Legacy copper/nickel mine tailings potentially harbor novel iron/sulfur cycling microorganisms within highly variable communities.

The oxidation of sulfide-bearing mine tailings catalyzed by acidophilic iron and sulfur-oxidizing bacteria releases toxic metals and other contaminants into soil and groundwater as acid mine drainage. Understanding the environmental variables that control the community structure and metabolic activity of microbes indigenous to tailings (especially the abiotic stressors of low pH and high dissolved metal content) is crucial to developing sustainable bioremediation strategies. We determined the microbial community composition along two continuous vertical gradients of Cu/Ni mine tailings at each of two tailings impoundments near Sudbury, Ontario. 16S rRNA amplicon data showed high variability in community diversity and composition between locations, as well as at different depths within each location. A temporal comparison for one tailings location showed low fluctuation in microbial communities across 2 years. Differences in community composition correlated most strongly with pore-water pH, Eh, alkalinity, salinity, and the concentration of several dissolved metals (including iron, but not copper or nickel). The relative abundances of individual genera differed in their degrees of correlation with geochemical factors. Several abundant lineages present at these locations have not previously been associated with mine tailings environments, including novel species predicted to be involved in iron and sulfur cycling.IMPORTANCEMine tailings represent a significant threat to North American freshwater, with legacy tailings areas generating acid mine drainage (AMD) that contaminates rivers, lakes, and aquifers. Microbial activity accelerates AMD formation through oxidative metabolic processes but may also ameliorate acidic tailings by promoting secondary mineral precipitation and immobilizing dissolved metals. Tailings exhibit high geochemical variation within and between mine sites and may harbor many novel extremophiles adapted to high concentrations of toxic metals. Characterizing the unique microbiomes associated with tailing environments is key to identifying consortia that may be used as the foundation for innovative mine-waste bioremediation strategies. We provide an in-depth analysis of microbial diversity at four copper/nickel mine tailings impoundments, describe how communities (and individual lineages) differ based on geochemical gradients, predict organisms involved in AMD transformations, and identify taxonomically novel groups present that have not previously been observed in mine tailings.

Environmental Potential for Microbial 1,4-Dioxane Degradation Is Sparse despite Mobile Elements Playing a Role in Trait Distribution.

1,4-Dioxane (dioxane) is an emerging contaminant of concern for which bioremediation is seen as a promising solution. To date, eight distinct gene families have been implicated in dioxane degradation, though only dioxane monooxygenase (DXMO) from Pseudonocardia dioxanivorans is routinely used as a biomarker in environmental surveys. In order to assess the functional and taxonomic diversity of bacteria capable of dioxane degradation, we collated existing, poorly-organized information on known biodegraders to create a curated suite of biomarkers with confidence levels for assessing 1,4-dioxane degradation potential. The characterized enzyme systems for dioxane degradation are frequently found on mobile elements, and we identified that many of the curated biomarkers are associated with other hallmarks of genomic rearrangements, indicating lateral gene transfer plays a role in dissemination of this trait. This is contrasted by the extremely limited phylogenetic distribution of known dioxane degraders, where all representatives belong to four classes within three bacterial phyla. Based on the curated set of expanded biomarkers, a search of more than 11,000 publicly available metagenomes identified a sparse and taxonomically limited distribution of potential dioxane degradation proteins. Our work provides an important and necessary structure to the current knowledge base for dioxane degradation and clarifies the potential for natural attenuation of dioxane across different environments. It further highlights a disconnect between the apparent mobility of these gene families and their limited distributions, indicating dioxane degradation may be difficult to integrate into a microorganism's metabolism. IMPORTANCE New regulatory limits for 1,4-dioxane in groundwater have been proposed or adopted in many countries, including the United States and Canada, generating a direct need for remediation options as well as better tools for assessing the fate of dioxane in an environment. A comprehensive suite of biomarkers associated with dioxane degradation was identified and then leveraged to examine the global potential for dioxane degradation in natural and engineered environments. We identified consistent differences in the dioxane-degrading gene families associated with terrestrial, aquatic, and wetland environments, indicating reliance on a single biomarker for assessing natural attenuation of dioxane is likely to miss key players. Most environments do not currently host the capacity for dioxane degradation-the sparse distribution of dioxane degradation potential highlights the need for bioaugmentation approaches over biostimulation of naturally occurring microbial communities.

