Novel endolithic bacteria of phylum Chloroflexota reveal a myriad of potential survival strategies in the Antarctic desert.

The ice-free McMurdo Dry Valleys of Antarctica are dominated by nutrient-poor mineral soil and rocky outcrops. The principal habitat for microorganisms is within rocks (endolithic). In this environment, microorganisms are provided with protection against sub-zero temperatures, rapid thermal fluctuations, extreme dryness, and ultraviolet and solar radiation. Endolithic communities include lichen, algae, fungi, and a diverse array of bacteria. Chloroflexota is among the most abundant bacterial phyla present in these communities. Among the Chloroflexota are four novel classes of bacteria, here named Candidatus Spiritibacteria class. nov. (=UBA5177), Candidatus Martimicrobia class. nov. (=UBA4733), Candidatus Tarhunnaeia class. nov. (=UBA6077), and Candidatus Uliximicrobia class. nov. (=UBA2235). We retrieved 17 high-quality metagenome-assembled genomes (MAGs) that represent these four classes. Based on genome predictions, all these bacteria are inferred to be aerobic heterotrophs that encode enzymes for the catabolism of diverse sugars. These and other organic substrates are likely derived from lichen, algae, and fungi, as metabolites (including photosynthate), cell wall components, and extracellular matrix components. The majority of MAGs encode the capacity for trace gas oxidation using high-affinity uptake hydrogenases, which could provide energy and metabolic water required for survival and persistence. Furthermore, some MAGs encode the capacity to couple the energy generated from H2 and CO oxidation to support carbon fixation (atmospheric chemosynthesis). All encode mechanisms for the detoxification and efflux of heavy metals. Certain MAGs encode features that indicate possible interactions with other organisms, such as Tc-type toxin complexes, hemolysins, and macroglobulins.IMPORTANCEThe ice-free McMurdo Dry Valleys of Antarctica are the coldest and most hyperarid desert on Earth. It is, therefore, the closest analog to the surface of the planet Mars. Bacteria and other microorganisms survive by inhabiting airspaces within rocks (endolithic). We identify four novel classes of phylum Chloroflexota, and, based on interrogation of 17 metagenome-assembled genomes, we predict specific metabolic and physiological adaptations that facilitate the survival of these bacteria in this harsh environment-including oxidation of trace gases and the utilization of nutrients (including sugars) derived from lichen, algae, and fungi. We propose that such adaptations allow these endolithic bacteria to eke out an existence in this cold and extremely dry habitat.

Population structure of an Antarctic aquatic cyanobacterium.

BACKGROUND: Ace Lake is a marine-derived, stratified lake in the Vestfold Hills of East Antarctica with an upper oxic and lower anoxic zone. Cyanobacteria are known to reside throughout the water column. A Synechococcus-like species becomes the most abundant member in the upper sunlit waters during summer while persisting annually even in the absence of sunlight and at depth in the anoxic zone. Here, we analysed ~ 300 Gb of Ace Lake metagenome data including 59 Synechococcus-like metagenome-assembled genomes (MAGs) to determine depth-related variation in cyanobacterial population structure. Metagenome data were also analysed to investigate viruses associated with this cyanobacterium and the host's capacity to defend against or evade viruses. RESULTS: A single Synechococcus-like species was found to exist in Ace Lake, Candidatus Regnicoccus frigidus sp. nov., consisting of one phylotype more abundant in the oxic zone and a second phylotype prevalent in the oxic-anoxic interface and surrounding depths. An important aspect of genomic variation pertained to nitrogen utilisation, with the capacity to perform cyanide assimilation and asparagine synthesis reflecting the depth distribution of available sources of nitrogen. Both specialist (host specific) and generalist (broad host range) viruses were identified with a predicted ability to infect Ca. Regnicoccus frigidus. Host-virus interactions were characterised by a depth-dependent distribution of virus type (e.g. highest abundance of specialist viruses in the oxic zone) and host phylotype capacity to defend against (e.g. restriction-modification, retron and BREX systems) and evade viruses (cell surface proteins and cell wall biosynthesis and modification enzymes). CONCLUSION: In Ace Lake, specific environmental factors such as the seasonal availability of sunlight affects microbial abundances and the associated processes that the microbial community performs. Here, we find that the population structure for Ca. Regnicoccus frigidus has evolved differently to the other dominant phototroph in the lake, Candidatus Chlorobium antarcticum. The geography (i.e. Antarctica), limnology (e.g. stratification) and abiotic (e.g. sunlight) and biotic (e.g. microbial interactions) factors determine the types of niches that develop in the lake. While the lake community has become increasingly well studied, metagenome-based studies are revealing that niche adaptation can take many paths; these paths need to be determined in order to make reasonable predictions about the consequences of future ecosystem perturbations. Video Abstract.

