Bacterial Community Dynamics in the Marine Sponge <Emphasis Type="Italic">Rhopaloeides odorabile</Emphasis> Under In Situ and Ex Situ Cultivation

Cultivation of sponges is being explored to supply biomaterial for the pharmaceutical and cosmetics industries. This study assesses the impact of various cultivation methods on the microbial community within the sponge Rhopaloeides odorabile during: (1) in situ cultivation under natural environmental conditions, (2) ex situ cultivation in small flow-through aquaria and (3) ex situ cultivation in large mesocosm systems. Principal components analysis of denaturing gradient gel electrophoresis profiles indicated a stable microbial community in sponges cultured in situ (grown in the wild) and in sponges cultured ex situ in small flow-through aquaria over 12 weeks. In contrast, a shift in the microbial community was detected in sponges cultivated ex situ in large mesocosm aquaria for 12 months. This shift included (1) a loss of some stable microbial inhabitants, including members of the Poribacteria, Chloroflexi and Acidobacteria and (2) the addition of new microbes not detected in the wild sponges. Many of these acquired bacteria had highest similarity to known sponge-associated microbes, indicating that the sponge may be capable of actively selecting its microbial community. Alternatively, long-term ex situ cultivation may cause a shift in the dominant microbes that facilitates the growth of the more rare species. The microbial community composition varied between sponges cultivated in mesocosm aquaria with different nutrient concentrations and seawater chemistry, suggesting that these variables play a role in structuring the sponge-associated microbes. The high growth and symbiont stability in R. odorabile cultured in situ confirm that this is the preferred method of aquaculture for this species at this time.     

Phylogenetic Analysis of a Microbialite-Forming Microbial Mat from a Hypersaline Lake of the Kiritimati Atoll,Central Pacific

On the Kiritimati atoll, several lakes exhibit microbial mat-formation under different hydrochemical conditions. Some of these lakes trigger microbialite formation such as Lake 21, which is an evaporitic, hypersaline lake (salinity of approximately 170‰). Lake 21 is completely covered with a thick multilayered microbial mat. This mat is associated with the formation of decimeter-thick highly porous microbialites, which are composed of aragonite and gypsum crystals. We assessed the bacterial and archaeal community composition and its alteration along the vertical stratification by large-scale analysis of 16S rRNA gene sequences of the nine different mat layers. The surface layers are dominated by aerobic, phototrophic, and halotolerant microbes. The bacterial community of these layers harbored Cyanobacteria (Halothece cluster), which were accompanied with known phototrophic members of the Bacteroidetes and Alphaproteobacteria. In deeper anaerobic layers more diverse communities than in the upper layers were present. The deeper layers were dominated by Spirochaetes, sulfate-reducing bacteria (Deltaproteobacteria), Chloroflexi (Anaerolineae and Caldilineae), purple non-sulfur bacteria (Alphaproteobacteria), purple sulfur bacteria (Chromatiales), anaerobic Bacteroidetes (Marinilabiacae), Nitrospirae (OPB95), Planctomycetes and several candidate divisions. The archaeal community, including numerous uncultured taxonomic lineages, generally changed from Euryarchaeota (mainly Halobacteria and Thermoplasmata) to uncultured members of the Thaumarchaeota (mainly Marine Benthic Group B) with increasing depth.     

A Winogradsky-based culture system shows an association between microbial fermentation and cystic fibrosis exacerbation

There is a poor understanding of how the physiology of polymicrobial communities in cystic fibrosis (CF) lungs contributes to pulmonary exacerbations and lung function decline. In this study, a microbial culture system based on the principles of the Winogradsky column (WinCF system) was developed to study the physiology of CF microbes. The system used glass capillary tubes filled with artificial sputum medium to mimic a clogged airway bronchiole. Chemical indicators were added to observe microbial physiology within the tubes. Characterization of sputum samples from seven patients showed variation in pH, respiration, biofilm formation and gas production, indicating that the physiology of CF microbial communities varied among patients. Incubation of homogenized tissues from an explant CF lung mirrored responses of a Pseudomonas aeruginosa pure culture, supporting evidence that end-stage lungs are dominated by this pathogen. Longitudinal sputum samples taken through two exacerbation events in a single patient showed that a two-unit drop in pH and a 30% increase in gas production occurred in the tubes prior to exacerbation, which was reversed with antibiotic treatment. Microbial community profiles obtained through amplification and sequencing of the 16S rRNA gene showed that fermentative anaerobes became more abundant during exacerbation and were then reduced during treatment where P. aeruginosa became the dominant bacterium. Results from the WinCF experiments support the model where two functionally different CF microbial communities exist, the persistent Climax Community and the acute Attack Community. Fermentative anaerobes are hypothesized to be the core members of the Attack Community and production of acidic and gaseous products from fermentation may drive developing exacerbations. Treatment targeting the Attack Community may better resolve exacerbations and resulting lung damage.     

