Metagenomic and Genomic Sequences from a Methanogenic Benzene-Degrading Consortium.

Draft and complete metagenome assembled genomes (MAGs) were created from multiple metagenomic assemblies of DGG-B, a strictly anaerobic, stable mixed microbial consortium that degrades benzene completely to methane and CO2. Our objective was to obtain closed genome sequences of benzene-fermenting bacteria to enable the elucidation of their elusive anaerobic benzene degradation pathway.

"Candidatus Nealsonbacteria" Are Likely Biomass Recycling Ectosymbionts of Methanogenic Archaea in a Stable Benzene-Degrading Enrichment Culture.

The Candidate Phyla Radiation (CPR), also referred to as superphylum Patescibacteria, is a very large group of bacteria with no pure culture representatives discovered by 16S rRNA sequencing or genome-resolved metagenomic analyses of environmental samples. Within the CPR, candidate phylum Parcubacteria, previously referred to as OD1, is prevalent in anoxic sediments and groundwater. Previously, we had identified a specific member of the Parcubacteria (referred to as DGGOD1a) as an important member of a methanogenic benzene-degrading consortium. Phylogenetic analyses herein place DGGOD1a within the clade "Candidatus Nealsonbacteria." Because of its persistence over many years, we hypothesized that "Ca. Nealsonbacteria" DGGOD1a must play an important role in sustaining anaerobic benzene metabolism in the consortium. To try to identify its growth substrate, we amended the culture with a variety of defined compounds (pyruvate, acetate, hydrogen, DNA, and phospholipid), as well as crude culture lysate and three subfractions thereof. We observed the greatest (10-fold) increase in the absolute abundance of "Ca. Nealsonbacteria" DGGOD1a only when the consortium was amended with crude cell lysate. These results implicate "Ca. Nealsonbacteria" in biomass recycling. Fluorescence in situ hybridization and cryogenic transmission electron microscope images revealed that "Ca. Nealsonbacteria" DGGOD1a cells were attached to larger archaeal Methanothrix cells. This apparent epibiont lifestyle was supported by metabolic predictions from a manually curated complete genome. This is one of the first examples of bacterial-archaeal episymbiosis and may be a feature of other "Ca. Nealsonbacteria" found in anoxic environments. IMPORTANCE An anaerobic microbial enrichment culture was used to study members of candidate phyla that are difficult to grow in the lab. We were able to visualize tiny "Candidatus Nealsonbacteria" cells attached to a large Methanothrix cell, revealing a novel episymbiosis.

Long-term survival of Dehalococcoides mccartyi strains in mixed cultures under electron acceptor and ammonium limitation.

Few strains of Dehalococcoides mccartyi harbour and express the vinyl chloride reductase (VcrA) that catalyzes the dechlorination of vinyl chloride (VC), a carcinogenic soil and groundwater contaminant. The vcrA operon is found on a Genomic Island (GI) and, therefore, believed to participate in horizontal gene transfer (HGT). To try to induce HGT of the vcrA-GI, we blended two enrichment cultures in medium without ammonium while providing VC. We hypothesized that these conditions would select for a mutant strain of D. mccartyi that could both fix nitrogen and respire VC. However, after more than 4 years of incubation, we found no evidence for HGT of the vcrA-GI. Rather, we observed VC-dechlorinating activity attributed to the trichloroethene reductase TceA. Sequencing and protein modelling revealed a mutation in the predicted active site of TceA, which may have influenced substrate specificity. We also identified two nitrogen-fixing D. mccartyi strains in the KB-1 culture. The presence of multiple strains of D. mccartyi with distinct phenotypes is a feature of natural environments and certain enrichment cultures (such as KB-1), and may enhance bioaugmentation success. The fact that multiple distinct strains persist in the culture for decades and that we could not induce HGT of the vcrA-GI suggests that it is not as mobile as predicted, or that mobility is restricted in ways yet to be discovered to specific subclades of Dehalococcoides.

Insights into origins and function of the unexplored majority of the reductive dehalogenase gene family as a result of genome assembly and ortholog group classification.

