Chlorinated electron acceptor availability selects for specific<i>Dehalococcoides</i>populations in dechlorinating enrichment cultures and in groundwater

Pérez‐de‐Mora, Alfredo; Lacourt, Anna; McMaster, Michaye L.; Liang, Xinghua; Dworatzek, Sandra; Edwards, Elizabeth A.

doi:10.1101/175182

Cited by 3 publications

(7 citation statements)

References 66 publications

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“…KB-1 TM differs from the D2 culture in that it contains multiple DMC strains in addition to an organohalide respiring species of Geobacter. Furthermore, distinct RDases are transcribed when the KB-1 lab cultures maintained at the University of Toronto (KB1-UT) are fed TCE, cis- DCE, VC, or 1,2-dichloroethane [ 11 , 17 , 27 ]. Notably, enzymatic activity of BvcA using native enzyme gels from KB-1 TM enrichment cultures was only detected in enrichment cultures maintained on cis -DCE, whereas VcrA activity was detected in all enrichment cultures including the culture maintained solely on VC [ 11 ].…”

Section: Introductionmentioning

confidence: 99%

Biomarkers’ Responses to Reductive Dechlorination Rates and Oxygen Stress in Bioaugmentation Culture KB-1TM

et al. 2018

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Using mRNA transcript levels for key functional enzymes as proxies for the organohalide respiration (OHR) rate, is a promising approach for monitoring bioremediation populations in situ at chlorinated solvent-contaminated field sites. However, to date, no correlations have been empirically derived for chlorinated solvent respiring, Dehalococcoides mccartyi (DMC) containing, bioaugmentation cultures. In the current study, genome-wide transcriptome and proteome data were first used to confirm the most highly expressed OHR-related enzymes in the bioaugmentation culture, KB-1TM, including several reductive dehalogenases (RDases) and a Ni-Fe hydrogenase, Hup. Different KB-1™ DMC strains could be resolved at the RNA and protein level through differences in the sequence of a common RDase (DET1545-like homologs) and differences in expression of their vinyl chloride-respiring RDases. The dominant strain expresses VcrA, whereas the minor strain utilizes BvcA. We then used quantitative reverse-transcriptase PCR (qRT-PCR) as a targeted approach for quantifying transcript copies in the KB-1TM consortium operated under a range of TCE respiration rates in continuously-fed, pseudo-steady-state reactors. These candidate biomarkers from KB-1TM demonstrated a variety of trends in terms of transcript abundance as a function of respiration rate over the range: 7.7 × 10−12 to 5.9 × 10−10 microelectron equivalents per cell per hour (μeeq/cell∙h). Power law trends were observed between the respiration rate and transcript abundance for the main DMC RDase (VcrA) and the hydrogenase HupL (R2 = 0.83 and 0.88, respectively), but not transcripts for 16S rRNA or three other RDases examined: TceA, BvcA or the RDase DET1545 homologs in KB1TM. Overall, HupL transcripts appear to be the most robust activity biomarker across multiple DMC strains and in mixed communities including DMC co-cultures such as KB1TM. The addition of oxygen induced cell stress that caused respiration rates to decline immediately (>95% decline within one hour). Although transcript levels did decline, they did so more slowly than the respiration rate observed (transcript decay rates between 0.02 and 0.03 per hour). Data from strain-specific probes on the pangenome array strains suggest that a minor DMC strain in KB-1™ that harbors a bvcA homolog preferentially recovered following oxygen stress relative to the dominant, vcrA-containing strain.

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Section: Introductionmentioning

confidence: 99%

Biomarkers’ Responses to Reductive Dechlorination Rates and Oxygen Stress in Bioaugmentation Culture KB-1TM

et al. 2018

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“…The 16S rRNA gene and RDase gene abundances at another bioaugmented, TCE-contaminated field site displayed similar results – the 16S rRNA, vcrA , and DET1545 homolog genes were the most abundant, with bvcA and tceA detected at lower levels (Lee et al, 2008). Varying levels of DNA biomarkers result from the presence of multiple strains of D. mccartyi at the ISSO site – including native populations and multiple strains of D. mccartyi in the KB-1 TM inoculum (Duhamel and Edwards, 2007; Wei et al, 2016; Pérez de Mora et al, 2017). Although hupL is present in all strains of D. mccartyi , the hupL primers initially used in this study were specific for D. mccartyi strain 195 which may explain the low ratio of gene copies of hupL versus 16S rRNA genes (Figure 1).…”

