Exploring Andean High-Altitude Lake Extremophiles through Advanced Proteotyping.

Quickly identifying and characterizing isolates from extreme environments is currently challenging while very important to explore the Earth's biodiversity. As these isolates may, in principle, be distantly related to known species, techniques are needed to reliably identify the branch of life to which they belong. Proteotyping these environmental isolates by tandem mass spectrometry offers a rapid and cost-effective option for their identification using their peptide profiles. In this study, we document the first high-throughput proteotyping approach for environmental extremophilic and halophilic isolates. Microorganisms were isolated from samples originating from high-altitude Andean lakes (3700-4300 m a.s.l.) in the Chilean Altiplano, which represent environments on Earth that resemble conditions on other planets. A total of 66 microorganisms were cultivated and identified by proteotyping and 16S rRNA gene amplicon sequencing. Both the approaches revealed the same genus identification for all isolates except for three isolates possibly representing not yet taxonomically characterized organisms based on their peptidomes. Proteotyping was able to indicate the presence of two potentially new genera from the families of Paracoccaceae and Chromatiaceae/Alteromonadaceae, which have been overlooked by 16S rRNA amplicon sequencing approach only. The paper highlights that proteotyping has the potential to discover undescribed microorganisms from extreme environments.

Questioning the radiation limits of life: Ignicoccus hospitalis between replication and VBNC.

Radiation of ionizing or non-ionizing nature has harmful effects on cellular components like DNA as radiation can compromise its proper integrity. To cope with damages caused by external stimuli including radiation, within living cells, several fast and efficient repair mechanisms have evolved. Previous studies addressing organismic radiation tolerance have shown that radiotolerance is a predominant property among extremophilic microorganisms including (hyper-) thermophilic archaea. The analysis of the ionizing radiation tolerance of the chemolithoautotrophic, obligate anaerobic, hyperthermophilic Crenarchaeon Ignicoccus hospitalis showed a D10-value of 4.7 kGy, fourfold exceeding the doses previously determined for other extremophilic archaea. The genome integrity of I. hospitalis after γ-ray exposure in relation to its survival was visualized by RAPD and qPCR. Furthermore, the discrimination between reproduction, and ongoing metabolic activity was possible for the first time indicating that a potential viable but non-culturable (VBNC) state may also account for I. hospitalis.

An overview of the bacterial microbiome of public transportation systems-risks, detection, and countermeasures.

When we humans travel, our microorganisms come along. These can be harmless but also pathogenic, and are spread by touching surfaces or breathing aerosols in the passenger cabins. As the pandemic with SARS-CoV-2 has shown, those environments display a risk for infection transmission. For a risk reduction, countermeasures such as wearing face masks and distancing were applied in many places, yet had a significant social impact. Nevertheless, the next pandemic will come and additional countermeasures that contribute to the risk reduction are needed to keep commuters safe and reduce the spread of microorganisms and pathogens, but also have as little impact as possible on the daily lives of commuters. This review describes the bacterial microbiome of subways around the world, which is mainly characterized by human-associated genera. We emphasize on healthcare-associated ESKAPE pathogens within public transport, introduce state-of-the art methods to detect common microbes and potential pathogens such as LAMP and next-generation sequencing. Further, we describe and discuss possible countermeasures that could be deployed in public transportation systems, as antimicrobial surfaces or air sterilization using plasma. Commuting in public transport can harbor risks of infection. Improving the safety of travelers can be achieved by effective detection methods, microbial reduction systems, but importantly by hand hygiene and common-sense hygiene guidelines.

Enabling deep-space experimentations on cyanobacteria by monitoring cell division resumption in dried Chroococcidiopsis sp. 029 with accumulated DNA damage.