Organohalide respiring bacteria (OHRB) express reductive dehalogenases for energy conservation and growth. Some of these enzymes catalyze the reductive dehalogenation of chlorinated and brominated pollutants in anaerobic subsurface environments, providing a valuable ecosystem service. Dehalococcoides mccartyi strains have been most extensively studied owing to their ability to dechlorinate all chlorinated ethenes - most notably carcinogenic vinyl chloride - to ethene. The genomes of OHRB, particularly obligate OHRB, often harbour multiple putative reductive dehalogenase genes (rdhA), most of which have yet to be characterized. We recently sequenced and closed the genomes of eight new strains, increasing the number of available D. mccartyi genomes in NCBI from 16 to 24. From all available OHRB genomes, we classified predicted translations of reductive dehalogenase genes using a previously established 90% amino acid pairwise identity cut-off to identify Ortholog Groups (OGs). Interestingly, the majority of D. mccartyi dehalogenase gene sequences, once classified into OGs, exhibited a remarkable degree of synteny (gene order) in all genomes sequenced to date. This organization was not apparent without the classification. A high degree of synteny indicates that differences arose from rdhA gene loss rather than recombination. Phylogenetic analysis suggests that most rdhA genes have a long evolutionary history in the Dehalococcoidia with origin prior to speciation of Dehalococcoides and Dehalogenimonas. We also looked for evidence of synteny in the genomes of other species of OHRB. Unfortunately, there are too few closed Dehalogenimonas genomes to compare at this time. There is some partial evidence for synteny in the Dehalobacter restrictus genomes, but here too more closed genomes are needed for confirmation. Interestingly, we found that the rdhA genes that encode enzymes that catalyze dehalogenation of industrial pollutants are the only rdhA genes with strong evidence of recent lateral transfer - at least in the genomes examined herein. Given the utility of the RdhA sequence classification to comparative analyses, we are building a public web server () for the community to use, which allows users to add and classify new sequences, and download the entire curated database of reductive dehalogenases.

Sustained Dechlorination of Vinyl Chloride to Ethene in Dehalococcoides-Enriched Cultures Grown without Addition of Exogenous Vitamins and at Low pH.

Trichloroethene (TCE) bioremediation has been demonstrated at field sites using microbial cultures harboring TCE-respiring Dehalococcoides whose growth is cobalamin (vitamin B12)-dependent. Bioaugmentation cultures grown ex situ with ample exogenous vitamins and at neutral pH may become vitamin-limited or inhibited by acidic pH once injected into field sites, resulting in incomplete TCE dechlorination and accumulation of vinyl chloride (VC). Here, we report growth of the Dehalococcoides-containing bioaugmentation culture KB-1 in a TCE-amended mineral medium devoid of vitamins and in a VC-amended mineral medium at low pH (6.0 and 5.5). In these cultures, Acetobacterium, which can synthesize 5,6-dimethylbenzimidazole (DMB), the lower ligand of cobalamin, and Sporomusa are dominant acetogens. At neutral pH, Acetobacterium supports complete TCE dechlorination by Dehalococcoides at millimolar levels with a substantial increase in cobalamin (∼20-fold). Sustained dechlorination of VC to ethene was achieved at pH as low as 5.5. Below pH 5.0, dechlorination was not stimulated by DMB supplementation but was restored by raising pH to neutral. Cell-extract assays revealed that vinyl chloride reductase activity declines significantly below pH 6.0 and is undetectable below pH 5.0. This study highlights the importance of cobamide-producing populations and pH in microbial dechlorinating communities for successful bioremediation at field sites.

Extrachromosomal circular elements targeted by CRISPR-Cas in Dehalococcoides mccartyi are linked to mobilization of reductive dehalogenase genes.

Dehalococcoides mccartyi are obligate organohalide-respiring bacteria that play an important detoxifying role in the environment. They have small genomes (~1.4 Mb) with a core region interrupted by two high plasticity regions (HPRs) containing dozens of genes encoding reductive dehalogenases involved in organohalide respiration. The genomes of eight new strains of D. mccartyi were closed from metagenomic data from a related set of enrichment cultures, bringing the total number of genomes to 24. Two of the newly sequenced strains and three previously sequenced strains contain CRISPR-Cas systems. These D. mccartyi CRISPR-Cas systems were found to primarily target prophages and genomic islands. The genomic islands were identified either as integrated into D. mccartyi genomes or as circular extrachromosomal elements. We observed active circularization of the integrated genomic island containing vcrABC operon encoding the dehalogenase (VcrA) responsible for the transformation of vinyl chloride to non-toxic ethene. We interrogated archived DNA from established enrichment cultures and found that the CRISPR array acquired three new spacers in 11 years. These data provide a glimpse into dynamic processes operating on the genomes distinct to D. mccartyi strains found in enrichment cultures and provide the first insights into possible mechanisms of lateral DNA exchange in D. mccartyi.