Composition of the North American Wood Frog (Rana sylvatica) Bacterial Skin Microbiome and Seasonal Variation in Community Structure.

While a number of amphibian skin microbiomes have been characterized, it is unclear how these communities might vary in response to seasonal changes in the environment and the corresponding behaviors that many amphibians exhibit. Given recent studies demonstrating the importance of the skin microbiome in frog innate immune defense against pathogens, investigating how changes in the environment impact the microbial species present will provide a better understanding of conditions that may alter host susceptibility to pathogens in their environment. We sampled the bacterial skin microbiome of North American wood frogs (Rana sylvatica) from two breeding ponds in the spring, along with the bacterial community present in their vernal breeding pools, and frogs from the nearby forest floor in the summer and fall to determine whether community composition differs by sex, vernal pond site, or temporally across season (spring, summer, fall). Taxon relative abundance data reveals a profile of bacterial phyla similar to those previously described on anuran skin, with Proteobacteria, Bacteroidetes, and Actinobacteria dominating the wood frog skin microbiome. Our results indicate that sex had no significant effect on skin microbiota diversity; however, this may be due to our limited female frog sample size. Vernal pool site had a small but significant effect on skin microbiota, but skin-associated communities were more similar to each other than to the communities observed in the frogs' respective pond water. Across seasons, diversity analyses suggest that there are significant differences between the bacterial skin microbiome of frogs from spring and summer/fall groups while the average α-diversity per frog remained consistent. These results illustrate seasonal variation in wood frog skin microbiome structure and highlight the importance of considering temporal trends in an amphibian microbiome, particularly for species whose life history requires recurrent shifts in habitat and behavior.

Unusual biology across a group comprising more than 15% of domain Bacteria.

A prominent feature of the bacterial domain is a radiation of major lineages that are defined as candidate phyla because they lack isolated representatives. Bacteria from these phyla occur in diverse environments and are thought to mediate carbon and hydrogen cycles. Genomic analyses of a few representatives suggested that metabolic limitations have prevented their cultivation. Here we reconstructed 8 complete and 789 draft genomes from bacteria representing >35 phyla and documented features that consistently distinguish these organisms from other bacteria. We infer that this group, which may comprise >15% of the bacterial domain, has shared evolutionary history, and describe it as the candidate phyla radiation (CPR). All CPR genomes are small and most lack numerous biosynthetic pathways. Owing to divergent 16S ribosomal RNA (rRNA) gene sequences, 50-100% of organisms sampled from specific phyla would evade detection in typical cultivation-independent surveys. CPR organisms often have self-splicing introns and proteins encoded within their rRNA genes, a feature rarely reported in bacteria. Furthermore, they have unusual ribosome compositions. All are missing a ribosomal protein often absent in symbionts, and specific lineages are missing ribosomal proteins and biogenesis factors considered universal in bacteria. This implies different ribosome structures and biogenesis mechanisms, and underlines unusual biology across a large part of the bacterial domain.

AnnoTree: visualization and exploration of a functionally annotated microbial tree of life.

Bacterial genomics has revolutionized our understanding of the microbial tree of life; however, mapping and visualizing the distribution of functional traits across bacteria remains a challenge. Here, we introduce AnnoTree-an interactive, functionally annotated bacterial tree of life that integrates taxonomic, phylogenetic and functional annotation data from over 27 000 bacterial and 1500 archaeal genomes. AnnoTree enables visualization of millions of precomputed genome annotations across the bacterial and archaeal phylogenies, thereby allowing users to explore gene distributions as well as patterns of gene gain and loss in prokaryotes. Using AnnoTree, we examined the phylogenomic distributions of 28 311 gene/protein families, and measured their phylogenetic conservation, patchiness, and lineage-specificity within bacteria. Our analyses revealed widespread phylogenetic patchiness among bacterial gene families, reflecting the dynamic evolution of prokaryotic genomes. Genes involved in phage infection/defense, mobile elements, and antibiotic resistance dominated the list of most patchy traits, as well as numerous intriguing metabolic enzymes that appear to have undergone frequent horizontal transfer. We anticipate that AnnoTree will be a valuable resource for exploring prokaryotic gene histories, and will act as a catalyst for biological and evolutionary hypothesis generation. AnnoTree is freely available at http://annotree.uwaterloo.ca.