Cyanobacteria are gaining considerable interest as a method of supporting the long-term presence of humans on the Moon and settlements on Mars due to their ability to produce oxygen and their potential as bio-factories for space biotechnology/synthetic biology and other applications. Since many unknowns remain in our knowledge to bridge the gap and move cyanobacterial bioprocesses from Earth to space, we investigated cell division resumption on the rehydration of dried Chroococcidiopsis sp. CCMEE 029 accumulated DNA damage while exposed to space vacuum, Mars-like conditions, and Fe-ion radiation. Upon rehydration, the monitoring of the ftsZ gene showed that cell division was arrested until DNA damage was repaired, which took 48 h under laboratory conditions. During the recovery, a progressive DNA repair lasting 48 h of rehydration was revealed by PCR-stop assay. This was followed by overexpression of the ftsZ gene, ranging from 7.5- to 9-fold compared to the non-hydrated samples. Knowing the time required for DNA repair and cell division resumption is mandatory for deep-space experiments that are designed to unravel the effects of reduced/microgravity on this process. It is also necessary to meet mission requirements for dried-sample implementation and real-time monitoring upon recovery. Future experiments as part of the lunar exploration mission Artemis and the lunar gateway station will undoubtedly help to move cyanobacterial bioprocesses beyond low Earth orbit. From an astrobiological perspective, these experiments will further our understanding of microbial responses to deep-space conditions.

Biosignature stability in space enables their use for life detection on Mars.

Two rover missions to Mars aim to detect biomolecules as a sign of extinct or extant life with, among other instruments, Raman spectrometers. However, there are many unknowns about the stability of Raman-detectable biomolecules in the martian environment, clouding the interpretation of the results. To quantify Raman-detectable biomolecule stability, we exposed seven biomolecules for 469 days to a simulated martian environment outside the International Space Station. Ultraviolet radiation (UVR) strongly changed the Raman spectra signals, but only minor change was observed when samples were shielded from UVR. These findings provide support for Mars mission operations searching for biosignatures in the subsurface. This experiment demonstrates the detectability of biomolecules by Raman spectroscopy in Mars regolith analogs after space exposure and lays the groundwork for a consolidated space-proven database of spectroscopy biosignatures in targeted environments.

Geomicrobiology of a seawater-influenced active sulfuric acid cave.

Fetida Cave is an active sulfuric acid cave influenced by seawater, showing abundant microbial communities that organize themselves under three main different morphologies: water filaments, vermiculations and moonmilk deposits. These biofilms/deposits have different cave distribution, pH, macro- and microelement and mineralogical composition, carbon and nitrogen content. In particular, water filaments and vermiculations had circumneutral and slightly acidic pH, respectively, both had abundant organic carbon and high microbial diversity. They were rich in macro- and microelements, deriving from mineral dissolution, and, in the case of water filaments, from seawater composition. Vermiculations had different color, partly associated with their mineralogy, and unusual minerals probably due to trapping capacities. Moonmilk was composed of gypsum, poor in organic matter, had an extremely low pH (0-1) and low microbial diversity. Based on 16S rRNA gene analysis, the microbial composition of the biofilms/deposits included autotrophic taxa associated with sulfur and nitrogen cycles and biomineralization processes. In particular, water filaments communities were characterized by bacterial taxa involved in sulfur oxidation and reduction in aquatic, aphotic, microaerophilic/anoxic environments (Campylobacterales, Thiotrichales, Arenicellales, Desulfobacterales, Desulforomonadales) and in chemolithotrophy in marine habitats (Oceanospirillales, Chromatiales). Their biodiversity was linked to the morphology of the water filaments and their collection site. Microbial communities within vermiculations were partly related to their color and showed high abundance of unclassified Betaproteobacteria and sulfur-oxidizing Hydrogenophilales (including Sulfuriferula), and Acidiferrobacterales (including Sulfurifustis), sulfur-reducing Desulfurellales, and ammonia-oxidizing Planctomycetes and Nitrospirae. The microbial community associated with gypsum moonmilk showed the strong dominance (>60%) of the archaeal genus Thermoplasma and lower abundance of chemolithotrophic Acidithiobacillus, metal-oxidizing Metallibacterium, Sulfobacillus, and Acidibacillus. This study describes the geomicrobiology of water filaments, vermiculations and gypsum moonmilk from Fetida Cave, providing insights into the microbial taxa that characterize each morphology and contribute to biogeochemical cycles and speleogenesis of this peculiar seawater-influenced sulfuric acid cave.

Biological Contamination Prevention for Outer Solar System Moons of Astrobiological Interest: What Do We Need to Know?

To ensure that scientific investments in space exploration are not compromised by terrestrial contamination of celestial bodies, special care needs to be taken to preserve planetary conditions for future astrobiological exploration. Significant effort has been made and is being taken to address planetary protection in the context of inner Solar System exploration. In particular for missions to Mars, detailed internationally accepted guidelines have been established. For missions to the icy moons in the outer Solar System, Europa and Enceladus, the planetary protection requirements are so far based on a probabilistic approach and a conservative estimate of poorly known parameters. One objective of the European Commission-funded project, Planetary Protection of Outer Solar System, was to assess the existing planetary protection approach, to identify inherent knowledge gaps, and to recommend scientific investigations necessary to update the requirements for missions to the icy moons.

