DNA repair enzymes of the Antarctic Dry Valley metagenome.

Microbiota inhabiting the Dry Valleys of Antarctica are subjected to multiple stressors that can damage deoxyribonucleic acid (DNA) such as desiccation, high ultraviolet light (UV) and multiple freeze-thaw cycles. To identify novel or highly-divergent DNA-processing enzymes that may enable effective DNA repair, we have sequenced metagenomes from 30 sample-sites which are part of the most extensive Antarctic biodiversity survey undertaken to date. We then used these to construct wide-ranging sequence similarity networks from protein-coding sequences and identified candidate genes involved in specialized repair processes including unique nucleases as well as a diverse range of adenosine triphosphate (ATP) -dependent DNA ligases implicated in stationary-phase DNA repair processes. In one of the first direct investigations of enzyme function from these unique samples, we have heterologously expressed and assayed a number of these enzymes, providing insight into the mechanisms that may enable resident microbes to survive these threats to their genomic integrity.

SSU-rRNA Gene Sequencing Survey of Benthic Microbial Eukaryotes from Guaymas Basin Hydrothermal Vent.

Microbial eukaryotes have important roles in marine food webs, but their diversity and activities in hydrothermal vent ecosystems are poorly characterized. In this study, we analyzed microbial eukaryotic communities associated with bacterial (Beggiatoa) mats in the 2,000 m deep-sea Guaymas Basin hydrothermal vent system using 18S rRNA gene high-throughput sequencing of the V4 region. We detected 6,954 distinct Operational Taxonomic Units (OTUs) across various mat systems. Of the sequences that aligned with known protistan phylotypes, most were affiliated with alveolates (especially dinoflagellates and ciliates) and cercozoans. OTU richness and community structure differed among sediment habitats (e.g. different mat types and cold sediments away from mats). Additionally, full-length 18S rRNA genes amplified and cloned from single cells revealed the identities of some of the most commonly encountered, active ciliates in this hydrothermal vent ecosystem. Observations and experiments were also conducted to demonstrate that ciliates were trophically active and ingesting fluorescent bacteria or Beggiatoa trichomes. Our work suggests that the active and diverse protistan community at the Guaymas Basin hydrothermal vent ecosystem likely consumes substantial amounts of bacterial biomass, and that the different habitats, often defined by distances of just a few 10s of cm, select for particular assemblages and levels of diversity.

Bacterial bioclusters relate to hydrochemistry in New Zealand groundwater.

Groundwater is a major source of New Zealand's water supply and supports base flows in rivers. Microbial communities in groundwater ecosystems mediate biogeochemical processes, and it is therefore crucial to understand microbial diversity in these ecosystems. We analysed bacterial assemblages from 35 New Zealand groundwater monitoring sites with varying hydrogeochemical conditions across the country. Proteobacteria was the most abundant phylum, and Variovorax represented the most common taxon. Pseudomonas, Burkholderia, Acidovorax, Janthinobacterium, Polaromonas and Caulobacter were the other common taxa. There was no Operational Taxonomic Unit (OTU) that was found in every one of the 35 samples. Here, we introduce a framework that has potential utility for groundwater ecosystem management, where the samples with similar microbial communities are grouped together into 'bioclusters'. Metabolic inferences derived from the taxonomic data were used to predict the oxygen requirements, metabolic potential and bacterial energy sources of each biocluster. Groundwater chemistry explains 59% of the variation in the relative abundance of all OTUs, with NO3-N, pH, DO, NH4-N, Fe, Br and SO4 displaying the strongest relationships to bioclusters. We propose that the biocluster framework, coupled with metabolic inferences derived from the taxonomic data, may have application outside New Zealand for on-going monitoring of the health of groundwater ecosystems.

Characterization of bacterial communities in lithobionts and soil niches from Victoria Valley, Antarctica.

Here we provide the first exploration of microbial diversity from three distinct Victoria Valley edaphic habitats, namely lithobionts (hypoliths, endoliths) and surface soils. Using a combination of terminal restriction fragment length polymorphism (T-RFLP) analysis and 16S rRNA gene amplicon pyrosequencing we assess community structure and diversity patterns, respectively. Our analysis revealed that habitat type (endolithic versus hypolithic versus surface soils) significantly influenced bacterial community composition, even though dominant phyla such as Actinobacteria (41% of total reads) were common to all samples. Consistent with previous surveys in other Dry Valley ecosystems, we found that lithobionts were colonized by a few highly dominant phylotypes (such asGemmatimonasandLeptolyngbya). Our analyses also show that soil bacteria were more diverse and evenly distributed than initially expected based on previous evidence. In contrast to total bacteria, the distribution of Cyanobacteria was not strongly influenced by habitat type, although soil- and endolith-specific cyanobacterial lineages were found. The detection of cyanobacterial lineages in these habitats appears to be influenced by the dispersal of aquatic inocula from lacustrine communities or benthic mats which are abundant in Victoria Valley. Together, our results provide insights into the phylogenetic variation and community structure across niche habitats in Victoria Valley.