Physiological and Transcriptional Responses of Different Industrial Microbes at Near-Zero Specific Growth Rates

The current knowledge of the physiology and gene expression of industrially relevant microorganisms is largely based on laboratory studies under conditions of rapid growth and high metabolic activity. However, in natural ecosystems and industrial processes, microbes frequently encounter severe calorie restriction. As a consequence, microbial growth rates in such settings can be extremely slow and even approach zero. Furthermore, uncoupling microbial growth from product formation, while cellular integrity and activity are maintained, offers perspectives that are economically highly interesting. Retentostat cultures have been employed to investigate microbial physiology at (near-)zero growth rates. This minireview compares information from recent physiological and gene expression studies on retentostat cultures of the industrially relevant microorganisms Lactobacillus plantarum, Lactococcus lactis, Bacillus subtilis, Saccharomyces cerevisiae, and Aspergillus niger. Shared responses of these organisms to (near-)zero growth rates include increased stress tolerance and a downregulation of genes involved in protein synthesis. Other adaptations, such as changes in morphology and (secondary) metabolite production, were species specific. This comparison underlines the industrial and scientific significance of further research on microbial (near-)zero growth physiology.     

Cultivation of methanogenic community from subseafloor sediments using a continuous-flow bioreactor

Microbial methanogenesis in subseafloor sediments is a key process in the carbon cycle on the Earth. However, the cultivation-dependent evidences have been poorly demonstrated. Here we report the cultivation of a methanogenic microbial consortium from subseafloor sediments using a continuous-flow-type bioreactor with polyurethane sponges as microbial habitats, called down-flow hanging sponge (DHS) reactor. We anaerobically incubated methane-rich core sediments collected from off Shimokita Peninsula, Japan, for 826 days in the reactor at 10 °C. Synthetic seawater supplemented with glucose, yeast extract, acetate and propionate as potential energy sources was provided into the reactor. After 289 days of operation, microbiological methane production became evident. Fluorescence in situ hybridization analysis revealed the presence of metabolically active microbial cells with various morphologies in the reactor. DNA- and RNA-based phylogenetic analyses targeting 16S rRNA indicated the successful growth of phylogenetically diverse microbial components during cultivation in the reactor. Most of the phylotypes in the reactor, once it made methane, were more closely related to culture sequences than to the subsurface environmental sequence. Potentially methanogenic phylotypes related to the genera Methanobacterium, Methanococcoides and Methanosarcina were predominantly detected concomitantly with methane production, while uncultured archaeal phylotypes were also detected. Using the methanogenic community enrichment as subsequent inocula, traditional batch-type cultivations led to the successful isolation of several anaerobic microbes including those methanogens. Our results substantiate that the DHS bioreactor is a useful system for the enrichment of numerous fastidious microbes from subseafloor sediments and will enable the physiological and ecological characterization of pure cultures of previously uncultivated subseafloor microbial life.     

Metagenomics--the key to the uncultured microbes

It is widely accepted that up to 99.8% of the microbes present in many environments are not readily culturable. 'Metagenome technology' tries to overcome this bottleneck by developing and using culture-independent approaches. From the outset, metagenome-based approaches have led to the accumulation of an increasing number of DNA sequences, but until this time the sequences retrieved have been those of uncultured microbes. These genomic sequences are currently exploited for novel biotechnological and pharmaceutical applications and to increase our knowledge on microbial ecology and physiology of these microbes. Using the metagenome sequences to fully understand how complex microbial communities function and how microbes interact within these niches represents a major challenge for microbiologists today.     

