ecological diversity compared to traditional, cultivation-based techniques. Based on rRNA gene phylogenetic trees, most of Earth’s biodiversity is microbial (Hugenholtz et al., 1998). These molecular studies have largely been based on the 16S rRNA gene, although other genes have also been used. It was through molecular studies that the Archaea were first realised to be distinct from the other prokaryotic Domain, the Bacteria; Woese and Fox in 1977 found that the universal phylogenetic tree did not correspond to the expected bifurcated Bacteria/Eukaryote pattern but instead consisted of the three Domains recognised today. (Woese et al.,1990). Archaea are now fully accepted as the third Domain of life alongside Bacteria and Eucarya, and exhibit features of both of the other Domains, as well as having their own unique characteristics. Current phylogenetic studies indicate that the Bacteria seceded from the Eucarya and Archaea, before the Archaea and Eucarya themselves diverged into the present tri-Domain arrangement.
On account of their unique evolutionary history, Archaea represent a remarkable opportunity to study evolutionary relationships and the development of life. Archaea are also, in their own right, a diverse and significant part of the biosphere, particularly in “extreme” conditions such as high-temperature, pressure, or salinity locales, although their distribution is otherwise as broad as Bacteria.
The separation of Domains is supported by other characteristics than just 16S rRNA gene sequence variation, including unique glycerol isopranyl ether-linked lipids specific to Archaeal cell envelopes (and absence of acyl ester lipids) (Boone and Castenholz, 2001, Seghal et al., 1962), as well as the nature of transcription systems, being dissimilar to both Bacteria and Eukarya, but with features of both (Soppa, 1999).
Ribosomal RNA gene sequencing revealed many of the shortcomings of purely morphological and biochemical based classifications, and as such, this form of phylogenetic classification has become the preferred method of assessing diversity, identity and evolutionary relationships in the majority of microbial communities and also for cultivated organisms. However, formal classification still requires a range of taxonomic features to be considered, including membrane lipids, cell morphology, ionic requirement (both H+ and salts) and growth characteristics, as 16S rRNA gene sequence alone is not necessarily indicative of phenotype.
While there have been some counterarguments against the monophyly or branch point of the Archaea (Gupta, 1998; Rivera and Lake, 1992), the current Domain divisions appear robust and are strongly supported by the currently available data.
Archaea: phylogeny
The Archaeal Domain consists of four phlya. The two major phyla are the Crenarchaeota and the Euryarchaeota. In general, they are separated by phenotype as well as by molecular means; the Crenarchaeota consist of the extremely thermophilic, sulphur metabolising groups such as Thermoproteales, Desulfurococcales and Sulfolobales. In contrast, the Euryarchaeota is a much more diverse phylum, including the methanogens, extreme halophiles and hyperthemophiles, represented by classes such as the Halobacteria, Archaeoglobi, Methanobacteria and Thermoplasmata.
The remaining two phyla contain very few known representatives; the Korachaeota and Nanoarchaeota. These phyla are both recent additions to the Archaea; Korarchaeota was recognised in 1996 (Barns et al., 1996) and does not contain any isolated organisms, and is only represented by 16S rRNA gene sequences recovered from the environment. Its distribution is very limited, appearing thus far only in high temperature springs and deep sea vents (Auchtung et al., 2006). The Nanoarchaeota includes only one organism, which has been isolated in binary culture with Ignicoccus spp. (Huber et al, 2003) However, the longevity of this phylum appears in doubt, with recent studies arguing the sole organism in the clade (Nanoarchaeum equitans) is in fact a fast-evolving member of the phylum Euryarchaeota (Brochier et al., 2005).
History
Haloarchaea, or halobacteria as they have been called, have been the subject of human interest for centuries. The colouration of brines was recorded by the ancient Chinese (Baas-Becking, 1931) and also the Romans (Jones, 1963). More recently, halobacteria were studied for their role in food spoilage; with salting of food a common method of preservation, these organisms were unusual in their ability to grow in these harsh conditions. The strain Halobacterium cutirubrum (“red skin”) was isolated in 1934 (Loackhead, 1934, Boone and Castenholz, 2001) and, along with other strains such as Pseudomonas salinaria (Harrison and Kennedy, 1922; Boone and Castenholz, 2001) and Halobacterium halobium (Petter, 1931; Boone and Castenholz, 2001), were in fact Halobacterium salinarum strains, a haloarchaeon that has a particular prevalence in proteinaceous salted products.