Limits of Life and the Habitability of Mars: The ESA Space Experiment BIOMEX on the ISS.

BIOMEX (BIOlogy and Mars EXperiment) is an ESA/Roscosmos space exposure experiment housed within the exposure facility EXPOSE-R2 outside the Zvezda module on the International Space Station (ISS). The design of the multiuser facility supports-among others-the BIOMEX investigations into the stability and level of degradation of space-exposed biosignatures such as pigments, secondary metabolites, and cell surfaces in contact with a terrestrial and Mars analog mineral environment. In parallel, analysis on the viability of the investigated organisms has provided relevant data for evaluation of the habitability of Mars, for the limits of life, and for the likelihood of an interplanetary transfer of life (theory of lithopanspermia). In this project, lichens, archaea, bacteria, cyanobacteria, snow/permafrost algae, meristematic black fungi, and bryophytes from alpine and polar habitats were embedded, grown, and cultured on a mixture of martian and lunar regolith analogs or other terrestrial minerals. The organisms and regolith analogs and terrestrial mineral mixtures were then exposed to space and to simulated Mars-like conditions by way of the EXPOSE-R2 facility. In this special issue, we present the first set of data obtained in reference to our investigation into the habitability of Mars and limits of life. This project was initiated and implemented by the BIOMEX group, an international and interdisciplinary consortium of 30 institutes in 12 countries on 3 continents. Preflight tests for sample selection, results from ground-based simulation experiments, and the space experiments themselves are presented and include a complete overview of the scientific processes required for this space experiment and postflight analysis. The presented BIOMEX concept could be scaled up to future exposure experiments on the Moon and will serve as a pretest in low Earth orbit.

Carotenoid analysis of halophilic archaea by resonance Raman spectroscopy.

Recently, halite and sulfate evaporate rocks have been discovered on Mars by the NASA rovers, Spirit and Opportunity. It is reasonable to propose that halophilic microorganisms could have potentially flourished in these settings. If so, biomolecules found in microorganisms adapted to high salinity and basic pH environments on Earth may be reliable biomarkers for detecting life on Mars. Therefore, we investigated the potential of Resonance Raman (RR) spectroscopy to detect biomarkers derived from microorganisms adapted to hypersaline environments. RR spectra were acquired using 488.0 and 514.5 nm excitation from a variety of halophilic archaea, including Halobacterium salinarum NRC-1, Halococcus morrhuae, and Natrinema pallidum. It was clearly demonstrated that RR spectra enhance the chromophore carotenoid molecules in the cell membrane with respect to the various protein and lipid cellular components. RR spectra acquired from all halophilic archaea investigated contained major features at approximately 1000, 1152, and 1505 cm(-1). The bands at 1505 cm(-1) and 1152 cm(-1) are due to in-phase C=C (nu(1) ) and C-C stretching ( nu(2) ) vibrations of the polyene chain in carotenoids. Additionally, in-plane rocking modes of CH(3) groups attached to the polyene chain coupled with C-C bonds occur in the 1000 cm(-1) region. We also investigated the RR spectral differences between bacterioruberin and bacteriorhodopsin as another potential biomarker for hypersaline environments. By comparison, the RR spectrum acquired from bacteriorhodopsin is much more complex and contains modes that can be divided into four groups: the C=C stretches (1600-1500 cm(-1)), the CCH in-plane rocks (1400-1250 cm(-1)), the C-C stretches (1250-1100 cm(-1)), and the hydrogen out-of-plane wags (1000-700 cm(-1)). RR spectroscopy was shown to be a useful tool for the analysis and remote in situ detection of carotenoids from halophilic archaea without the need for large sample sizes and complicated extractions, which are required by analytical techniques such as high performance liquid chromatography and mass spectrometry.

The Effects of HZE Particles, γ and X-ray Radiation on the Survival and Genetic Integrity of Halobacterium salinarum NRC-1, Halococcus hamelinensis, and Halococcus morrhuae.