The role of host promiscuity in the invasion process of a seaweed holobiont

Invasive species are co-introduced with microbiota from their native range and also interact with microbiota found in the novel environment to which they are introduced. Host flexibility toward microbiota, or host promiscuity, is an important trait underlying terrestrial plant invasions. To test whether host promiscuity may be important in macroalgal invasions, we experimentally simulated an invasion in a common garden setting, using the widespread invasive macroalga Agarophyton vermiculophyllum as a model invasive seaweed holobiont. After disturbing the microbiota of individuals from native and non-native populations with antibiotics, we monitored the microbial succession trajectories in the presence of a new source of microbes. Microbial communities were strongly impacted by the treatment and changed compositionally and in terms of diversity but recovered functionally by the end of the experiment in most respects. Beta-diversity in disturbed holobionts strongly decreased, indicating that different populations configure more similar –or more common– microbial communities when exposed to the same conditions. This decline in beta-diversity occurred not only more rapidly, but was also more pronounced in non-native populations, while individuals from native populations retained communities more similar to those observed in the field. This study demonstrates that microbial communities of non-native A. vermiculophyllum are more flexibly adjusted to the environment and suggests that an intraspecific increase in host promiscuity has promoted the invasion process of A. vermiculophyllum. This phenomenon may be important among invasive macroalgal holobionts in general.Subject terms: Symbiosis, Molecular ecology, Microbial ecology     

Epilithic Cyanobacterial Communities of a Marine Tropical Beach Rock (Heron Island, Great Barrier Reef): Diversity and Diazotrophy

The diversity and nitrogenase activity of epilithic marine microbes in a Holocene beach rock (Heron Island, Great Barrier Reef, Australia) with a proposed biological calcification “microbialite” origin were examined. Partial 16S rRNA gene sequences from the dominant mat (a coherent and layered pink-pigmented community spread over the beach rock) and biofilms (nonstratified, differently pigmented microbial communities of small shallow depressions) were retrieved using denaturing gradient gel electrophoresis (DGGE), and a clone library was retrieved from the dominant mat. The 16S rRNA gene sequences and morphological analyses revealed heterogeneity in the cyanobacterial distribution patterns. The nonheterocystous filamentous genus Blennothrix sp., phylogenetically related to Lyngbya, dominated the mat together with unidentified nonheterocystous filaments of members of the Pseudanabaenaceae and the unicellular genus Chroococcidiopsis. The dominance and three-dimensional intertwined distribution of these organisms were confirmed by nonintrusive scanning microscopy. In contrast, the less pronounced biofilms were dominated by the heterocystous cyanobacterial genus Calothrix, two unicellular Entophysalis morphotypes, Lyngbya spp., and members of the Pseudanabaenaceae family. Cytophaga-Flavobacterium-Bacteroides and Alphaproteobacteria phylotypes were also retrieved from the beach rock. The microbial diversity of the dominant mat was accompanied by high nocturnal nitrogenase activities (as determined by in situ acetylene reduction assays). A new DGGE nifH gene optimization approach for cyanobacterial nitrogen fixers showed that the sequences retrieved from the dominant mat were related to nonheterocystous uncultured cyanobacterial phylotypes, only distantly related to sequences of nitrogen-fixing cultured cyanobacteria. These data stress the occurrence and importance of nonheterocystous epilithic cyanobacteria, and it is hypothesized that such epilithic cyanobacteria are the principal nitrogen fixers of the Heron Island beach rock.     

Novel clades of soil biphenyl degraders revealed by integrating isotope probing,multi-omics,and single-cell analyses

Most microorganisms in the biosphere remain uncultured and poorly characterized. Although the surge in genome sequences has enabled insights into the genetic and metabolic properties of uncultured microorganisms, their physiology and ecological roles cannot be determined without direct probing of their activities in natural habitats. Here we employed an experimental framework coupling genome reconstruction and activity assays to characterize the largely uncultured microorganisms responsible for aerobic biodegradation of biphenyl as a proxy for a large class of environmental pollutants, polychlorinated biphenyls. We used ¹³C-labeled biphenyl in contaminated soils and traced the flow of pollutant-derived carbon into active cells using single-cell analyses and protein–stable isotope probing. The detection of ¹³C-enriched proteins linked biphenyl biodegradation to the uncultured Alphaproteobacteria clade {"type":"entrez-protein","attrs":{"text":"UBA11222","term_id":"2096922005"}}UBA11222, which we found to host a distinctive biphenyl dioxygenase gene widely retrieved from contaminated environments. The same approach indicated the capacity of Azoarcus species to oxidize biphenyl and suggested similar metabolic abilities for species of Rugosibacter. Biphenyl oxidation would thus represent formerly unrecognized ecological functions of both genera. The quantitative role of these microorganisms in pollutant degradation was resolved using single-cell-based uptake measurements. Our strategy advances our understanding of microbially mediated biodegradation processes and has general application potential for elucidating the ecological roles of uncultured microorganisms in their natural habitats.Subject terms: Microbial ecology, Biogeochemistry, Biogeochemistry, Soil microbiology, Biogeochemistry     