A Need for Improved Cellulase Identification from Metagenomic Sequence Data.

Improved sequencing technologies and the maturation of metagenomic approaches allow the identification of gene variants with potential industrial applications, including cellulases. Cellulase identification from metagenomic environmental surveys is complicated by inconsistent nomenclature and multiple categorization systems. Here, we summarize the current classification and nomenclature systems, with recommendations for improvements to these systems. Addressing the issues described will strengthen the annotation of cellulose-active enzymes from environmental sequence data sets-a rapidly growing resource in environmental and applied microbiology.

Accurate, multi-kb reads resolve complex populations and detect rare microorganisms.

Accurate evaluation of microbial communities is essential for understanding global biogeochemical processes and can guide bioremediation and medical treatments. Metagenomics is most commonly used to analyze microbial diversity and metabolic potential, but assemblies of the short reads generated by current sequencing platforms may fail to recover heterogeneous strain populations and rare organisms. Here we used short (150-bp) and long (multi-kb) synthetic reads to evaluate strain heterogeneity and study microorganisms at low abundance in complex microbial communities from terrestrial sediments. The long-read data revealed multiple (probably dozens of) closely related species and strains from previously undescribed Deltaproteobacteria and Aminicenantes (candidate phylum OP8). Notably, these are the most abundant organisms in the communities, yet short-read assemblies achieved only partial genome coverage, mostly in the form of short scaffolds (N50 = ∼ 2200 bp). Genome architecture and metabolic potential for these lineages were reconstructed using a new synteny-based method. Analysis of long-read data also revealed thousands of species whose abundances were <0.1% in all samples. Most of the organisms in this "long tail" of rare organisms belong to phyla that are also represented by abundant organisms. Genes encoding glycosyl hydrolases are significantly more abundant than expected in rare genomes, suggesting that rare species may augment the capability for carbon turnover and confer resilience to changing environmental conditions. Overall, the study showed that a diversity of closely related strains and rare organisms account for a major portion of the communities. These are probably common features of many microbial communities and can be effectively studied using a combination of long and short reads.

Novel Hydrogenosomes in the Microaerophilic Jakobid Stygiella incarcerata.

Mitochondrion-related organelles (MROs) have arisen independently in a wide range of anaerobic protist lineages. Only a few of these organelles and their functions have been investigated in detail, and most of what is known about MROs comes from studies of parasitic organisms such as the parabasalid Trichomonas vaginalis Here, we describe the MRO of a free-living anaerobic jakobid excavate, Stygiella incarcerata We report an RNAseq-based reconstruction of S. incarcerata's MRO proteome, with an associated biochemical map of the pathways predicted to be present in this organelle. The pyruvate metabolism and oxidative stress response pathways are strikingly similar to those found in the MROs of other anaerobic protists, such as Pygsuia and Trichomonas This elegant example of convergent evolution is suggestive of an anaerobic biochemical 'module' of prokaryotic origins that has been laterally transferred among eukaryotes, enabling them to adapt rapidly to anaerobiosis. We also identified genes corresponding to a variety of mitochondrial processes not found in Trichomonas, including intermembrane space components of the mitochondrial protein import apparatus, and enzymes involved in amino acid metabolism and cardiolipin biosynthesis. In this respect, the MROs of S. incarcerata more closely resemble those of the much more distantly related free-living organisms Pygsuia biforma and Cantina marsupialis, likely reflecting these organisms' shared lifestyle as free-living anaerobes.

Genomic resolution of a cold subsurface aquifer community provides metabolic insights for novel microbes adapted to high CO2 concentrations.