Purinyl-cobamide is a native prosthetic group of reductive dehalogenases.

Cobamides such as vitamin B12 are structurally conserved, cobalt-containing tetrapyrrole biomolecules that have essential biochemical functions in all domains of life. In organohalide respiration, a vital biological process for the global cycling of natural and anthropogenic organohalogens, cobamides are the requisite prosthetic groups for carbon-halogen bond-cleaving reductive dehalogenases. This study reports the biosynthesis of a new cobamide with unsubstituted purine as the lower base and assigns unsubstituted purine a biological function by demonstrating that Coα-purinyl-cobamide (purinyl-Cba) is the native prosthetic group in catalytically active tetrachloroethene reductive dehalogenases of Desulfitobacterium hafniense. Cobamides featuring different lower bases are not functionally equivalent, and purinyl-Cba elicits different physiological responses in corrinoid-auxotrophic, organohalide-respiring bacteria. Given that cobamide-dependent enzymes catalyze key steps in essential metabolic pathways, the discovery of a novel cobamide structure and the realization that lower bases can effectively modulate enzyme activities generate opportunities to manipulate functionalities of microbiomes.

Complete Genome Sequence of Dehalococcoides mccartyi Strain WBC-2, Capable of Anaerobic Reductive Dechlorination of Vinyl Chloride.

Dehalococcoides mccartyi strain WBC-2 dechlorinates carcinogen vinyl chloride to ethene in the West Branch Canal Creek (WBC-2) microbial consortium used for bioaugmentation. We assembled and closed the complete genome sequence of this prokaryote using metagenomic sequencing from an enrichment culture.

1,2-Dichloroethane Exposure Alters the Population Structure, Metabolism, and Kinetics of a Trichloroethene-Dechlorinating Dehalococcoides mccartyi Consortium.

Bioremediation of groundwater contaminated with chlorinated aliphatic hydrocarbons such as perchloroethene and trichloroethene can result in the accumulation of the undesirable intermediate vinyl chloride. Such accumulation can either be due to the absence of specific vinyl chloride respiring Dehalococcoides mccartyi or to the inhibition of such strains by the metabolism of other microorganisms. The fitness of vinyl chloride respiring Dehalococcoides mccartyi subpopulations is particularly uncertain in the presence of chloroethene/chloroethane cocontaminant mixtures, which are commonly found in contaminated groundwater. Therefore, we investigated the structure of Dehalococcoides populations in a continuously fed reactor system under changing chloroethene/ethane influent conditions. We observed that increasing the influent ratio of 1,2-dichloroethane to trichloroethene was associated with ecological selection of a tceA-containing Dehalococcoides population relative to a vcrA-containing Dehalococcoides population. Although both vinyl chloride and 1,2-dichloroethane could be simultaneously transformed to ethene, prolonged exposure to 1,2-dichloroethane diminished the vinyl chloride transforming capacity of the culture. Kinetic tests revealed that dechlorination of 1,2-dichloroethane by the consortium was strongly inhibited by cis-dichloroethene but not vinyl chloride. Native polyacrylamide gel electrophoresis and mass spectrometry revealed that a trichloroethene reductive dehalogenase (TceA) homologue was the most consistently expressed of four detectable reductive dehalogenases during 1,2-dichloroethane exposure, suggesting that it catalyzes the reductive dihaloelimination of 1,2-dichloroethane to ethene.

Improved biomass productivity in algal biofilms through synergistic interactions between photon flux density and carbon dioxide concentration.

