Falicia Goh

Bacterial, archaeal and eukaryotic diversity of smooth and pustular microbial mat communities in the hypersaline lagoon of Shark Bay.

The bacterial, archaeal and eukaryotic populations of nonlithifying mats with pustular and smooth morphology from Hamelin Pool, Shark Bay were characterised using small subunit rRNA gene analysis and microbial isolation. A highly diverse bacterial population was detected for each mat, with 16S rDNA clones related to Actinobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, Gemmatimonas, Planctomycetes, Alphaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Verrucomicrobia and candidate division TM6 present in each mat. Spirochaetes were detected in the smooth mat only, whereas candidate division OP11 was only detected in the pustular mat. Targeting populations with specific primers revealed additional cyanobacterial diversity. The archaeal population of the pustular mat was comprised purely of Halobacteriales, whereas the smooth mat contained 16S rDNA clones from the Halobacteriales, two groups of Euryarchaea with no close characterised matches, and the Thaumarchaea. Nematodes and fungi were present in each mat type, with diatom 18S rDNA clones only obtained from the smooth mat, and tardigrade and microalgae clones only retrieved from the pustular mat. Cultured isolates belonged to the Firmicutes, Gammaproteobacteria, Alphaproteobacteria, Bacteroidetes, Actinobacteria, Cyanobacteria, and Halobacteriales. The mat populations were significantly more diverse than those previously reported for Hamelin Pool stromatolites, suggesting specific microbial populations may be associated with the nonlithifying and lithifying microbial communities of Hamelin Pool.

Determining the specific microbial populations and their spatial distribution within the stromatolite ecosystem of Shark Bay

The stromatolites at Shark Bay, Western Australia, are analogues of some of the oldest evidence of life on Earth. The aim of this study was to identify and spatially characterize the specific microbial communities associated with Shark Bay intertidal columnar stromatolites. Conventional culturing methods and construction of 16S rDNA clone libraries from community genomic DNA with both universal and specific PCR primers were employed. The estimated coverage, richness and diversity of stromatolite microbial populations were compared with earlier studies on these ecosystems. The estimated coverage for all clone libraries indicated that population coverage was comprehensive. Phylogenetic analyses of stromatolite and surrounding seawater sequences were performed in ARB with the Greengenes database of full-length non-chimaeric 16S rRNA genes. The communities identified exhibited extensive diversity. The most abundant sequences from the stromatolites were α- and γ-proteobacteria (58%), whereas the cyanobacterial community was characterized by sequences related to the genera Euhalothece, Gloeocapsa, Gloeothece, Chroococcidiopsis, Dermocarpella, Acaryochloris, Geitlerinema and Schizothrix. All clones from the archaeal-specific clone libraries were related to the halophilic archaea; however, no archaeal sequence was identified from the surrounding seawater. Fluorescence in situ hybridization also revealed stromatolite surfaces to be dominated by unicellular cyanobacteria, in contrast to the sub-surface archaea and sulphate-reducing bacteria. This study is the first to compare the microbial composition of morphologically similar stromatolites over time and examine the spatial distribution of specific microorganismic groups in these intertidal structures and the surrounding seawater at Shark Bay. The results provide a platform for identifying the key microbial physiology groups and their potential roles in modern stromatolite morphogenesis and ecology.

Haloferax elongans sp. nov. and Haloferax mucosum sp. nov., isolated from microbial mats from Hamelin Pool, Shark Bay, Australia

Extremely halophilic archaea were cultivated from smooth and pustular microbial mats collected from Hamelin Pool, Shark Bay, Western Australia. On the basis of morphology, two phenotypes were present and 16S rRNA gene sequence analysis indicated that all strains were most closely related to members of the genus Haloferax (98.1-99.4 % similarity). One representative strain from each phenotype was selected for further taxonomic characterization. Strain SA5T, isolated from the smooth mat, formed small ( approximately 1 mm diameter), red, translucent colonies on agar medium and strain PA12T, isolated from the pustular mat, formed large (3-5 mm diameter), pink, mucoid, domed colonies. Both strains grew in media with 1.7-5.1 M NaCl, required at least 0.2 M Mg2+ for growth and had pH optima of 7.4. The 16S rRNA gene similarity between strains SA5T and PA12T was 97.1 %. Physiological properties, G+C content and polar lipid composition supported placement of both strains in the genus Haloferax. Phenotypic analysis indicated that the two strains were distinct from each other and from all other members of the genus. This was confirmed by the low DNA-DNA relatedness between strains SA5T and PA12T (18-30 %) and between both strains and all other recognized Haloferax species. Two novel species of the genus Haloferax are proposed to accommodate these novel isolates, Haloferax elongans sp. nov. (type strain SA5T=JCM 14791T=ATCC BAA-1513T=UNSW 104100T) and Haloferax mucosum sp. nov. (type strain PA12T=JCM 14792T=ATCC BAA-1512T=UNSW 104200T).