Three halophilic archaea, Halobacterium salinarum NRC-1, Halococcus hamelinensis, and Halococcus morrhuae, have been exposed to different regimes of simulated outer space ionizing radiation. Strains were exposed to high-energy heavy ion (HZE) particles, namely iron and argon ions, as well as to γ radiation (60Co) and X-rays, and the survival and the genetic integrity of the 16S rRNA gene were evaluated. Exposure to 1 kGy of argon or iron ions at the Heavy Ion Medical Accelerator in Chiba (HIMAC) facility at the National Institute for Radiological Sciences (NIRS) in Japan did not lead to a detectable loss in viability; only after exposure to 2 kGy of iron ions a decline in survival was observed. Furthermore, a delay in growth was manifested following exposure to 2 kGy iron ions. DNA integrity of the 16S rRNA was not compromised up to 1 kGy, with the exception of Hcc. hamelinensis following exposure to argon particles. All three strains showed a high resistance toward X-rays (exposed at the DLR in Cologne, Germany), where Hcc. hamelinensis and Hcc. morrhuae displayed better survival compared to Hbt. salinarum NRC-1. In all three organisms the DNA damage increased in a dose-dependent manner. To determine a biological endpoint for survival following exposure to γ radiation, strains were exposed to up to 112 kGy at the Beta-Gamma-Service GmbH (BGS) in Germany. Although all strains were incubated for up to 4 months, only Hcc. hamelinensis and Hcc. morrhuae recovered from 6 kGy of γ radiation. In comparison, Hbt. salinarum NRC-1 did not recover. The 16S rRNA gene integrity stayed remarkably well preserved up to 48 kGy for both halococci. This research presents novel data on the survival and genetic stability of three halophilic archaea following exposure to simulated outer space radiation. Key Words: Halophilic archaea-Radiation-Survival. Astrobiology 17, 110-117.

STARLIFE-An International Campaign to Study the Role of Galactic Cosmic Radiation in Astrobiological Model Systems.

In-depth knowledge regarding the biological effects of the radiation field in space is required for assessing the radiation risks in space. To obtain this knowledge, a set of different astrobiological model systems has been studied within the STARLIFE radiation campaign during six irradiation campaigns (2013-2015). The STARLIFE group is an international consortium with the aim to investigate the responses of different astrobiological model systems to the different types of ionizing radiation (X-rays, γ rays, heavy ions) representing major parts of the galactic cosmic radiation spectrum. Low- and high-energy charged particle radiation experiments have been conducted at the Heavy Ion Medical Accelerator in Chiba (HIMAC) facility at the National Institute of Radiological Sciences (NIRS) in Chiba, Japan. X-rays or γ rays were used as reference radiation at the German Aerospace Center (DLR, Cologne, Germany) or Beta-Gamma-Service GmbH (BGS, Wiehl, Germany) to derive the biological efficiency of different radiation qualities. All samples were exposed under identical conditions to the same dose and qualities of ionizing radiation (i) allowing a direct comparison between the tested specimens and (ii) providing information on the impact of the space radiation environment on currently used astrobiological model organisms. Key Words: Space radiation environment-Sparsely ionizing radiation-Densely ionizing radiation-Heavy ions-Gamma radiation-Astrobiological model systems. Astrobiology 17, 101-109.

The influence of human exploration on the microbial community structure and ammonia oxidizing potential of the Su Bentu limestone cave in Sardinia, Italy.

The bacterial diversity in the Su Bentu Cave in Sardinia was investigated by means of 16S rRNA gene-based analysis. This 15 km long cave, carved in Jurassic limestone, hosts a variety of calcite speleothems, and a long succession of subterranean lakes with mixed granite and carbonate sands. The lower level is occasionally flooded by a rising groundwater level, but with only scarce input of organic remains (leaves and charcoal fragments). On the quiet cave pools there are visible calcite rafts, whereas walls are locally coated with manganese deposits. In the drier upper levels, where organic input is much more subdued, moonmilk-a hydrated calcium-magnesium carbonate speleothem-can be found. Relative humidity approaches 100% and the measured mean annual cave air temperature is 14.8°C. Samples were obtained in 2014 from calcite rafts, moonmilk, manganese oxide deposits and soil (limestone and granite grains). Microclimatic conditions in the cave near the sampling sites, sample properties, physico-chemical parameters of water, and sediment composition were determined. The microbial community of this system is predominately composed of the phyla Proteobacteria, Actinobacteria, Acidobacteria, Nitrospirae, and Firmicutes. Sampling sites near the entrance of the cave and in close proximity of the underground campsite-located 500 meters deep into the cave-revealed the highest diversity as well as the highest number of human associated microorganisms. Two samples obtained in very close proximity of each other near the campsite, indicate that the human impact is localized and is not distributed freely within the system. Analysis of the abundance of bacterial and archaeal amoA genes revealed a far greater abundance of archaeal amoA genes compared to bacterial representatives. The results of this study highlight that human impact is confined to locations that are utilized as campsites and that exploration leaves little microbial trails. Furthermore, we uncovered a highly specialized microbiome, which is perfectly adapted to survive and thrive in an environment with low nutrient availability.