Into the darkness: the ecologies of novel 'microbial dark matter' phyla in an Antarctic lake.

Uncultivated microbial clades ('microbial dark matter') are inferred to play important but uncharacterized roles in nutrient cycling. Using Antarctic lake (Ace Lake, Vestfold Hills) metagenomes, 12 metagenome-assembled genomes (MAGs; 88%-100% complete) were generated for four 'dark matter' phyla: six MAGs from Candidatus Auribacterota (=Aureabacteria, SURF-CP-2), inferred to be hydrogen- and sulfide-producing fermentative heterotrophs, with individual MAGs encoding bacterial microcompartments (BMCs), gas vesicles, and type IV pili; one MAG (100% complete) from Candidatus Hinthialibacterota (=OLB16), inferred to be a facultative anaerobe capable of dissimilatory nitrate reduction to ammonia, specialized for mineralization of complex organic matter (e.g. sulfated polysaccharides), and encoding BMCs, flagella, and Tad pili; three MAGs from Candidatus Electryoneota (=AABM5-125-24), previously reported to include facultative anaerobes capable of dissimilatory sulfate reduction, and here inferred to perform sulfite oxidation, reverse tricarboxylic acid cycle for autotrophy, and possess numerous proteolytic enzymes; two MAGs from Candidatus Lernaellota (=FEN-1099), inferred to be capable of formate oxidation, amino acid fermentation, and possess numerous enzymes for protein and polysaccharide degradation. The presence of 16S rRNA gene sequences in public metagenome datasets (88%-100% identity) suggests these 'dark matter' phyla contribute to sulfur cycling, degradation of complex organic matter, ammonification and/or chemolithoautotrophic CO2 fixation in diverse global environments.

Remarkably coherent population structure for a dominant Antarctic Chlorobium species.

BACKGROUND: In Antarctica, summer sunlight enables phototrophic microorganisms to drive primary production, thereby "feeding" ecosystems to enable their persistence through the long, dark winter months. In Ace Lake, a stratified marine-derived system in the Vestfold Hills of East Antarctica, a Chlorobium species of green sulphur bacteria (GSB) is the dominant phototroph, although its seasonal abundance changes more than 100-fold. Here, we analysed 413 Gb of Antarctic metagenome data including 59 Chlorobium metagenome-assembled genomes (MAGs) from Ace Lake and nearby stratified marine basins to determine how genome variation and population structure across a 7-year period impacted ecosystem function. RESULTS: A single species, Candidatus Chlorobium antarcticum (most similar to Chlorobium phaeovibrioides DSM265) prevails in all three aquatic systems and harbours very little genomic variation (≥ 99% average nucleotide identity). A notable feature of variation that did exist related to the genomic capacity to biosynthesize cobalamin. The abundance of phylotypes with this capacity changed seasonally ~ 2-fold, consistent with the population balancing the value of a bolstered photosynthetic capacity in summer against an energetic cost in winter. The very high GSB concentration (> 108 cells ml-1 in Ace Lake) and seasonal cycle of cell lysis likely make Ca. Chlorobium antarcticum a major provider of cobalamin to the food web. Analysis of Ca. Chlorobium antarcticum viruses revealed the species to be infected by generalist (rather than specialist) viruses with a broad host range (e.g., infecting Gammaproteobacteria) that were present in diverse Antarctic lakes. The marked seasonal decrease in Ca. Chlorobium antarcticum abundance may restrict specialist viruses from establishing effective lifecycles, whereas generalist viruses may augment their proliferation using other hosts. CONCLUSION: The factors shaping Antarctic microbial communities are gradually being defined. In addition to the cold, the annual variation in sunlight hours dictates which phototrophic species can grow and the extent to which they contribute to ecosystem processes. The Chlorobium population studied was inferred to provide cobalamin, in addition to carbon, nitrogen, hydrogen, and sulphur cycling, as critical ecosystem services. The specific Antarctic environmental factors and major ecosystem benefits afforded by this GSB likely explain why such a coherent population structure has developed in this Chlorobium species. Video abstract.