As in many deep underground environments, the microbial communities in subsurface high-CO2 ecosystems remain relatively unexplored. Recent investigations based on single-gene assays revealed a remarkable variety of organisms from little studied phyla in Crystal Geyser (Utah, USA), a site where deeply sourced CO2 -saturated fluids are erupted at the surface. To provide genomic resolution of the metabolisms of these organisms, we used a novel metagenomic approach to recover 227 high-quality genomes from 150 microbial species affiliated with 46 different phylum-level lineages. Bacteria from two novel phylum-level lineages have the capacity for CO2 fixation. Analyses of carbon fixation pathways in all studied organisms revealed that the Wood-Ljungdahl pathway and the Calvin-Benson-Bassham Cycle occurred with the highest frequency, whereas the reverse TCA cycle was little used. We infer that this, and selection for form II RuBisCOs, are adaptions to high CO2 -concentrations. However, many autotrophs can also grow mixotrophically, a strategy that confers metabolic versatility. The assignment of 156 hydrogenases to 90 different organisms suggests that H2 is an important inter-species energy currency even under gaseous CO2 -saturation. Overall, metabolic analyses at the organism level provided insight into the biochemical cycles that support subsurface life under the extreme condition of CO2 saturation.

Critical biogeochemical functions in the subsurface are associated with bacteria from new phyla and little studied lineages.

Nitrogen, sulfur and carbon fluxes in the terrestrial subsurface are determined by the intersecting activities of microbial community members, yet the organisms responsible are largely unknown. Metagenomic methods can identify organisms and functions, but genome recovery is often precluded by data complexity. To address this limitation, we developed subsampling assembly methods to re-construct high-quality draft genomes from complex samples. We applied these methods to evaluate the interlinked roles of the most abundant organisms in biogeochemical cycling in the aquifer sediment. Community proteomics confirmed these activities. The eight most abundant organisms belong to novel lineages, and two represent phyla with no previously sequenced genome. Four organisms are predicted to fix carbon via the Calvin-Benson-Bassham, Wood-Ljungdahl or 3-hydroxyproprionate/4-hydroxybutarate pathways. The profiled organisms are involved in the network of denitrification, dissimilatory nitrate reduction to ammonia, ammonia oxidation and sulfate reduction/oxidation, and require substrates supplied by other community members. An ammonium-oxidizing Thaumarchaeote is the most abundant community member, despite low ammonium concentrations in the groundwater. This organism likely benefits from two other relatively abundant organisms capable of producing ammonium from nitrate, which is abundant in the groundwater. Overall, dominant members of the microbial community are interconnected through exchange of geochemical resources.

Metatranscriptome of an anaerobic benzene-degrading, nitrate-reducing enrichment culture reveals involvement of carboxylation in benzene ring activation.

The enzymes involved in the initial steps of anaerobic benzene catabolism are not known. To try to elucidate this critical step, a metatranscriptomic analysis was conducted to compare the genes transcribed during the metabolism of benzene and benzoate by an anaerobic benzene-degrading, nitrate-reducing enrichment culture. RNA was extracted from the mixed culture and sequenced without prior mRNA enrichment, allowing simultaneous examination of the active community composition and the differential gene expression between the two treatments. Ribosomal and mRNA sequences attributed to a member of the family Peptococcaceae from the order Clostridiales were essentially only detected in the benzene-amended culture samples, implicating this group in the initial catabolism of benzene. Genes similar to each of two subunits of a proposed benzene-carboxylating enzyme were transcribed when the culture was amended with benzene. Anaerobic benzoate degradation genes from strict anaerobes were transcribed only when the culture was amended with benzene. Genes for other benzoate catabolic enzymes and for nitrate respiration were transcribed in both samples, with those attributed to an Azoarcus species being most abundant. These findings indicate that the mineralization of benzene starts with its activation by a strict anaerobe belonging to the Peptococcaceae, involving a carboxylation step to form benzoate. These data confirm the previously hypothesized syntrophic association between a benzene-degrading Peptococcaceae strain and a benzoate-degrading denitrifying Azoarcus strain for the complete catabolism of benzene with nitrate as the terminal electron acceptor.

Discarded diversity: Novel megaphages, auxiliary metabolic genes, and virally encoded CRISPR-Cas systems in landfills.