Interwoven Biology of the Tsetse Holobiont

Microbial symbionts can be instrumental to the evolutionary success of their hosts. Here, we discuss medically significant tsetse flies (Diptera: Glossinidae), a group comprised of over 30 species, and their use as a valuable model system to study the evolution of the holobiont (i.e., the host and associated microbes). We first describe the tsetse microbiota, which, despite its simplicity, harbors a diverse range of associations. The maternally transmitted microbes consistently include two Gammaproteobacteria, the obligate mutualists Wigglesworthia spp. and the commensal Sodalis glossinidius, along with the parasitic Alphaproteobacteria Wolbachia. These associations differ in their establishment times, making them unique and distinct from previously characterized symbioses, where multiple microbial partners have associated with their host for a significant portion of its evolution. We then expand into discussing the functional roles and intracommunity dynamics within this holobiont, which enhances our understanding of tsetse biology to encompass the vital functions and interactions of the microbial community. Potential disturbances influencing the tsetse microbiome, including salivary gland hypertrophy virus and trypanosome infections, are highlighted. While previous studies have described evolutionary consequences of host association for symbionts, the initial steps facilitating their incorporation into a holobiont and integration of partner biology have only begun to be explored. Research on the tsetse holobiont will contribute to the understanding of how microbial metabolic integration and interdependency initially may develop within hosts, elucidating mechanisms driving adaptations leading to cooperation and coresidence within the microbial community. Lastly, increased knowledge of the tsetse holobiont may also contribute to generating novel African trypanosomiasis disease control strategies.     

Prospecting for Novel Biocatalysts in a Soil Metagenome

The metagenomes of complex microbial communities are rich sources of novel biocatalysts. We exploited the metagenome of a mixed microbial population for isolation of more than 15 different genes encoding novel biocatalysts by using a combined cultivation and direct cloning strategy. A 16S rRNA sequence analysis revealed the presence of hitherto uncultured microbes closely related to the genera Pseudomonas, Agrobacterium, Xanthomonas, Microbulbifer, and Janthinobacterium. Total genomic DNA from this bacterial community was used to construct cosmid DNA libraries, which were functionally searched for novel enzymes of biotechnological value. Our searches in combination with cosmid sequencing resulted in identification of four clones encoding 12 putative agarase genes, most of which were organized in clusters consisting of two or three genes. Interestingly, nine of these agarase genes probably originated from gene duplications. Furthermore, we identified by DNA sequencing several other biocatalyst-encoding genes, including genes encoding a putative stereoselective amidase (amiA), two cellulases (gnuB and uvs080), an α-amylase (amyA), a 1,4-α-glucan branching enzyme (amyB), and two pectate lyases (pelA and uvs119). Also, a conserved cluster of two lipase genes was identified, which was linked to genes encoding a type I secretion system. The novel gene aguB was overexpressed in Escherichia coli, and the enzyme activities were determined. Finally, we describe more than 162 kb of DNA sequence that provides a strong platform for further characterization of this microbial consortium.     

Detection and Differentiation of Chlamydiae by Fluorescence In Situ Hybridization

Chlamydiae are important pathogens of humans and animals but diagnosis of chlamydial infections is still hampered by inadequate detection methods. Fluorescence in situ hybridization (FISH) using rRNA-targeted oligonucleotide probes is widely used for the investigation of uncultured bacteria in complex microbial communities and has recently also been shown to be a valuable tool for the rapid detection of various bacterial pathogens in clinical specimens. Here we report on the development and evaluation of a hierarchic probe set for the specific detection and differentiation of chlamydiae, particularly C. pneumoniae, C. trachomatis, C. psittaci, and the recently described chlamydia-like bacteria comprising the novel genera Neochlamydia and Parachlamydia. The specificity of the nine newly developed probes was successfully demonstrated by in situ hybridization of experimentally infected amoebae and HeLa 229 cells, including HeLa 229 cells coinfected with C. pneumoniae and C. trachomatis. FISH reliably stained chlamydial inclusions as early as 12 h postinfection. The sensitivity of FISH was further confirmed by combination with direct fluorescence antibody staining. In contrast to previously established detection methods for chlamydiae, FISH was not susceptible to false-positive results and allows the detection of all recognized chlamydiae in one single step.     