Shedding Light on Microbial "Dark Matter": Insights Into Novel Cloacimonadota and Omnitrophota From an Antarctic Lake.

The potential metabolism and ecological roles of many microbial taxa remain unknown because insufficient genomic data are available to assess their functional potential. Two such microbial "dark matter" taxa are the Candidatus bacterial phyla Cloacimonadota and Omnitrophota, both of which have been identified in global anoxic environments, including (but not limited to) organic-carbon-rich lakes. Using 24 metagenome-assembled genomes (MAGs) obtained from an Antarctic lake (Ace Lake, Vestfold Hills), novel lineages and novel metabolic traits were identified for both phyla. The Cloacimonadota MAGs exhibited a capacity for carbon fixation using the reverse tricarboxylic acid cycle driven by oxidation of hydrogen and sulfur. Certain Cloacimonadota MAGs encoded proteins that possess dockerin and cohesin domains, which is consistent with the assembly of extracellular cellulosome-like structures that are used for degradation of polypeptides and polysaccharides. The Omnitrophota MAGs represented phylogenetically diverse taxa that were predicted to possess a strong biosynthetic capacity for amino acids, nucleosides, fatty acids, and essential cofactors. All of the Omnitrophota were inferred to be obligate fermentative heterotrophs that utilize a relatively narrow range of organic compounds, have an incomplete tricarboxylic acid cycle, and possess a single hydrogenase gene important for achieving redox balance in the cell. We reason that both Cloacimonadota and Omnitrophota form metabolic interactions with hydrogen-consuming partners (methanogens and Desulfobacterota, respectively) and, therefore, occupy specific niches in Ace Lake.

Linking genomic and physiological characteristics of psychrophilic Arthrobacter to metagenomic data to explain global environmental distribution.

BACKGROUND: Microorganisms drive critical global biogeochemical cycles and dominate the biomass in Earth's expansive cold biosphere. Determining the genomic traits that enable psychrophiles to grow in cold environments informs about their physiology and adaptive responses. However, defining important genomic traits of psychrophiles has proven difficult, with the ability to extrapolate genomic knowledge to environmental relevance proving even more difficult. RESULTS: Here we examined the bacterial genus Arthrobacter and, assisted by genome sequences of new Tibetan Plateau isolates, defined a new clade, Group C, that represents isolates from polar and alpine environments. Group C had a superior ability to grow at -1°C and possessed genome G+C content, amino acid composition, predicted protein stability, and functional capacities (e.g., sulfur metabolism and mycothiol biosynthesis) that distinguished it from non-polar or alpine Group A Arthrobacter. Interrogation of nearly 1000 metagenomes identified an over-representation of Group C in Canadian permafrost communities from a simulated spring-thaw experiment, indicative of niche adaptation, and an under-representation of Group A in all polar and alpine samples, indicative of a general response to environmental temperature. CONCLUSION: The findings illustrate a capacity to define genomic markers of specific taxa that potentially have value for environmental monitoring of cold environments, including environmental change arising from anthropogenic impact. More broadly, the study illustrates the challenges involved in extrapolating from genomic and physiological data to an environmental setting. Video Abstract.

Genome Analysis of a Verrucomicrobial Endosymbiont With a Tiny Genome Discovered in an Antarctic Lake.

