Biology subjects

Molenda, O.

Publications and source records attributed to Molenda, O..

6 recordsLinked to original sources

bioRxiv2026

A novel virus lineage is abundant in metaviromes from Dehalococcoides-containing mixed cultures

Dehalococcoides mccartyi are obligately anaerobic organohalide-respiring bacteria that play important roles in the detoxification of chlorinated pollutants in groundwater and sediments. Despite having small genomes, they host a diverse set of mobile elements. Here we characterize a family of mobile elements, termed integrative and mobilizable element 1 or IME1, comprising 20 from Dehalococcoides and one from Dehalogenimonas alkenigignens. IME1s are 20,930 - 28,058 bp and are found both integrated in the genomes and as circular episomes. Bioinformatic characterization of IME1 encoded proteins revealed a highly conserved structure with 14 hierarchical orthologous groups (HOGs) found in all 21 IME1s. IME1s lack recognizable hallmark proteins of tailed bacterial viruses (or tailed phages) but encode proteins with similarities to those of filamentous bacterial viruses. In particular, one conserved HOG shows sequence similarity to the pI-like ATPase, the only conserved marker protein identified across filamentous bacterial viruses. Additionally, IME1s encode several small proteins with predicted transmembrane domains and signal peptides, another feature used to identify filamentous bacterial viruses. Both features are also found in budding archaeal viruses of various morphotypes. IME1s dominated metaviromes obtained from the Dehalococcoides-containing KB-1 mixed culture and electron micrographs of the corresponding viral fractions revealed abundant filamentous virus-like particles. We therefore propose that the IME1s represent a novel lineage of double stranded budding, likely filamentous, viruses. Database searches suggest IME1s are found in Dehalococcodia and other Chlorofexota but are so far restricted to this phylum.

microbiology↗

bioRxiv2024

Identification of a Cluster of Benzene Activation Enzymes in a Strictly Anoxic Methanogenic Consortium

The Oil Refinery (OR) consortium is a model methanogenic enrichment culture used to study anaerobic benzene degradation. Over half of the cultures bacterial community consists of two closely related Desulfobacterota strains, designated ORM2a and ORM2b, whose mechanisms of benzene activation are unknown. Two new metagenomes, including a complete circularized metagenome-assembled genome (MAG) for ORM2a, enabled a thorough investigation of this cultures proteome. Among the proteins identified were Bam-like subunits of an ATP-independent benzoyl-CoA degradation pathway, as well as downstream {beta}-oxidation proteins yielding acetate. The most abundant proteins identified mapped to two ORM2a gene clusters of unknown function. Homologous and syntenic gene clusters were identified in genomes of ORM2b and a sulfate-reducing Pelotomaculum that also degrades benzene, as well as in nine contigs assembled from hydrothermal vent metagenomes. Extensive homology and structural predictions suggest that the first cluster - termed the "Magic" gene cluster - encodes for enzymes catalyzing the chemically challenging activation of benzene and subsequent transformation steps yielding benzoyl-CoA. The second ("Nanopod") gene cluster encodes a transmembrane complex that may facilitate benzene transport across the cell membrane. Phylogenomic analyses place ORM2a and ORM2b within a novel genus of strict anaerobes specialized for benzene degradation, which we propose naming "Candidatus Anaerobenzenivorax". IMPORTANCEBenzene is a widespread, persistent and toxic pollutant that can accumulate in anoxic environments such as groundwater and sediments. Despite decades of study, the biochemical mechanisms by which benzene is activated under anaerobic conditions remain unproven. This study provides strong genetic and proteomic evidence for a new class of enzymes that initiate anaerobic benzene activation and proposes a preliminary model for their underlying biochemistry. These findings lay a foundation for future biochemical studies and expand our understanding of how microbes carry out extreme redox chemistry in the absence of oxygen.

microbiology↗

bioRxiv2022

Candidatus Nealsonbacteria (OD1) in a methanogenic benzene-degrading enrichment culture is likely an ectosymbiotic biomass recycler

The Candidate Phyla Radiation (CPR) is a very large group of bacteria with no pure culture representatives, first discovered by metagenomic analyses. Within the CPR, candidate phylum Parcubacteria (previously referred to as OD1) within the candidate superphylum Patescibacteria 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 Candidate clade Nealsonbacteria. Because of its persistence over many years, we hypothesized that Ca. Nealsonbacteria DGGOD1a must serve 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, 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. Fluorescent 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.

microbiology↗

bioRxiv2020

Longitudinal study of vinyl chloride degrading Dehalococcoides mccartyi-containing cultures to promote horizontal gene transfer