Microbial Communities from Methane Hydrate-Bearing Deep Marine Sediments in a Forearc Basin

Microbial communities in cores obtained from methane hydrate-bearing deep marine sediments (down to more than 300 m below the seafloor) in the forearc basin of the Nankai Trough near Japan were characterized with cultivation-dependent and -independent techniques. Acridine orange direct count data indicated that cell numbers generally decreased with sediment depth. Lipid biomarker analyses indicated the presence of viable biomass at concentrations greater than previously reported for terrestrial subsurface environments at similar depths. Archaeal lipids were more abundant than bacterial lipids. Methane was produced from both acetate and hydrogen in enrichments inoculated with sediment from all depths evaluated, at both 10 and 35°C. Characterization of 16S rRNA genes amplified from the sediments indicated that archaeal clones could be discretely grouped within the Euryarchaeota and Crenarchaeota domains. The bacterial clones exhibited greater overall diversity than the archaeal clones, with sequences related to the Bacteroidetes, Planctomycetes, Actinobacteria, Proteobacteria, and green nonsulfur groups. The majority of the bacterial clones were either members of a novel lineage or most closely related to uncultured clones. The results of these analyses suggest that the microbial community in this environment is distinct from those in previously characterized methane hydrate-bearing sediments.     

Cell-Specific Thioautotrophic Productivity of Epsilon-Proteobacterial Epibionts Associated with Shinkaia crosnieri

In this study, we report experimental evidence of the thioautotrophic activity of the epibiotic microbial community associated with the setae of Shinkaia crosnieri, a galatheid crab that is endemic to deep-sea hydrothermal systems in the Okinawa Trough in Japan. Microbial consumption of reduced sulfur compounds under in situ hydrostatic and atmospheric pressure provided evidence of sulfur-oxidizing activity by the epibiotic microbial community; the rate of sulfur oxidation was similar under in situ and decompressed conditions. Results of the microbial consumption of reduced sulfur compounds and tracer experiments using ¹³C-labeled bicarbonate in the presence and absence of thiosulfate (used as a thioautotrophic substrate) convincingly demonstrated that the epibiotic microbial community on S. crosnieri drove primary production via an energy metabolism that was coupled with the oxidation of reductive sulfur compounds. A combination of tracer experiments, fluorescence in situ hybridization (FISH) and nano-scale secondary ion mass spectrometry (Nano-SIMS) indicated that the filamentous cells of the genus Sulfurovum belonging to the class Epsilonproteobacteria were thioautotrophs in the epibiotic community of S. crosnieri. In conclusion, our results strongly suggest that thioautotrophic production by Sulfurovum members present as the epibiotic microbial community play a predominant role in a probable nutritional ectosymbiosis with S. crosnieri.     

Microbial Community in High Arsenic Shallow Groundwater Aquifers in Hetao Basin of Inner Mongolia,China

A survey was carried out on the microbial community of 20 groundwater samples (4 low and 16 high arsenic groundwater) and 19 sediments from three boreholes (two high arsenic and one low arsenic boreholes) in a high arsenic groundwater system located in Hetao Basin, Inner Mongolia, using the 454 pyrosequencing approach. A total of 233,704 sequence reads were obtained and classified into 12–267 operational taxonomic units (OTUs). Groundwater and sediment samples were divided into low and high arsenic groups based on measured geochemical parameters and microbial communities, by hierarchical clustering and principal coordinates analysis. Richness and diversity of the microbial communities in high arsenic sediments are higher than those in high arsenic groundwater. Microbial community structure was significantly different either between low and high arsenic samples or between groundwater and sediments. Acinetobacter, Pseudomonas, Psychrobacter and Alishewanella were the top four genera in high arsenic groundwater, while Thiobacillus, Pseudomonas, Hydrogenophaga, Enterobacteriaceae, Sulfuricurvum and Arthrobacter dominated high arsenic sediments. Archaeal sequences in high arsenic groundwater were mostly related to methanogens. Biota-environment matching and co-inertia analyses showed that arsenic, total organic carbon, SO₄^2-, SO₄^2-/total sulfur ratio, and Fe²⁺ were important environmental factors shaping the observed microbial communities. The results of this study expand our current understanding of microbial ecology in high arsenic groundwater aquifers and emphasize the potential importance of microbes in arsenic transformation in the Hetao Basin, Inner Mongolia.     