Later researchers realised haloarchaea could be isolated from almost any hypersaline environment, including salt mines and deposits (McGenity et al., 2000), on beach sand particles (Onishi et al., 1985), and from crude solar salt, as well as the aforementioned salted products (hides and other food products) (Boone and Castenholz, 2001).
Haloarchaea, and particularly, the family Halobacteriaceae are members of the Domain Archaea, and comprise the majority of the prokaryotic population in hypersaline environments (Oren 2002). There are currently 26 recognised genera in the family (Gutierrez et al., 2002; Euzéby, 2007). The domain Bacteria can comprise up to 25% of the prokaryotic community, but is more commonly a much lower percentage of the overall population (Antón et al., 2000). At times, the alga Dunaliella salina can also proliferate in this environment (Casamayor et al., 2002).
A comparatively wide range of taxa have been isolated from saltern crystalliser ponds, including members of the following genera: Haloferax, Halogeometricum, Halococcus, Haloterrigena, Halorubrum, Haloarcula and Halobacterium families (Oren 2002). However, the viable counts in these studies have been small when compared to total counts, and the numerical significance of these isolates has been unclear. Recently it has become possible to determine the identities and relative abundances of organisms in natural populations, typically using PCR-based strategies that target 16S rRNA genes. While comparatively few studies of this type have been performed, results from the majority of these suggest that some of the most readily isolated and studied genera may not in fact be significant in the in-situ community. This is seen in cases such as the genus Haloarcula, which is estimated to make up less than 0.1% of the in situ community (Antón et al., 1999) but commonly appears in isolation studies. An exception to this was seen in an Australian saltern, in which it was found to be possible to isolate and cultivate the majority of the diversity observed by 16S rRNA gene libraries (Burns et al., 2004). Even this study, however, failed to isolate one of the more abundant organisms in this lake, the Square Haloarchea of Walsby.
Halobacteria
The Halobacteriaceae are by no means the only prokaryotic representatives at saturating salinities. The halophilic bacterium Salinibacter ruber can make up from 5-27% of the prokaryotic population (Antón et al., 2000). While at saturation, particularly for thassohaline lakes, this is generally the only or dominant member of the Bacterial community, in some hypersaline environments Salicola marasensis can make up to 10% of the prokaryotic community (Maturrano et al., 2006). At lower salinities, the Domain Bacteria are more abundant than Archaea, although both halophilic Archaea and Bacteria are represented across the entire salinity range from seawater to saturation.
Hypersaline Lakes
The oceans are about 3.5% (w/v) salt, mainly sodium chloride (NaCl), and contain the majority of halophilic microorganisms, including a wide variety of Bacteria and Eukarya (eg. protists and algae), and some Archaea. Moderate halophiles (both Bacteria and Archaea) exist in the range from above seawater to approximately 15% salts, while the extremely halophilic microrganisms grow from this point up to saturation (around 37%). Previous studies have demonstrated that the dominant microorganisms in lakes with salinities approaching saturation are haloarchaea from the Family Halobacteriaceae (Benlloch et al., 2002; Oren 2002).
While the diversity is low in these environments, the productivity and cell densities can be quite high. The distinctive pink colouration of these lakes is reflective of the fact that rather than being hostile to life, hypersaline lakes harbour large and active populations of microorganisms. The colour in these lakes is brought about from pigment contained in the haloarchaea, and to a lesser extent, halophilic algae and halobacteria. Even in lakes with saturated salt concentrations (when not dried out to salt pans, or after recent rains, salt lakes are usually near saturation, with a thick layer of crystalline salt as the lake bed) microbial cell densities can reach up to 107 to 108 cells per millilitre. These populations reflect a lack of predation and often quite high nutrient levels (Oren, 2002). Saline lakes are widely recognized as highly productive aquatic habitats, harbouring specialised assemblages of species and often supporting large populations of both migrating and breeding birds (Rodrìguez-Valera, 1988).
Natural hypersaline environments are a significant part of Australia’s landscape and ecology. Of Australia’s largest five lakes, four are salt lakes - Lake Eyre is 9500 km2 when full, and the next two largest salt lakes in Australia, Lake Torrens and Lake Gairdner are 5745 and 4351 km2 respectively. The Northern Territory’s (salt) Lake Amadeus is the fifth largest lake on the continent at 1032 km2 (Geoscience, 2002). In addition, artificial hypersaline lakes are abundant, being created to alleviate the country’s salinity problems and also for economic reasons, such as salt harvesting. It is important to have an understanding of the microbial ecology of these environments, given the increasing occurrence, and importance of these environments in Australia, and also internationally; we note that salt lakes make up about the same volume of the world’s bodies of water as freshwater systems (excluding oceans).