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. To try to induce horizontal gene transfer 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 horizontal gene transfer 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 horizontal gene transfer 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 sub-clades of Dehalococcoides. TOC Art O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/423565v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@80423forg.highwire.dtl.DTLVardef@7d6484org.highwire.dtl.DTLVardef@5223b2org.highwire.dtl.DTLVardef@a58aab_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2019

Sustained dechlorination of vinyl chloride to ethene in Dehalococcoides-enriched cultures grown without addition of exogenous vitamins and at low pH

Trichloroethene (TCE) is a ubiquitous groundwater pollutant. Successful TCE bioremediation has been demonstrated at field sites using specialized microbial consortia harboring TCE-respiring Dehaloccocoides whose growth is cobalamin (vitamin B12)-dependent. Bioaugmentation cultures grown ex situ with ample exogenous vitamins in the medium 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 more toxic 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 cultures grown without exogenous vitamins or cobalamin, Acetobacterium, which can synthesize 5,6-dimethylbenzimidazole (DMB), the lower ligand of cobalamin, and Sporomusa are the dominant acetogens. At neutral pH, a growing Acetobacterium population supports complete TCE dechlorination by Dehalococcoides at millimolar levels with a substantial increase in the amount of measured cobalamin (~20-fold). Sustained dechlorination of VC to ethene was achieved at a pH as low as 5.5, yet at low pH Acetobacterium is less abundant, potentially affecting the production of DMB and/or cobalamin. However, dechlorination activity at very low pH (< 5.0) was not stimulated by DMB supplementation, but was restored by raising pH to neutral. Assays in cell extracts revealed that vinyl chloride reductase (VcrA) activity declines significantly below pH 6.0 and is undetectable below pH 5.0. This study highlights the roles of and interplay between vitamin-producing populations and pH in microbial dechlorinating communities, and their importance for successful chlorinated ethenes bioremediation at field sites.

microbiology↗

bioRxiv2018

Eight new genomes of organohalide-respiring Dehalococcoides mccartyi reveal evolutionary trends in reductive dehalogenase enzymes

BackgroundBioaugmentation is now a well-established approach for attenuating toxic groundwater and soil contaminants, particularly for chlorinated ethenes and ethanes. The KB-1 and WBC-2 consortia are cultures used for this purpose. These consortia contain organisms belonging to the Dehalococcoidia, including strains of Dehalococcoides mccartyi in KB-1 and of both D. mccartyi and Dehalogenimonas in WBC-2. These tiny anaerobic bacteria couple respiratory reductive dechlorination to growth and harbour multiple reductive dehalogenase genes (rdhA) in their genomes, the majority of which have yet to be characterized.\n\nResultsUsing a combination of Illumina mate-pair and paired-end sequencing we closed the genomes of eight new strains of Dehalococcoides mccartyi found in three related KB-1 sub-cultures that were enriched on trichloroethene (TCE), 1,2-dichloroethane (1,2-DCA) and vinyl chloride (VC), bringing the total number of genomes available in NCBI to 24. A pangenome analysis was conducted on 24 Dehalococcoides genomes and five Dehalogenimonas genomes (2 in draft) currently available in NCBI. This Dehalococcoidia pangenome generated 2875 protein families comprising of 623 core, 2203 accessory, and 49 unique protein families. In Dehalococcoides mccartyi the complement of reductive dehalogenase genes varies by strain, but what was most surprising was how the majority of rdhA sequences actually exhibit a remarkable degree of synteny across all D. mccartyi genomes. Several homologous sequences are also shared with Dehalogenimonas genomes. Nucleotide and predicted protein sequences for all reductive dehalogenases were aligned to begin to decode the evolutionary history of reductive dehalogenases in the Dehalococcoidia.\n\nConclusionsThe conserved synteny of the rdhA genes observed across Dehalococcoides genomes indicates that the major differences between strain rdhA gene complement has resulted from gene loss rather than recombination. These rdhA have a long evolutionary history and trace their origin in the Dehalococcoidia prior to the speciation of Dehalococcoides and Dehalogenimonas. The only rdhA genes suspected to have been acquired by lateral gene transfer are protein-coding rdhA that have been identified to catalyze dehalogenation of industrial pollutants. Sequence analysis suggests that evolutionary pressures resulting in new rdhA genes involve adaptation of existing dehalogenases to new substrates, mobilization of rdhA between genomes or within a genome, and to a lesser degree manipulation of regulatory regions to alter expression.

bioinformatics↗