INDIGO – INtegrated Data Warehouse of MIcrobial GenOmes with Examples from the Red Sea Extremophiles

Background

The next generation sequencing technologies substantially increased the throughput of microbial genome sequencing. To functionally annotate newly sequenced microbial genomes, a variety of experimental and computational methods are used. Integration of information from different sources is a powerful approach to enhance such annotation. Functional analysis of microbial genomes, necessary for downstream experiments, crucially depends on this annotation but it is hampered by the current lack of suitable information integration and exploration systems for microbial genomes.

Results

We developed a data warehouse system (INDIGO) that enables the integration of annotations for exploration and analysis of newly sequenced microbial genomes. INDIGO offers an opportunity to construct complex queries and combine annotations from multiple sources starting from genomic sequence to protein domain, gene ontology and pathway levels. This data warehouse is aimed at being populated with information from genomes of pure cultures and uncultured single cells of Red Sea bacteria and Archaea. Currently, INDIGO contains information from Salinisphaera shabanensis, Haloplasma contractile, and Halorhabdus tiamatea - extremophiles isolated from deep-sea anoxic brine lakes of the Red Sea. We provide examples of utilizing the system to gain new insights into specific aspects on the unique lifestyle and adaptations of these organisms to extreme environments.

Conclusions

We developed a data warehouse system, INDIGO, which enables comprehensive integration of information from various resources to be used for annotation, exploration and analysis of microbial genomes. It will be regularly updated and extended with new genomes. It is aimed to serve as a resource dedicated to the Red Sea microbes. In addition, through INDIGO, we provide our Automatic Annotation of Microbial Genomes (AAMG) pipeline. The INDIGO web server is freely available at http://www.cbrc.kaust.edu.sa/indigo.     

Functional Basis of Microorganism Classification

Correctly identifying nearest “neighbors” of a given microorganism is important in industrial and clinical applications where close relationships imply similar treatment. Microbial classification based on similarity of physiological and genetic organism traits (polyphasic similarity) is experimentally difficult and, arguably, subjective. Evolutionary relatedness, inferred from phylogenetic markers, facilitates classification but does not guarantee functional identity between members of the same taxon or lack of similarity between different taxa. Using over thirteen hundred sequenced bacterial genomes, we built a novel function-based microorganism classification scheme, functional-repertoire similarity-based organism network (FuSiON; flattened to fusion). Our scheme is phenetic, based on a network of quantitatively defined organism relationships across the known prokaryotic space. It correlates significantly with the current taxonomy, but the observed discrepancies reveal both (1) the inconsistency of functional diversity levels among different taxa and (2) an (unsurprising) bias towards prioritizing, for classification purposes, relatively minor traits of particular interest to humans. Our dynamic network-based organism classification is independent of the arbitrary pairwise organism similarity cut-offs traditionally applied to establish taxonomic identity. Instead, it reveals natural, functionally defined organism groupings and is thus robust in handling organism diversity. Additionally, fusion can use organism meta-data to highlight the specific environmental factors that drive microbial diversification. Our approach provides a complementary view to cladistic assignments and holds important clues for further exploration of microbial lifestyles. Fusion is a more practical fit for biomedical, industrial, and ecological applications, as many of these rely on understanding the functional capabilities of the microbes in their environment and are less concerned with phylogenetic descent.     

Highly Divergent Genes for Methanopterin-Linked C1 Transfer Reactions in Lake Washington, Assessed via Metagenomic Analysis and mRNA Detection

The origins and the evolutionary history of tetrahydromethanopterin-linked C₁ transfer reactions that are part of two environmentally important biotransformations, methylotrophy and methanogenesis, are still not well understood. In previous studies, we have expanded the known phylogenetic diversity of these reactions by identifying genes highly diverging from the ones associated with cultivated Proteobacteria, Planctomycetes, or Archaea (M. G. Kalyuzhnaya, M. E. Lidstrom, and L. Chistoserdova, Microb. Ecol. 48:463-472, 2004; M. G. Kalyuzhnaya, O. Nercessian, M. E. Lidstrom, and L. Chistoserdova, Environ. Microbiol. 7:1269-1274, 2005). Here we used a metagenomic approach to demonstrate that these divergent genes are present with high abundance in the microbial community inhabiting Lake Washington sediment. We also gained preliminary insights into the genomic composition of the organisms possessing these genes by sequencing genomic fragments from three uncultured microbes possessing the genes of interest. Phylogenetic analyses suggested that, although distantly related to each other, these organisms deeply diverge from known Bacteria and Archaea, with more relation to the former, suggesting their affiliation with a new bacterial phylum. We also demonstrate, via specific mRNA detection, that these divergent genes are expressed in the environment, pointing toward their potential role in local carbon cycling.