Background: Viruses are the most abundant microbial entity on the planet, impacting microbial community structure and ecosystem services. Despite outnumbering Bacteria and Archaea by an order of magnitude, viruses have been comparatively underrepresented in reference databases. Metagenomic examinations have illustrated that viruses of Bacteria and Archaea have been specifically understudied in engineered environments. Here we employed metagenomic and computational biology methods to examine the diversity, host interactions, and genetic systems of viruses predicted from 27 samples taken from three municipal landfills across North America. Results: We identified numerous viruses that are not represented in reference databases, including the third largest bacteriophage genome identified to date (~678 kbp), and note a cosmopolitan diversity of viruses in landfills that are distinct from viromes in other systems. Host-virus interactions were examined via host CRISPR spacer to viral protospacer mapping which captured hyper-targeted viral populations and six viral populations predicted to infect across multiple phyla. Virally-encoded auxiliary metabolic genes (AMGs) were identified with the potential to augment hosts' methane, sulfur, and contaminant degradation metabolisms, including AMGs not previously reported in literature. CRISPR arrays and CRISPR-Cas systems were identified from predicted viral genomes, including the two largest bacteriophage genomes to contain these genetic features. Some virally encoded Cas effector proteins appear distinct relative to previously reported Cas systems and are interesting targets for potential genome editing tools. Conclusions: Our observations indicate landfills, as heterogeneous contaminated sites with unique selective pressures, are key locations for diverse viruses and atypical virus-host dynamics.

CRISPR-resolved virus-host interactions in a municipal landfill include non-specific viruses, hyper-targeted viral populations, and interviral conflicts.

Viruses are the most abundant microbial guild on the planet, impacting microbial community structure and ecosystem services. Viruses are specifically understudied in engineered environments, including examinations of their host interactions. We examined host-virus interactions via host CRISPR spacer to viral protospacer mapping in a municipal landfill across two years. Viruses comprised ~ 4% of both the unassembled reads and assembled basepairs. A total of 458 unique virus-host connections captured hyper-targeted viral populations and host CRISPR array adaptation over time. Four viruses were predicted to infect across multiple phyla, suggesting that some viruses are far less host-specific than is currently understood. We detected 161 viral elements that encode CRISPR arrays, including one with 187 spacers, the longest virally-encoded CRISPR array described to date. Virally-encoded CRISPR arrays targeted other viral elements in interviral conflicts. CRISPR-encoding proviruses integrated into host chromosomes were latent examples of CRISPR-immunity-based superinfection exclusion. The bulk of the observed virus-host interactions fit the one-virus-one-host paradigm, but with limited geographic specificity. Our networks highlight rare and previously undescribed complex interactions influencing the ecology of this dynamic engineered system. Our observations indicate landfills, as heterogeneous contaminated sites with unique selective pressures, are key locations for atypical virus-host dynamics.

MT-MAG: Accurate and interpretable machine learning for complete or partial taxonomic assignments of metagenomeassembled genomes.

We propose MT-MAG, a novel machine learning-based software tool for the complete or partial hierarchically-structured taxonomic classification of metagenome-assembled genomes (MAGs). MT-MAG is alignment-free, with k-mer frequencies being the only feature used to distinguish a DNA sequence from another (herein k = 7). MT-MAG is capable of classifying large and diverse metagenomic datasets: a total of 245.68 Gbp in the training sets, and 9.6 Gbp in the test sets analyzed in this study. In addition to complete classifications, MT-MAG offers a "partial classification" option, whereby a classification at a higher taxonomic level is provided for MAGs that cannot be classified to the Species level. MT-MAG outputs complete or partial classification paths, and interpretable numerical classification confidences of its classifications, at all taxonomic ranks. To assess the performance of MT-MAG, we define a "weighted classification accuracy," with a weighting scheme reflecting the fact that partial classifications at different ranks are not equally informative. For the two benchmarking datasets analyzed (genomes from human gut microbiome species, and bacterial and archaeal genomes assembled from cow rumen metagenomic sequences), MT-MAG achieves an average of 87.32% in weighted classification accuracy. At the Species level, MT-MAG outperforms DeepMicrobes, the only other comparable software tool, by an average of 34.79% in weighted classification accuracy. In addition, MT-MAG is able to completely classify an average of 67.70% of the sequences at the Species level, compared with DeepMicrobes which only classifies 47.45%. Moreover, MT-MAG provides additional information for sequences that it could not classify at the Species level, resulting in the partial or complete classification of 95.13%, of the genomes in the datasets analyzed. Lastly, unlike other taxonomic assignment tools (e.g., GDTB-Tk), MT-MAG is an alignment-free and genetic marker-free tool, able to provide additional bioinformatics analysis to confirm existing or tentative taxonomic assignments.

Microbial methane cycling in a landfill on a decadal time scale.