Organic Lake in Antarctica is a marine-derived, cold (-13∘C), stratified (oxic-anoxic), hypersaline (>200 gl-1) system with unusual chemistry (very high levels of dimethylsulfide) that supports the growth of phylogenetically and metabolically diverse microorganisms. Symbionts are not well characterized in Antarctica. However, unicellular eukaryotes are often present in Antarctic lakes and theoretically could harbor endosymbionts. Here, we describe Candidatus Organicella extenuata, a member of the Verrucomicrobia with a highly reduced genome, recovered as a metagenome-assembled genome with genetic code 4 (UGA-to-Trp recoding) from Organic Lake. It is closely related to Candidatus Pinguicocccus supinus (163,218 bp, 205 genes), a newly described cytoplasmic endosymbiont of the freshwater ciliate Euplotes vanleeuwenhoeki (Serra et al., 2020). At 158,228 bp (encoding 194 genes), the genome of Ca. Organicella extenuata is among the smallest known bacterial genomes and similar to the genome of Ca. Pinguicoccus supinus (163,218 bp, 205 genes). Ca. Organicella extenuata retains a capacity for replication, transcription, translation, and protein-folding while lacking any capacity for the biosynthesis of amino acids or vitamins. Notably, the endosymbiont retains a capacity for fatty acid synthesis (type II) and iron-sulfur (Fe-S) cluster assembly. Metagenomic analysis of 150 new metagenomes from Organic Lake and more than 70 other Antarctic aquatic locations revealed a strong correlation in abundance between Ca. Organicella extenuata and a novel ciliate of the genus Euplotes. Like Ca. Pinguicoccus supinus, we infer that Ca. Organicella extenuata is an endosymbiont of Euplotes and hypothesize that both Ca. Organicella extenuata and Ca. Pinguicocccus supinus provide fatty acids and Fe-S clusters to their Euplotes host as the foundation of a mutualistic symbiosis. The discovery of Ca. Organicella extenuata as possessing genetic code 4 illustrates that in addition to identifying endosymbionts by sequencing known symbiotic communities and searching metagenome data using reference endosymbiont genomes, the potential exists to identify novel endosymbionts by searching for unusual coding parameters.

Ecology and molecular targets of hypermutation in the global microbiome.

Changes in the sequence of an organism's genome, i.e., mutations, are the raw material of evolution. The frequency and location of mutations can be constrained by specific molecular mechanisms, such as diversity-generating retroelements (DGRs). DGRs have been characterized from cultivated bacteria and bacteriophages, and perform error-prone reverse transcription leading to mutations being introduced in specific target genes. DGR loci were also identified in several metagenomes, but the ecological roles and evolutionary drivers of these DGRs remain poorly understood. Here, we analyze a dataset of >30,000 DGRs from public metagenomes, establish six major lineages of DGRs including three primarily encoded by phages and seemingly used to diversify host attachment proteins, and demonstrate that DGRs are broadly active and responsible for >10% of all amino acid changes in some organisms. Overall, these results highlight the constraints under which DGRs evolve, and elucidate several distinct roles these elements play in natural communities.

Influence of the polar light cycle on seasonal dynamics of an Antarctic lake microbial community.

BACKGROUND: Cold environments dominate the Earth's biosphere and microbial activity drives ecosystem processes thereby contributing greatly to global biogeochemical cycles. Polar environments differ to all other cold environments by experiencing 24-h sunlight in summer and no sunlight in winter. The Vestfold Hills in East Antarctica contains hundreds of lakes that have evolved from a marine origin only 3000-7000 years ago. Ace Lake is a meromictic (stratified) lake from this region that has been intensively studied since the 1970s. Here, a total of 120 metagenomes representing a seasonal cycle and four summers spanning a 10-year period were analyzed to determine the effects of the polar light cycle on microbial-driven nutrient cycles. RESULTS: The lake system is characterized by complex sulfur and hydrogen cycling, especially in the anoxic layers, with multiple mechanisms for the breakdown of biopolymers present throughout the water column. The two most abundant taxa are phototrophs (green sulfur bacteria and cyanobacteria) that are highly influenced by the seasonal availability of sunlight. The extent of the Chlorobium biomass thriving at the interface in summer was captured in underwater video footage. The Chlorobium abundance dropped from up to 83% in summer to 6% in winter and 1% in spring, before rebounding to high levels. Predicted Chlorobium viruses and cyanophage were also abundant, but their levels did not negatively correlate with their hosts. CONCLUSION: Over-wintering expeditions in Antarctica are logistically challenging, meaning insight into winter processes has been inferred from limited data. Here, we found that in contrast to chemolithoautotrophic carbon fixation potential of Southern Ocean Thaumarchaeota, this marine-derived lake evolved a reliance on photosynthesis. While viruses associated with phototrophs also have high seasonal abundance, the negative impact of viral infection on host growth appeared to be limited. The microbial community as a whole appears to have developed a capacity to generate biomass and remineralize nutrients, sufficient to sustain itself between two rounds of sunlight-driven summer-activity. In addition, this unique metagenome dataset provides considerable opportunity for future interrogation of eukaryotes and their viruses, abundant uncharacterized taxa (i.e. dark matter), and for testing hypotheses about endemic species in polar aquatic ecosystems. Video Abstract.