Lysis efficiency of standard DNA extraction methods for Halococcus spp. in an organic rich environment

The extraction of nucleic acids from a given environment marks a crucial and essential starting point in any molecular investigation. Members of Halococcus spp. are known for their rigid cell walls, and are thus difficult to lyse and could potentially be overlooked in an environment. Furthermore, the lack of a suitable lysis method hinders subsequent molecular analysis. The effects of six different DNA extraction methods were tested on Halococcus hamelinensis, Halococcus saccharolyticus and Halobacterium salinarum NRC-1 as well as on an organic rich, highly carbonated sediment from stromatolites spiked with Halococcus hamelinensis. The methods tested were based on physical disruption (boiling and freeze/thawing), chemical lysis (Triton X-100, potassium ethyl xanthogenate (XS) buffer and CTAB) and on enzymatic lysis (lysozyme). Results showed that boiling and freeze/thawing had little effect on the lysis of both Halococcus strains. Methods based on chemical lysis (Triton X-100, XS-buffer, and CTAB) showed the best results, however, Triton X-100 treatment failed to produce visible DNA fragments. Using a combination of bead beating, chemical lysis with lysozyme, and thermal shock, lysis of cells was achieved however DNA was badly sheared. Lysis of cells and DNA extraction of samples from spiked sediment proved to be difficult, with the XS-buffer method indicating the best results. This study provides an evaluation of six commonly used methods of cell lysis and DNA extraction of Halococcus spp., and the suitability of the resulting DNA for molecular analysis.

Identification and regulation of novel compatible solutes from hypersaline stromatolite-associated cyanobacteria

Cyanobacteria are able to survive in various extreme environments via the production of organic compounds known as compatible solutes. In particular, cyanobacteria are capable of inhabiting hypersaline environments such as those found in intertidal regions. Cyanobacteria in these environments must possess regulatory mechanisms for surviving the changing osmotic pressure as a result of desiccation, rainfall and tidal fluxes. The objective of this study was to determine the compatible solutes that are accumulated by cyanobacteria from hypersaline regions, and specifically, the stromatolite ecosystems of Shark Bay, Western Australia. Previously, the cyanobacterial populations associated with these stromatolites were characterized in two separate studies. Compatible solutes were extracted from isolated cyanobacteria here and identified by nuclear magnetic resonance. As the media of isolation contained no complex carbon source, the solutes accumulated were likely synthesized by the cyanobacteria. The data indicate that from this one habitat taxonomically distinct cyanobacteria exposed to varying salinities accumulate a range of known compatible solutes. In addition, taxonomically similar cyanobacteria do not necessarily accumulate the same compatible solutes. Glucosylglycerol, a compatible solute unique to marine cyanobacteria was not detected; however, various saccharides, glycine betaine, and trimethylamine-N-oxide were identified as the predominant solutes. We conclude that the cyanobacterial communities from these hypersaline stromatolites are likely to possess more complex mechanisms of adaptation to osmotic stress than previously thought. The characterization of osmoregulatory properties of stromatolite microorganisms provides further insight into how life can thrive in such extreme environments.

Stromatolites as a Resource for Novel Natural Products

Cyanobacteria produce numerous secondary metabolites, including peptides and depsipeptides, and certain cyanobacterial genera are especially rich sources of bioactive compounds. In a recent study, we characterised the microbial diversity of stromatolites in the hypersaline marine setting of Shark Bay in Western Australia (Burns et al. 2004), identifying a number of cyanobacteria that may be novel to this niche. These stromatolites are analogues of some of the earliest life on Earth (Walter et al. 1980), and their substantial biodiversity may also reflect significant chemical diversity. We hypothesised that the cyanobacterial communities associated with stromatolites surviving in extreme habitats are a potentially rich source of bioactive secondary metabolites. We screened for the potential for production of bioactive metabolites in diverse species of cyanobacteria isolated from stromatolites in Hamelin Pool, Shark Bay, Australia. Using degenerate primer sets, putative peptide synthetase and polyketide synthase genes were detected from strains of Symploca, Leptolyngybya, Microcoleus, Pleuorocapsa, and Plectonema sp. Sequence analysis indicates the enzymes encoded by these genes may be responsible for the production of different secondary metabolites, such as hepatotoxins and antibiotics. Computer modelling was also conducted to predict the putative amino acid recognised by the unknown adenylation domain in the NRPS sequences. Mass spectral analysis also allowed the putative identification of the cyclic peptides cyanopeptolin S and 21-bromo-oscillatoxin A in two of the isolates. Of further significance, preliminary bioassays have identified putative anti-proliferative and anti-protozoal activity in two stromatolite isolates. This is the first time evidence of secondary metabolite production and bioactivity has been shown in stromatolite-associated microorganisms. Further investigation may allow the discovery of unique biochemical pathways that could lead to the discovery and production of novel secondary metabolites. In particular we will employ recent principles of functional metagenomic analysis to rationally assess the biotechnological potential of unculturable stromatolite microorganOrig Life Evol Biosph (2006) 36:623–624 DOI 10.1007/s11084-006-9047-0