Algal biofilms were grown to investigate the interaction effects of bulk medium CO2 concentration and photon flux density (PFD) on biomass productivities. When increasing the CO2 concentration from 0.04% to 2%, while maintaining a PFD of 100µmol/m(2)/s, biomass productivities increased from ∼0.5 to 2.0g/m(2)/d; however, the productivities plateaued when CO2 concentrations were incrementally increased above 2-12%. Statistical analysis demonstrates that there is a significant interaction between PFD and CO2 concentrations on biomass productivities. By simultaneously increasing PFD and CO2 concentrations, biomass productivities were significantly increased to 4.0 and 4.1g/m(2)/d in the experimental and modeled data, respectively. The second order model predicted increases in biomass productivities as both PFD and CO2 simultaneously increased yielding an optimum at 440µmol/m(2)/s and 7.1%; however, when conditions were extended to the highest end of their respective ranges, the conditions were detrimental to growth and productivities decreased.

Long-Term Field Study of Microbial Community and Dechlorinating Activity Following Carboxymethyl Cellulose-Stabilized Nanoscale Zero-Valent Iron Injection.

Nanoscale zerovalent iron (nZVI) is an emerging technology for the remediation of contaminated sites. However, there are concerns related to the impact of nZVI on in situ microbial communities. In this study, the microbial community composition at a contaminated site was monitored over two years following the injection of nZVI stabilized with carboxymethyl cellulose (nZVI-CMC). Enhanced dechlorination of chlorinated ethenes to nontoxic ethene was observed long after the expected nZVI oxidation. The abundance of Dehalococcoides (Dhc) and vinyl chloride reductase (vcrA) genes, monitored using qPCR, increased by over an order of magnitude in nZVI-CMC-impacted wells. The entire microbial community was tracked using 16S rRNA gene amplicon pyrosequencing. Following nZVI-CMC injection, a clear shift in microbial community was observed, with most notable increases in the dechlorinating genera Dehalococcoides and Dehalogenimonas. This study suggests that coupled abiotic degradation (i.e., from reaction with nZVI) and biotic degradation fueled by CMC led to the long-term degradation of chlorinated ethenes at this field site. Furthermore, nZVI-CMC addition stimulated dehalogenator growth (e.g., Dehalococcoides) and biotic degradation of chlorinated ethenes.

Dehalogenimonas sp. Strain WBC-2 Genome and Identification of Its trans-Dichloroethene Reductive Dehalogenase, TdrA.

The Dehalogenimonas population in a dechlorinating enrichment culture referred to as WBC-2 was previously shown to be responsible for trans-dichloroethene (tDCE) hydrogenolysis to vinyl chloride (VC). In this study, blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by enzymatic assays and protein identification using liquid chromatography coupled with mass spectrometry (LC-MS/MS) led to the functional characterization of a novel dehalogenase, TdrA. This new reductive dehalogenase (RDase) catalyzes the dechlorination of tDCE to VC. A metagenome of the WBC-2 culture was sequenced, and a complete Dehalogenimonas genome, only the second Dehalogenimonas genome to become publicly available, was closed. The tdrA dehalogenase found within the Dehalogenimonas genome appears to be on a genomic island similar to genomic islands found in Dehalococcoides. TdrA itself is most similar to TceA from Dehalococcoides sp. strain FL2 with 76.4% amino acid pairwise identity. It is likely that the horizontal transfer of rdhA genes is not only a feature of Dehalococcoides but also a feature of other Dehalococcoidia, including Dehalogenimonas. A set of primers was developed to track tdrA in WBC-2 subcultures maintained on different electron acceptors. This newest dehalogenase is an addition to the short list of functionally defined RDases sharing the usual characteristic motifs (including an AB operon, a TAT export sequence, two iron-sulfur clusters, and a corrinoid binding domain), substrate flexibility, and evidence for horizontal gene transfer within the Dehalococcoidia.

Identity and Substrate Specificity of Reductive Dehalogenases Expressed in Dehalococcoides-Containing Enrichment Cultures Maintained on Different Chlorinated Ethenes.