Unexpected host dependency of Antarctic Nanohaloarchaeota.

In hypersaline environments, Nanohaloarchaeota (Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, Nanohaloarchaeota [DPANN] superphylum) are thought to be free-living microorganisms. We report cultivation of 2 strains of Antarctic Nanohaloarchaeota and show that they require the haloarchaeon Halorubrum lacusprofundi for growth. By performing growth using enrichments and fluorescence-activated cell sorting, we demonstrated successful cultivation of Candidatus Nanohaloarchaeum antarcticus, purification of Ca. Nha. antarcticus away from other species, and growth and verification of Ca. Nha. antarcticus with Hrr. lacusprofundi; these findings are analogous to those required for fulfilling Koch's postulates. We use fluorescent in situ hybridization and transmission electron microscopy to assess cell structures and interactions; metagenomics to characterize enrichment taxa, generate metagenome assembled genomes, and interrogate Antarctic communities; and proteomics to assess metabolic pathways and speculate about the roles of certain proteins. Metagenome analysis indicates the presence of a single species, which is endemic to Antarctic hypersaline systems that support the growth of haloarchaea. The presence of unusually large proteins predicted to function in attachment and invasion of hosts plus the absence of key biosynthetic pathways (e.g., lipids) in metagenome assembled genomes of globally distributed Nanohaloarchaeota indicate that all members of the lineage have evolved as symbionts. Our work expands the range of archaeal symbiotic lifestyles and provides a genetically tractable model system for advancing understanding of the factors controlling microbial symbiotic relationships.

Sucrose Metabolism in Haloarchaea: Reassessment Using Genomics, Proteomics, and Metagenomics.

The canonical pathway for sucrose metabolism in haloarchaea utilizes a modified Embden-Meyerhof-Parnas pathway (EMP), in which ketohexokinase and 1-phosphofructokinase phosphorylate fructose released from sucrose hydrolysis. However, our survey of haloarchaeal genomes determined that ketohexokinase and 1-phosphofructokinase genes were not present in all species known to utilize fructose and sucrose, thereby indicating that alternative mechanisms exist for fructose metabolism. A fructokinase gene was identified in the majority of fructose- and sucrose-utilizing species, whereas only a small number possessed a ketohexokinase gene. Analysis of a range of hypersaline metagenomes revealed that haloarchaeal fructokinase genes were far more abundant (37 times) than haloarchaeal ketohexokinase genes. We used proteomic analysis of Halohasta litchfieldiae (which encodes fructokinase) and identified changes in protein abundance that relate to growth on sucrose. Proteins inferred to be involved in sucrose metabolism included fructokinase, a carbohydrate primary transporter, a putative sucrose hydrolase, and two uncharacterized carbohydrate-related proteins encoded in the same gene cluster as fructokinase and the transporter. Homologs of these proteins were present in the genomes of all haloarchaea that use sugars for growth. Enzymes involved in the semiphosphorylative Entner-Doudoroff pathway also had higher abundances in sucrose-grown H. litchfieldiae cells, consistent with this pathway functioning in the catabolism of the glucose moiety of sucrose. The study revises the current understanding of fundamental pathways for sugar utilization in haloarchaea and proposes alternatives to the modified EMP pathway used by haloarchaea for sucrose and fructose utilization.IMPORTANCE Our ability to infer the function that microorganisms perform in the environment is predicated on assumptions about metabolic capacity. When genomic or metagenomic data are used, metabolic capacity is inferred from genetic potential. Here, we investigate the pathways by which haloarchaea utilize sucrose. The canonical haloarchaeal pathway for fructose metabolism involving ketohexokinase occurs only in a small proportion of haloarchaeal genomes and is underrepresented in metagenomes. Instead, fructokinase genes are present in the majority of genomes/metagenomes. In addition to genomic and metagenomic analyses, we used proteomic analysis of Halohasta litchfieldiae (which encodes fructokinase but lacks ketohexokinase) and identified changes in protein abundance that related to growth on sucrose. In this way, we identified novel proteins implicated in sucrose metabolism in haloarchaea, comprising a transporter and various catabolic enzymes (including proteins that are annotated as hypothetical).