Microbial community composition of transiently wetted Antarctic Dry Valley soils.

During the summer months, wet (hyporheic) soils associated with ephemeral streams and lake edges in the Antarctic Dry Valleys (DVs) become hotspots of biological activity and are hypothesized to be an important source of carbon and nitrogen for arid DV soils. Recent research in the DV has focused on the geochemistry and microbial ecology of lakes and arid soils, with substantially less information being available on hyporheic soils. Here, we determined the unique properties of hyporheic microbial communities, resolved their relationship to environmental parameters and compared them to archetypal arid DV soils. Generally, pH increased and chlorophyll a concentrations decreased along transects from wet to arid soils (9.0 to ~7.0 for pH and ~0.8 to ~5 µg/cm(3) for chlorophyll a, respectively). Soil water content decreased to below ~3% in the arid soils. Community fingerprinting-based principle component analyses revealed that bacterial communities formed distinct clusters specific to arid and wet soils; however, eukaryotic communities that clustered together did not have similar soil moisture content nor did they group together based on sampling location. Collectively, rRNA pyrosequencing indicated a considerably higher abundance of Cyanobacteria in wet soils and a higher abundance of Acidobacterial, Actinobacterial, Deinococcus/Thermus, Bacteroidetes, Firmicutes, Gemmatimonadetes, Nitrospira, and Planctomycetes in arid soils. The two most significant differences at the genus level were Gillisia signatures present in arid soils and chloroplast signatures related to Streptophyta that were common in wet soils. Fungal dominance was observed in arid soils and Viridiplantae were more common in wet soils. This research represents an in-depth characterization of microbial communities inhabiting wet DV soils. Results indicate that the repeated wetting of hyporheic zones has a profound impact on the bacterial and eukaryotic communities inhabiting in these areas.

Characterisation of bacterioplankton communities in the meltwater ponds of Bratina Island, Victoria Land, Antarctica.

A unique collection of Antarctic aquatic environments (meltwater ponds) lies in close proximity on the rock and sediment-covered undulating surface of the McMurdo Ice Shelf, near Bratina Island (Victoria Land, Antarctica). During the 2009-10 mid-austral summer, sets of discrete water samples were collected across the vertical geochemical gradients of five meltwater ponds (Egg, P70E, Legin, Salt and Orange) for geochemical and microbial community structure analysis. Bacterial DNA fingerprints (using Automated Ribosomal Intergenic Spacer Analysis) statistically clustered communities within ponds based on anosim (R = 0.766, P = 0.001); however, one highly stratified pond (Egg) had two distinct depth-related bacterial communities (R = 0.975, P = 0.008). 454 pyrosequencing at three depths within Egg also identified phylum level shifts and increased diversity with depth, Bacteroidetes being the dominant phyla in the surface sample and Proteobacteria being dominant in the bottom two depths. best analysis, which attempts to link community structure and the geochemistry of a pond, identified conductivity and pH individually, and to a lesser extent Ag(109) , NO2 and V(51) as dominant influences to the microbial community structure in these ponds. Increasing abundances of major halo-tolerant OTUs across the strong conductivity gradient reinforce it as the primary driver of community structure in this study.

Influence of soil properties on archaeal diversity and distribution in the McMurdo Dry Valleys, Antarctica.

Archaea are the least understood members of the microbial community in Antarctic mineral soils. Although their occurrence in Antarctic coastal soils has been previously documented, little is known about their distribution in soils across the McMurdo Dry Valleys, Victoria Land. In this study, terminal-restriction fragment length polymorphism (t-RFLP) analysis and 454 pyrosequencing were coupled with a detailed analysis of soil physicochemical properties to characterize archaeal diversity and identify environmental factors that might shape and maintain archaeal communities in soils of the three southern most McMurdo Dry Valleys (Garwood, Marshall, and Miers Valley). Archaea were successfully detected in all inland and coastal mineral soils tested, revealing a low overall richness (mean of six operational taxonomic units [OTUs] per sample site). However, OTU richness was higher in some soils and this higher richness was positively correlated with soil water content, indicating water as a main driver of archaeal community richness. In total, 18 archaeal OTUs were detected, predominately Thaumarchaeota affiliated with Marine Group 1.1b (> 80% of all archaeal sequences recovered). Less abundant OTUs (2% of all archaeal sequences) were loosely related to members of the phylum Euryarchaeota. This is the first comprehensive study showing a widespread presence and distribution of Archaea in inland Antarctic soils.