On the Stability of Deinoxanthin Exposed to Mars Conditions during a Long-Term Space Mission and Implications for Biomarker Detection on Other Planets.

Outer space, the final frontier, is a hostile and unforgiving place for any form of life as we know it. The unique environment of space allows for a close simulation of Mars surface conditions that cannot be simulated as accurately on the Earth. For this experiment, we tested the resistance of Deinococcus radiodurans to survive exposure to simulated Mars-like conditions in low-Earth orbit for a prolonged period of time as part of the Biology and Mars experiment (BIOMEX) project. Special focus was placed on the integrity of the carotenoid deinoxanthin, which may serve as a potential biomarker to search for remnants of life on other planets. Survival was investigated by evaluating colony forming units, damage inflicted to the 16S rRNA gene by quantitative PCR, and the integrity and detectability of deinoxanthin by Raman spectroscopy. Exposure to space conditions had a strong detrimental effect on the survival of the strains and the 16S rRNA integrity, yet results show that deinoxanthin survives exposure to conditions as they prevail on Mars. Solar radiation is not only strongly detrimental to the survival and 16S rRNA integrity but also to the Raman signal of deinoxanthin. Samples not exposed to solar radiation showed only minuscule signs of deterioration. To test whether deinoxanthin is able to withstand the tested parameters without the protection of the cell, it was extracted from cell homogenate and exposed to high/low temperatures, vacuum, germicidal UV-C radiation, and simulated solar radiation. Results obtained by Raman investigations showed a strong resistance of deinoxanthin against outer space and Mars conditions, with the only exception of the exposure to simulated solar radiation. Therefore, deinoxanthin proved to be a suitable easily detectable biomarker for the search of Earth-like organic pigment-containing life on other planets.

AstRoMap European Astrobiology Roadmap.

The European AstRoMap project (supported by the European Commission Seventh Framework Programme) surveyed the state of the art of astrobiology in Europe and beyond and produced the first European roadmap for astrobiology research. In the context of this roadmap, astrobiology is understood as the study of the origin, evolution, and distribution of life in the context of cosmic evolution; this includes habitability in the Solar System and beyond. The AstRoMap Roadmap identifies five research topics, specifies several key scientific objectives for each topic, and suggests ways to achieve all the objectives. The five AstRoMap Research Topics are • Research Topic 1: Origin and Evolution of Planetary Systems • Research Topic 2: Origins of Organic Compounds in Space • Research Topic 3: Rock-Water-Carbon Interactions, Organic Synthesis on Earth, and Steps to Life • Research Topic 4: Life and Habitability • Research Topic 5: Biosignatures as Facilitating Life Detection It is strongly recommended that steps be taken towards the definition and implementation of a European Astrobiology Platform (or Institute) to streamline and optimize the scientific return by using a coordinated infrastructure and funding system.

On the response of halophilic archaea to space conditions.

Microorganisms are ubiquitous and can be found in almost every habitat and ecological niche on Earth. They thrive and survive in a broad spectrum of environments and adapt to rapidly changing external conditions. It is of great interest to investigate how microbes adapt to different extreme environments and with modern human space travel, we added a new extreme environment: outer space. Within the last 50 years, technology has provided tools for transporting microbial life beyond Earth's protective shield in order to study in situ responses to selected conditions of space. This review will focus on halophilic archaea, as, due to their ability to survive in extremes, they are often considered a model group of organisms to study responses to the harsh conditions associated with space. We discuss ground-based simulations, as well as space experiments, utilizing archaea, examining responses and/or resistance to the effects of microgravity and UV in particular. Several halophilic archaea (e.g., Halorubrum chaoviator) have been exposed to simulated and actual space conditions and their survival has been determined as well as the protective effects of halite shown. Finally, the intriguing potential of archaea to survive on other planets or embedded in a meteorite is postulated.