Many reductive dehalogenases (RDases) have been identified in organohalide-respiring microorganisms, and yet their substrates, specific activities, and conditions for expression are not well understood. We tested whether RDase expression varied depending on the substrate-exposure history of reductive dechlorinating communities. For this purpose, we used the enrichment culture KB-1 maintained on trichloroethene (TCE), as well as subcultures maintained on the intermediates cis-dichloroethene (cDCE) and vinyl chloride (VC). KB-1 contains a TCE-to-cDCE dechlorinating Geobacter and several Dehalococcoides strains that together harbor many of the known chloroethene reductases. Expressed RDases were identified using blue native polyacrylamide gel electrophoresis, enzyme assays in gel slices, and peptide sequencing. As anticipated but never previously quantified, the RDase from Geobacter was only detected transiently at the beginning of TCE dechlorination. The Dehalococcoides RDase VcrA and smaller amounts of TceA were expressed in the parent KB-1 culture during complete dechlorination of TCE to ethene regardless of time point or amended substrate. The Dehalococcoides RDase BvcA was only detected in enrichments maintained on cDCE as growth substrates, in roughly equal abundance to VcrA. Only VcrA was detected in subcultures enriched on VC. Enzyme assays revealed that 1,1-DCE, a substrate not used for culture enrichment, afforded the highest specific activity. trans-DCE was substantially dechlorinated only by extracts from cDCE enrichments expressing BvcA. RDase gene distribution indicated enrichment of different strains of Dehalococcoides as a function of electron acceptor TCE, cDCE, or VC. Each chloroethene reductase has distinct substrate preferences leading to strain selection in mixed communities.

Ecological implications of reduced pollen deposition in alpine plants: a case study using a dominant cushion plant species.

The reproductive assurance hypothesis states that self-incompatible female plants must produce twice the number of seeds relative to their self-compatible hermaphroditic counterparts to persist in gynodioecious populations. This is a viable life-history strategy, provided that pollination rates are sufficiently high. However, reduced pollination rates in alpine plants are likely due to climate induced plant-pollinator mismatches and general declines in pollinators. Using a gynodioecious population of the dominant plant Silene acaulis (Caryophyllaceae), we tested the reproductive assurance hypothesis and also the stress gradient hypothesis with a series of pollinator exclusion trials and extensive measurements of subsequent reproductive output (gender ratio, plant size, percent fruit-set, fruit weight, seeds per fruit, total seeds, seed weight, and seed germination). The reproductive assurance hypothesis was supported with female plants being more sensitive to and less likely to be viable under reductions in pollination rates. These findings are the first to show that the stress gradient hypothesis is also supported under a gradient of pollen supply instead of environmental limitations. Beneficiary abundance was negatively correlated to percent fruit-set under current pollen supply, but became positive under reduced pollen supply suggesting that there are important plant-plant-pollinator interactions related to reproduction in these alpine plant species.

The alpine cushion plant Silene acaulis as foundation species: a bug's-eye view to facilitation and microclimate.

Alpine ecosystems are important globally with high levels of endemic and rare species. Given that they will be highly impacted by climate change, understanding biotic factors that maintain diversity is critical. Silene acaulis is a common alpine nurse plant shown to positively influence the diversity and abundance of organisms--predominantly other plant species. The hypothesis that cushion or nurse plants in general are important to multiple trophic levels has been proposed but rarely tested. Alpine arthropod diversity is also largely understudied worldwide, and the plant-arthropod interactions reported are mostly negative, that is,. herbivory. Plant and arthropod diversity and abundance were sampled on S. acaulis and at paired adjacent microsites with other non-cushion forming vegetation present on Whistler Mountain, B.C., Canada to examine the relative trophic effects of cushion plants. Plant species richness and abundance but not Simpson's diversity index was higher on cushion microsites relative to other vegetation. Arthropod richness, abundance, and diversity were all higher on cushion microsites relative to other vegetated sites. On a microclimatic scale, S. acaulis ameliorated stressful conditions for plants and invertebrates living inside it, but the highest levels of arthropod diversity were observed on cushions with tall plant growth. Hence, alpine cushion plants can be foundation species not only for other plant species but other trophic levels, and these impacts are expressed through both direct and indirect effects associated with altered environmental conditions and localized productivity. Whilst this case study tests a limited subset of the membership of alpine animal communities, it clearly demonstrates that cushion-forming plant species are an important consideration in understanding resilience to global changes for many organisms in addition to other plants.