Genomic variation and biogeography of Antarctic haloarchaea.

BACKGROUND: The genomes of halophilic archaea (haloarchaea) often comprise multiple replicons. Genomic variation in haloarchaea has been linked to viral infection pressure and, in the case of Antarctic communities, can be caused by intergenera gene exchange. To expand understanding of genome variation and biogeography of Antarctic haloarchaea, here we assessed genomic variation between two strains of Halorubrum lacusprofundi that were isolated from Antarctic hypersaline lakes from different regions (Vestfold Hills and Rauer Islands). To assess variation in haloarchaeal populations, including the presence of genomic islands, metagenomes from six hypersaline Antarctic lakes were characterised. RESULTS: The sequence of the largest replicon of each Hrr. lacusprofundi strain (primary replicon) was highly conserved, while each of the strains' two smaller replicons (secondary replicons) were highly variable. Intergenera gene exchange was identified, including the sharing of a type I-B CRISPR system. Evaluation of infectivity of an Antarctic halovirus provided experimental evidence for the differential susceptibility of the strains, bolstering inferences that strain variation is important for modulating interactions with viruses. A relationship was found between genomic structuring and the location of variation within replicons and genomic islands, demonstrating that the way in which haloarchaea accommodate genomic variability relates to replicon structuring. Metagenome read and contig mapping and clustering and scaling analyses demonstrated biogeographical patterning of variation consistent with environment and distance effects. The metagenome data also demonstrated that specific haloarchaeal species dominated the hypersaline systems indicating they are endemic to Antarctica. CONCLUSION: The study describes how genomic variation manifests in Antarctic-lake haloarchaeal communities and provides the basis for future assessments of Antarctic regional and global biogeography of haloarchaea.

Microbial communities of aquatic environments on Heard Island characterized by pyrotag sequencing and environmental data.

Heard Island in the Southern Ocean is a biological hotspot that is suffering the effects of climate change. Significant glacier retreat has generated proglacial lagoons, some of which are open to the ocean. We used pyrotag sequencing of SSU rRNA genes and environmental data to characterize microorganisms from two pools adjacent to animal breeding areas, two glacial lagoons and Atlas Cove (marine site). The more abundant taxa included Actinobacteria, Bacteroidetes and Proteobacteria, ciliates and picoflagellates (e.g. Micromonas), and relatively few Archaea. Seal Pool, which is rich in organic matter, was characterized by a heterotrophic degradative community, while the less eutrophic Atlas Pool had more eucaryotic primary producers. Brown Lagoon, with the lowest nutrient levels, had Eucarya and Bacteria predicted to be oligotrophs, possess small cell sizes, and have the ability to metabolize organic matter. The marine influence on Winston Lagoon was evident by its salinity and the abundance of marine-like Gammaproteobacteria, while also lacking typical marine eucaryotes indicating the system was still functioning as a distinct niche. This is the first microbiology study of Heard Island and revealed that communities are distinct at each location and heavily influenced by local environmental factors.

Ecophysiological Distinctions of Haloarchaea from a Hypersaline Antarctic Lake as Determined by Metaproteomics.