Landfills generate outsized environmental footprints due to microbial degradation of organic matter in municipal solid waste, which produces the potent greenhouse gas methane. With global solid waste production predicted to increase substantially in the next few decades, there is a pressing need to better understand the temporal dynamics of biogeochemical processes that control methane cycling in landfills. Here, we use metagenomic approaches to characterize microbial methane cycling in waste that was landfilled over 39 years. Our analyses indicate that newer waste supports more diverse communities with similar composition compared to older waste, which contains lower diversity and more varied communities. Older waste contains primarily autotrophic organisms with versatile redox metabolisms, whereas newer waste is dominated by anaerobic fermenters. Methane-producing microbes are more abundant, diverse, and metabolically versatile in new waste compared to old waste. Our findings indicate that predictive models for methane emission in landfills overlook methane oxidation in the absence of oxygen, as well as certain microbial lineages that can potentially contribute to methane sinks in diverse habitats.

Comparative metagenomics of three Dehalococcoides-containing enrichment cultures: the role of the non-dechlorinating community.

BACKGROUND: The Dehalococcoides are strictly anaerobic bacteria that gain metabolic energy via the oxidation of H2 coupled to the reduction of halogenated organic compounds. Dehalococcoides spp. grow best in mixed microbial consortia, relying on non-dechlorinating members to provide essential nutrients and maintain anaerobic conditions.A metagenome sequence was generated for the dechlorinating mixed microbial consortium KB-1. A comparative metagenomic study utilizing two additional metagenome sequences for Dehalococcoides-containing dechlorinating microbial consortia was undertaken to identify common features that are provided by the non-dechlorinating community and are potentially essential to Dehalococcoides growth. RESULTS: The KB-1 metagenome contained eighteen novel homologs to reductive dehalogenase genes. The metagenomes obtained from the three consortia were automatically annotated using the MG-RAST server, from which statistically significant differences in community composition and metabolic profiles were determined. Examination of specific metabolic pathways, including corrinoid synthesis, methionine synthesis, oxygen scavenging, and electron-donor metabolism identified the Firmicutes, methanogenic Archaea, and the ∂-Proteobacteria as key organisms encoding these pathways, and thus potentially producing metabolites required for Dehalococcoides growth. CONCLUSIONS: Comparative metagenomics of the three Dehalococcoides-containing consortia identified that similarities across the three consortia are more apparent at the functional level than at the taxonomic level, indicating the non-dechlorinating organisms' identities can vary provided they fill the same niche within a consortium. Functional redundancy was identified in each metabolic pathway of interest, with key processes encoded by multiple taxonomic groups. This redundancy likely contributes to the robust growth and dechlorination rates in dechlorinating enrichment cultures.

Genomic determinants of organohalide-respiration in Geobacter lovleyi, an unusual member of the Geobacteraceae.

BACKGROUND: Geobacter lovleyi is a unique member of the Geobacteraceae because strains of this species share the ability to couple tetrachloroethene (PCE) reductive dechlorination to cis-1,2-dichloroethene (cis-DCE) with energy conservation and growth (i.e., organohalide respiration). Strain SZ also reduces U(VI) to U(IV) and contributes to uranium immobilization, making G. lovleyi relevant for bioremediation at sites impacted with chlorinated ethenes and radionuclides. G. lovleyi is the only fully sequenced representative of this distinct Geobacter clade, and comparative genome analyses identified genetic elements associated with organohalide respiration and elucidated genome features that distinguish strain SZ from other members of the Geobacteraceae. RESULTS: Sequencing the G. lovleyi strain SZ genome revealed a 3.9 Mbp chromosome with 54.7% GC content (i.e., the percent of the total guanines (Gs) and cytosines (Cs) among the four bases within the genome), and average amino acid identities of 53-56% compared to other sequenced Geobacter spp. Sequencing also revealed the presence of a 77 kbp plasmid, pSZ77 (53.0% GC), with nearly half of its encoded genes corresponding to chromosomal homologs in other Geobacteraceae genomes. Among these chromosome-derived features, pSZ77 encodes 15 out of the 24 genes required for de novo cobalamin biosynthesis, a required cofactor for organohalide respiration. A plasmid with 99% sequence identity to pSZ77 was subsequently detected in the PCE-dechlorinating G. lovleyi strain KB-1 present in the PCE-to-ethene-dechlorinating consortium KB-1. Additional PCE-to-cis-DCE-dechlorinating G. lovleyi strains obtained from the PCE-contaminated Fort Lewis, WA, site did not carry a plasmid indicating that pSZ77 is not a requirement (marker) for PCE respiration within this species. Chromosomal genomic islands found within the G. lovleyi strain SZ genome encode two reductive dehalogenase (RDase) homologs and a putative conjugative pilus system. Despite the loss of many c-type cytochrome and oxidative-stress-responsive genes, strain SZ retained the majority of Geobacter core metabolic capabilities, including U(VI) respiration. CONCLUSIONS: Gene acquisitions have expanded strain SZ's respiratory capabilities to include PCE and TCE as electron acceptors. Respiratory processes core to the Geobacter genus, such as metal reduction, were retained despite a substantially reduced number of c-type cytochrome genes. pSZ77 is stably maintained within its host strains SZ and KB-1, likely because the replicon carries essential genes including genes involved in cobalamin biosynthesis and possibly corrinoid transport. Lateral acquisition of the plasmid replicon and the RDase genomic island represent unique genome features of the PCE-respiring G. lovleyi strains SZ and KB-1, and at least the latter signifies adaptation to PCE contamination.