Niche-dependent genetic diversity in Antarctic metaviromes.

The metaviromes from 2 different Antarctic terrestrial soil niches have been analyzed. Both hypoliths (microbial assemblages beneath transluscent rocks) and surrounding open soils showed a high level diversity of tailed phages, viruses of algae and amoeba, and virophage sequences. Comparisons of other global metaviromes with the Antarctic libraries showed a niche-dependent clustering pattern, unrelated to the geographical origin of a given metavirome. Within the Antarctic open soil metavirome, a putative circularly permuted, ∼42kb dsDNA virus genome was annotated, showing features of a temperate phage possessing a variety of conserved protein domains with no significant taxonomic affiliations in current databases.

Airborne bacterial populations above desert soils of the McMurdo Dry Valleys, Antarctica.

Bacteria are assumed to disperse widely via aerosolized transport due to their small size and resilience. The question of microbial endemicity in isolated populations is directly related to the level of airborne exogenous inputs, yet this has proven hard to identify. The ice-free terrestrial ecosystem of Antarctica, a geographically and climatically isolated continent, was used to interrogate microbial bio-aerosols in relation to the surrounding ecology and climate. High-throughput sequencing of bacterial ribosomal RNA (rRNA) genes was combined with analyses of climate patterns during an austral summer. In general terms, the aerosols were dominated by Firmicutes, whereas surrounding soils supported Actinobacteria-dominated communities. The most abundant taxa were also common to aerosols from other continents, suggesting that a distinct bio-aerosol community is widely dispersed. No evidence for significant marine input to bioaerosols was found at this maritime valley site, instead local influence was largely from nearby volcanic sources. Back trajectory analysis revealed transport of incoming regional air masses across the Antarctic Plateau, and this is envisaged as a strong selective force. It is postulated that local soil microbial dispersal occurs largely via stochastic mobilization of mineral soil particulates.

Diversity and distributional patterns of ciliates in Guaymas Basin hydrothermal vent sediments.

Little is known about protists at deep-sea hydrothermal vents. The vent sites at Guaymas Basin in the Gulf of California are characterized by dense mats of filamentous pigmented or nonpigmented Beggiatoa that serve as markers of subsurface thermochemical gradients. We constructed 18S rRNA libraries to investigate ciliate assemblages in Beggiatoa mats and from bare sediments at the Guaymas vent site. Results indicated a high diversity of ciliates, with 156 operational taxonomic units identified in 548 sequences. Comparison between mat environments demonstrated that ciliate and bacterial assemblages from pigmented mats, nonpigmented mats, and bare sediments were significantly different and highly correlated with bacterial assemblages. Neither bacterial nor ciliate assemblages were correlated with environmental factors. The most abundant ciliates at Guaymas were more likely to be represented in clone libraries from other hydrothermal, deep-sea, and/or anoxic or microaerophilic environments, supporting the hypothesis that these ciliate species are broadly distributed. The orange mat environment included a higher proportion of ciliate sequences that were more similar to those from other environmental studies than to cultured ciliate species, whereas clone libraries from bare sediments included sequences that were the most highly divergent from all other sequences and may represent species that are endemic to Guaymas.

Evidence for successional development in Antarctic hypolithic bacterial communities.

Hypoliths (cryptic microbial assemblages that develop on the undersides of translucent rocks) are significant contributors to regional C and N budgets in both hot and cold deserts. Previous studies in the Dry Valleys of Eastern Antarctica have reported three morphologically distinct hypolithic community types: cyanobacteria dominated (type I), fungus dominated (type II) and moss dominated (type III). Here we present terminal-restriction fragment length polymorphism analyses to elucidate the bacterial community structure in hypolithons and the surrounding soils. We show clear and robust distinction in bacterial composition between bulk surface soils and hypolithons. Moreover, the bacterial assemblages were similar in types II and III hypolithons and clearly distinct from those found in type I. Through 16S rRNA gene 454 pyrosequencing, we show that Proteobacteria dominated all three types of hypolithic communities. As expected, Cyanobacteria were more abundant in type I hypolithons, whereas Actinobacteria were relatively more abundant in types II and III hypolithons, and were the dominant group in soils. Using a probabilistic dissimilarity metric and random sampling, we demonstrate that deterministic processes are more important in shaping the structure of the bacterial community found in types II and III hypolithons. Most notably, the data presented in this study suggest that hypolithic bacterial communities establish via a successional model, with the type I hypolithons acting as the basal development state.