UNLABELLED: Deep Lake in the Vestfold Hills is hypersaline and the coldest system in Antarctica known to support microbial growth (temperatures as low as -20°C). It represents a strong experimental model because the lake supports a low-complexity community of haloarchaea, with the three most abundant species totaling ∼72%. Moreover, the dominant haloarchaea are cultivatable, and their genomes are sequenced. Here we use metaproteomics linked to metagenome data and the genome sequences of the isolates to characterize the main pathways, trophic strategies, and interactions associated with resource utilization. The dominance of the most abundant member, Halohasta litchfieldiae, appears to be predicated on competitive utilization of substrates (e.g., starch, glycerol, and dihydroxyacetone) produced by Dunaliella, the lake's primary producer, while also possessing diverse mechanisms for acquiring nitrogen and phosphorus. The second most abundant member, strain DL31, is proficient in degrading complex proteinaceous matter. Hht. litchfieldiae and DL31 are inferred to release labile substrates that are utilized by Halorubrum lacusprofundi, the third most abundant haloarchaeon in Deep Lake. The study also linked genome variation to specific protein variants or distinct genetic capacities, thereby identifying strain-level variation indicative of specialization. Overall, metaproteomics revealed that rather than functional differences occurring at different lake depths or through size partitioning, the main lake genera possess major trophic distinctions, and phylotypes (e.g., strains of Hht. litchfieldiae) exhibit a more subtle level of specialization. This study highlights the extent to which the lake supports a relatively uniform distribution of taxa that collectively possess the genetic capacity to effectively exploit available nutrients throughout the lake. IMPORTANCE: Life on Earth has evolved to colonize a broad range of temperatures, but most of the biosphere (∼85%) exists at low temperatures (≤5°C). By performing unique roles in biogeochemical cycles, environmental microorganisms perform functions that are critical for the rest of life on Earth to survive. Cold environments therefore make a particularly important contribution to maintaining healthy, stable ecosystems. Here we describe the main physiological traits of the dominant microorganisms that inhabit Deep Lake in Antarctica, the coldest aquatic environment known to support life. The hypersaline system enables the growth of halophilic members of the Archaea: haloarchaea. By analyzing proteins of samples collected from the water column, we determined the functions that the haloarchaea were likely to perform. This study showed that the dominant haloarchaea possessed distinct lifestyles yet formed a uniform community throughout the lake that was collectively adept at using available light energy and diverse organic substrates for growth.

Antarctic archaea-virus interactions: metaproteome-led analysis of invasion, evasion and adaptation.

Despite knowledge that viruses are abundant in natural ecosystems, there is limited understanding of which viruses infect which hosts, and how both hosts and viruses respond to those interactions-interactions that ultimately shape community structure and dynamics. In Deep Lake, Antarctica, intergenera gene exchange occurs rampantly within the low complexity, haloarchaea-dominated community, strongly balanced by distinctions in niche adaptation which maintain sympatric speciation. By performing metaproteomics for the first time on haloarchaea, genomic variation of S-layer, archaella and other cell surface proteins was linked to mechanisms of infection evasion. CRISPR defense systems were found to be active, with haloarchaea responding to at least eight distinct types of viruses, including those infecting between genera. The role of BREX systems in defending against viruses was also examined. Although evasion and defense were evident, both hosts and viruses also may benefit from viruses carrying and expressing host genes, thereby potentially enhancing genetic variation and phenotypic differences within populations. The data point to a complex inter-play leading to a dynamic optimization of host-virus interactions. This comprehensive overview was achieved only through the integration of results from metaproteomics, genomics and metagenomics.

Microbial ecology of an Antarctic hypersaline lake: genomic assessment of ecophysiology among dominant haloarchaea.

Deep Lake in Antarctica is a cold, hypersaline system where four types of haloarchaea representing distinct genera comprise >70% of the lake community: strain tADL ∼44%, strain DL31 ∼18%, Halorubrum lacusprofundi ∼10% and strain DL1 ∼0.3%. By performing comparative genomics, growth substrate assays, and analyses of distribution by lake depth, size partitioning and lake nutrient composition, we were able to infer important metabolic traits and ecophysiological characteristics of the four Antarctic haloarchaea that contribute to their hierarchical persistence and coexistence in Deep Lake. tADL is characterized by a capacity for motility via flagella (archaella) and gas vesicles, a highly saccharolytic metabolism, a preference for glycerol, and photoheterotrophic growth. In contrast, DL31 has a metabolism specialized in processing proteins and peptides, and appears to prefer an association with particulate organic matter, while lacking the genomic potential for motility. H. lacusprofundi is the least specialized, displaying a genomic potential for the utilization of diverse organic substrates. The least abundant species, DL1, is characterized by a preference for catabolism of amino acids, and is the only one species that lacks genes needed for glycerol degradation. Despite the four haloarchaea being distributed throughout the water column, our analyses describe a range of distinctive features, including preferences for substrates that are indicative of ecological niche partitioning. The individual characteristics could be responsible for shaping the composition of the haloarchaeal community throughout the lake by enabling selection of ecotypes and maintaining sympatric speciation.

High level of intergenera gene exchange shapes the evolution of haloarchaea in an isolated Antarctic lake.