Analysis of DNA repair and protection in the Tardigrade Ramazzottius varieornatus and Hypsibius dujardini after exposure to UVC radiation.

Tardigrades inhabiting terrestrial environments exhibit extraordinary resistance to ionizing radiation and UV radiation although little is known about the mechanisms underlying the resistance. We found that the terrestrial tardigrade Ramazzottius varieornatus is able to tolerate massive doses of UVC irradiation by both being protected from forming UVC-induced thymine dimers in DNA in a desiccated, anhydrobiotic state as well as repairing the dimers that do form in the hydrated animals. In R. varieornatus accumulation of thymine dimers in DNA induced by irradiation with 2.5 kJ/m(2) of UVC radiation disappeared 18 h after the exposure when the animals were exposed to fluorescent light but not in the dark. Much higher UV radiation tolerance was observed in desiccated anhydrobiotic R. varieornatus compared to hydrated specimens of this species. On the other hand, the freshwater tardigrade species Hypsibius dujardini that was used as control, showed much weaker tolerance to UVC radiation than R. varieornatus, and it did not contain a putative phrA gene sequence. The anhydrobiotes of R. varieornatus accumulated much less UVC-induced thymine dimers in DNA than hydrated one. It suggests that anhydrobiosis efficiently avoids DNA damage accumulation in R. varieornatus and confers better UV radiation tolerance on this species. Thus we propose that UV radiation tolerance in tardigrades is due to the both high capacities of DNA damage repair and DNA protection, a two-pronged survival strategy.

Global protein-level responses of Halobacterium salinarum NRC-1 to prolonged changes in external sodium chloride concentrations.

Responses to changes in external salinity were examined in Halobacterium salinarum NRC-1. H. salinarum NRC-1 grows optimally at 4.3 M NaCl and is capable of growth between 2.6 and 5.1 M NaCl. Physiological changes following incubation at 2.6 M NaCl were investigated with respect to growth behavior and proteomic changes. Initial observations indicated delayed growth at low NaCl concentrations (2.6 M NaCl), and supplementation with different sugars, amino acids, or KCl to increase external osmotic pressure did not reverse these growth perturbations. To gain a more detailed insight into the adaptive responses of H. salinarum NRC-1 to changes in salinity, the proteome was characterized using iTRAQ (amine specific isobaric tagging reagents). Three hundred and nine differentially expressed proteins were shown to be associated with changes in the external sodium chloride concentration, with proteins associated with metabolism revealing the greatest response.

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 alpha- and gamma-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).

Halococcus hamelinensis sp. nov., a novel halophilic archaeon isolated from stromatolites in Shark Bay, Australia.

Several halophilic archaea belonging to the genus Halococcus were isolated from stromatolites from Hamelin Pool, Shark Bay, Western Australia, collected during field trips in 1996 and 2002. This is the first incidence of halophilic archaea being isolated from this environment. Stromatolites are biosedimentary structures that have been formed throughout the earth's evolutionary history and have been preserved in the geological record for over 3 billion years. The stromatolites from Hamelin Pool, Western Australia, are the only known example of extant stromatolites forming in hypersaline coastal environments. Based on their 16S rRNA gene sequences and morphology, the isolates belong to the genus Halococcus. Strain 100NA1, isolated from stromatolites collected in 2002, was closely related to strain 100A6(T) that was isolated from the stromatolites collected in 1996, with a DNA-DNA hybridization value of 94 +/- 8 %. DNA-DNA hybridization values of strain 100A6(T) with Halococcus morrhuae NRC 16008 and Halococcus saccharolyticus ATCC 49257(T) were 17 +/- 6 and 11 +/-7 %, respectively. The DNA G + C content of strain 100A6(T) was 60.5 mol% (T(m)). The main polar lipid was S-DGA-1, a sulphated glycolipid that has been detected in all strains of the genus Halococcus. Whole-cell protein profiles, enzyme composition and utilization of various carbon sources were distinct from those of all previously characterized Halococcus species. The recognition of this strain as representing a novel species within the genus Halococcus is justified, and the name Halococcus hamelinensis sp. nov. is proposed. The type strain is 100A6(T) (=JCM 12892(T) = ACM 5227(T)).