Diverse and highly active diazotrophic assemblages inhabit ephemerally wetted soils of the Antarctic Dry Valleys.

Eolian transport of biomass from ephemerally wetted soils, associated with summer glacial meltwater runoffs and lake edges, to low-productivity areas of the Antarctic Dry Valleys (DV) has been postulated to be an important source of organic matter (fixed nitrogen and fixed carbon) to the entire DV ecosystem. However, descriptions and identification of the microbial members responsible for N(2) fixation within these wetted sites are limited. In this study, N(2) fixers from wetted soils were identified by direct nifH gene sequencing and their in situ N(2) fixation activities documented via acetylene reduction and RNA-based quantitative PCR assays. Shannon-index nifH diversity levels ranged between 1.8 and 2.6 and included the expected cyanobacterial signatures and a large number of phylotypes related to the gamma-, beta-, alpha-, and delta-proteobacteria. N(2) fixation rates ranged between approximately 0.5 and 6 nmol N cm(-3) h(-1) with measurements indicating that approximately 50% of this activity was linked with sulfate reduction at some sites. Comparisons with proximal dry soils also suggested that these communities are not ubiquitously distributed, and conditions unrelated to moisture content may define the composition, diversity, or habitat suitability of the microbial communities within wetted soils of the DVs.

EnGenIUS -- Environmental Genome Informational Utility System.

Short-insert shotgun sequencing approaches have been applied in recent years to environmental genomic libraries. In the case of complex multispecies microbial communities, there can be many sequence reads that are not incorporated into assemblies, and thus need to be annotated and accessible as single reads. Most existing annotation systems and genome databases accommodate assembled genomes containing contiguous gene-encoding sequences. Thus, a solution is required that can work effectively with environmental genomic annotation information to facilitate data analysis. The Environmental Genome Informational Utility System (EnGenIUS) is a comprehensive environmental genome (metagenome) research toolset that was specifically designed to accommodate the needs of large (> 250 K sequence reads) environmental genome sequencing efforts. The core EnGenIUS modules consist of a set of UNIX scripts and PHP programs used for data preprocessing, an annotation pipeline with accompanying analysis tools, two entity relational databases, and a graphical user interface. The annotation pipeline has a modular structure and can be customized to best fit input data set properties. The integrated entity relational databases store raw data and annotation analysis results. Access to the underlying databases and services is facilitated through a web-based graphical user interface. Users have the ability to browse, upload, download, and analyze preprocessed data, based on diverse search criteria. The EnGenIUS toolset was successfully tested using the Alvinella pompejana epibiont environmental genome data set, which comprises more than 300 K sequence reads. A fully browsable EnGenIUS portal is available at (http://ocean.dbi.udel.edu/) (access code: "guest"). The scope of this paper covers the implementation details and technical aspects of the EnGenIUS toolset.

Metagenome analysis of an extreme microbial symbiosis reveals eurythermal adaptation and metabolic flexibility.

Hydrothermal vent ecosystems support diverse life forms, many of which rely on symbiotic associations to perform functions integral to survival in these extreme physicochemical environments. Epsilonproteobacteria, found free-living and in intimate associations with vent invertebrates, are the predominant vent-associated microorganisms. The vent-associated polychaete worm, Alvinella pompejana, is host to a visibly dense fleece of episymbionts on its dorsal surface. The episymbionts are a multispecies consortium of Epsilonproteobacteria present as a biofilm. We unraveled details of these enigmatic, uncultivated episymbionts using environmental genome sequencing. They harbor wide-ranging adaptive traits that include high levels of strain variability analogous to Epsilonproteobacteria pathogens such as Helicobacter pylori, metabolic diversity of free-living bacteria, and numerous orthologs of proteins that we hypothesize are each optimally adapted to specific temperature ranges within the 10-65 degrees C fluctuations characteristic of the A. pompejana habitat. This strategic combination enables the consortium to thrive under diverse thermal and chemical regimes. The episymbionts are metabolically tuned for growth in hydrothermal vent ecosystems with genes encoding the complete rTCA cycle, sulfur oxidation, and denitrification; in addition, the episymbiont metagenome also encodes capacity for heterotrophic and aerobic metabolisms. Analysis of the environmental genome suggests that A. pompejana may benefit from the episymbionts serving as a stable source of food and vitamins. The success of Epsilonproteobacteria as episymbionts in hydrothermal vent ecosystems is a product of adaptive capabilities, broad metabolic capacity, strain variance, and virulent traits in common with pathogens.