Deep Lake in Antarctica is a globally isolated, hypersaline system that remains liquid at temperatures down to -20 °C. By analyzing metagenome data and genomes of four isolates we assessed genome variation and patterns of gene exchange to learn how the lake community evolved. The lake is completely and uniformly dominated by haloarchaea, comprising a hierarchically structured, low-complexity community that differs greatly to temperate and tropical hypersaline environments. The four Deep Lake isolates represent distinct genera (∼85% 16S rRNA gene similarity and ∼73% genome average nucleotide identity) with genomic characteristics indicative of niche adaptation, and collectively account for ∼72% of the cellular community. Network analysis revealed a remarkable level of intergenera gene exchange, including the sharing of long contiguous regions (up to 35 kb) of high identity (∼100%). Although the genomes of closely related Halobacterium, Haloquadratum, and Haloarcula (>90% average nucleotide identity) shared regions of high identity between species or strains, the four Deep Lake isolates were the only distantly related haloarchaea to share long high-identity regions. Moreover, the Deep Lake high-identity regions did not match to any other hypersaline environment metagenome data. The most abundant species, tADL, appears to play a central role in the exchange of insertion sequences, but not the exchange of high-identity regions. The genomic characteristics of the four haloarchaea are consistent with a lake ecosystem that sustains a high level of intergenera gene exchange while selecting for ecotypes that maintain sympatric speciation. The peculiarities of this polar system restrict which species can grow and provide a tempo and mode for accentuating gene exchange.

Key microbial drivers in Antarctic aquatic environments.

Antarctica is arguably the world's most important continent for influencing the Earth's climate and ocean ecosystem function. The unique physico-chemical properties of the Southern Ocean enable high levels of microbial primary production to occur. This not only forms the base of a significant fraction of the global oceanic food web, but leads to the sequestration of anthropogenic CO2 and its transport to marine sediments, thereby removing it from the atmosphere; the Southern Ocean accounts for ~ 30% of global ocean uptake of CO2 despite representing ~ 10% of the total surface area of the global ocean. The Antarctic continent itself harbors some liquid water, including a remarkably diverse range of surface and subglacial lakes. Being one of the remaining natural frontiers, Antarctica delivers the paradox of needing to be protected from disturbance while requiring scientific endeavor to discover what is indigenous and learn how best to protect it. Moreover, like many natural environments on Earth, in Antarctica, microorganisms dominate the genetic pool and biomass of the colonizable niches and play the key roles in maintaining proper ecosystem function. This review puts into perspective insight that has been and can be gained about Antarctica's aquatic microbiota using molecular biology, and in particular, metagenomic approaches.

The genome sequence of the psychrophilic archaeon, Methanococcoides burtonii: the role of genome evolution in cold adaptation.

Psychrophilic archaea are abundant and perform critical roles throughout the Earth's expansive cold biosphere. Here we report the first complete genome sequence for a psychrophilic methanogenic archaeon, Methanococcoides burtonii. The genome sequence was manually annotated including the use of a five-tiered evidence rating (ER) system that ranked annotations from ER1 (gene product experimentally characterized from the parent organism) to ER5 (hypothetical gene product) to provide a rapid means of assessing the certainty of gene function predictions. The genome is characterized by a higher level of aberrant sequence composition (51%) than any other archaeon. In comparison to hyper/thermophilic archaea, which are subject to selection of synonymous codon usage, M. burtonii has evolved cold adaptation through a genomic capacity to accommodate highly skewed amino-acid content, while retaining codon usage in common with its mesophilic Methanosarcina cousins. Polysaccharide biosynthesis genes comprise at least 3.3% of protein coding genes in the genome, and Cell wall, membrane, envelope biogenesis COG genes are overrepresented. Likewise, signal transduction (COG category T) genes are overrepresented and M. burtonii has a high 'IQ' (a measure of adaptive potential) compared to many methanogens. Numerous genes in these two overrepresented COG categories appear to have been acquired from epsilon- and delta-Proteobacteria, as do specific genes involved in central metabolism such as a novel B form of aconitase. Transposases also distinguish M. burtonii from other archaea, and their genomic characteristics indicate they have an important role in evolving the M. burtonii genome. Our study reveals a capacity for this model psychrophile to evolve through genome plasticity (including nucleotide skew, horizontal gene transfer and transposase activity) that enables adaptation to the cold, and to the biological and physical changes that have occurred over the last several thousand years as it adapted from a marine to an Antarctic lake environment.

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.