Discovery of a trans-dichloroethene-respiring Dehalogenimonas species in the 1,1,2,2-tetrachloroethane-dechlorinating WBC-2 consortium.

The WBC-2 consortium is an organohalide-respiring anaerobic microbial enrichment culture capable of dechlorinating 1,1,2,2-tetrachloroethane (TeCA) to ethene. In the WBC-2 culture, TeCA is first transformed to trans-dichloroethene (tDCE) by dichloroelimination; tDCE is subsequently transformed to vinyl chloride (VC) and then to ethene by hydrogenolysis. Analysis of 16S rRNA gene clone libraries from culture DNA revealed sequences from three putative dechlorinating organisms belonging to Dehalococcoides, Dehalobacter, and Dehalogenimonas genera. Quantitative PCR primers were designed for each of these sequences, and their abundance was quantified in enrichment cultures over time. These data revealed that complete dechlorination of TeCA to ethene involves all three organisms. Dehalobacter spp. grew during the dihaloelimination of TeCA to tDCE, while Dehalococcoides and Dehalogenimonas spp. grew during hydrogenolysis of tDCE to ethene. This is the first time a genus other than Dehalococcoides has been implicated in dechlorination of tDCE to VC.

Transcriptional analysis of a Dehalococcoides-containing microbial consortium reveals prophage activation.

Chlorinated solvents are among the most prevalent groundwater contaminants in the industrialized world. Biodegradation with Dehalococcoides-containing mixed cultures is an effective remediation technology. To elucidate transcribed genes in a Dehalococcoides-containing mixed culture, a shotgun metagenome microarray was created and used to investigate gene transcription during vinyl chloride (VC) dechlorination and during starvation (no chlorinated compounds) by a microbial enrichment culture called KB-1. In both treatment conditions, methanol was amended as an electron donor. Subsequently, spots were sequenced that contained the genes most differentially transcribed between the VC-degrading and methanol-only conditions, as well as spots with the highest intensities. Sequencing revealed that during VC degradation Dehalococcoides genes involved in transcription, translation, metabolic energy generation, and amino acid and lipid metabolism and transport were overrepresented in the transcripts compared to the average Dehalococcoides genome. KB-1 rdhA14 (vcrA) was the only reductive dehalogenase homologous (RDH) gene with higher transcript levels during VC degradation, while multiple RDH genes had higher transcript levels in the absence of VC. Numerous hypothetical genes from Dehalococcoides also had higher transcript levels in methanol-only treatments, indicating that many uncharacterized proteins are involved in cell maintenance in the absence of chlorinated substrates. In addition, microarray results prompted biological experiments confirming that electron acceptor limiting conditions activated a Dehalococcoides prophage. Transcripts from Spirochaetes, Chloroflexi, Geobacter, and methanogens demonstrate the importance of non-Dehalococcoides organisms to the culture, and sequencing of identified shotgun clones of interest provided information for follow-on targeted studies.