Section: Resultsmentioning

confidence: 99%

“…Details of the site have previously been published (Mundle et al, 2012; Pérez-de-Mora et al, 2014, Pérez de Mora et al, 2017). The EISB system was installed about 3 years prior to sampling.…”

Section: Methodsmentioning

confidence: 99%

“…In this study, groundwater samples were collected from a well-characterized, TCE-contaminated industrial site in southern Ontario, Canada (ISSO) (Mundle et al, 2012; Pérez-de-Mora et al, 2014, Pérez de Mora et al, 2017). The presence of cis -DCE and VC in the site groundwater indicated that TCE-dechlorination by native microorganisms was occurring, albeit at slow rates.…”

Section: Introductionmentioning

confidence: 99%

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Detection of Organohalide-Respiring Enzyme Biomarkers at a Bioaugmented TCE-Contaminated Field Site

et al. 2019

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RNA-based biomarkers have been successfully detected at field sites undergoing in situ bioremediation, but the detection of expressed enzymes is a more direct way to prove activity for a particular biocatalytic process of interest since they provide evidence of potential in situ activity rather than simply confirming presence and abundance of genes in a given population by measurement of DNA copies using qPCR. Here we successfully applied shotgun proteomics to field samples from a trichloroethene (TCE)-contaminated industrial site in southern Ontario, Canada that had been bio-augmented with the commercially available KB-1 TM microbial culture. The KB-1 TM culture contains multiple strains of Dehalococcoides mccartyi ( D. mccartyi ) as well as an organohalide respiring Geobacter species. The relative abundances of specific enzymatic proteins were subsequently compared to corresponding qPCR-derived levels of DNA and RNA biomarkers in the same samples. Samples were obtained from two wells with high hydraulic connectivity to the KB-1 TM -bioaugemented enhanced in situ bioremediation system, and two control wells that showed evidence of low levels of native organohalide respiring bacteria (OHRB), Dehalococcoides and Geobacter . Enzymes involved in organohalide respiration were detected in the metaproteomes of all four field samples, as were chaperonins of D. mccartyi , chemotaxis proteins, and ATPases. The most highly expressed RDase in the bioaugmentation culture (VcrA) was the most highly detected enzyme overall in the bioaugmented groundwater samples. In one background groundwater well, we found high expression of the Geobacter pceA RDase. The DNA and RNA biomarkers detected using qPCR-based assays were a set of orthologs of Dehalococcoides reductive dehalogenases (VcrA, TceA, BvcA, dehalogenase “DET1545”), and the Ni-Fe uptake hydrogenase, HupL. Within a sample, RNA levels for key enzymes correlated with relative protein abundance. These results indicate that laboratory observations of TCE-bioremediation biomarker protein expression are recapitulated in field environmental systems and that both RNA and protein biomarker monitoring hold promise for activity monitoring of in situ populations of OHRB.

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“…In the KB-1™ culture, chlorinated ethenes, such as PCE and TCE, are sequentially dechlorinated via cis -dichloroethene (cDCE) and VC to ethene (Duhamel et al 2002; Perez-de-Mora et al 2017; Molenda et al 2020). Reductive dechlorination in KB-1™ is primarily performed by multiple Dehalococcoides mccartyi strains in a growth-linked process called organohalide respiration (Duhamel et al 2002; Perez-de-Mora et al 2017; Molenda et al 2020). Based on 16S rRNA sequence similarity, all D. mccartyi strains are categorized into three phylogenetic clades or sub-groups: Pinellas, Victoria, and Cornell.…”

Section: Introductionmentioning

confidence: 99%

Long term survival ofDehalococcoides mccartyistrains in mixed cultures under electron acceptor and ammonium limitation

Morson

Molenda

Picott

et al. 2020

Preprint

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Vinyl chloride (VC) is a human carcinogen that accumulates in soil and groundwater due to incomplete dechlorination of chlorinated ethenes. Some strains of obligate organohalide respiring Dehalococcoides mccartyi can synthesize the VC reductase that catalyzes the dechlorination of VC to ethene. The gene encoding the VC reductase, vcrA, is found on a mobile genetic element called the vcrA-Genomic Island (GI) that may participate in horizontal gene transfer. We designed an experiment to try to induce horizontal gene transfer of the vcrA-GI by mixing two enrichment cultures: one containing the donor D. mccartyi strain with the vcrA-GI that could not fix nitrogen and the second containing the recipient strain devoid of the vcrA-GI that could fix nitrogen. Therefore, mixing the two cultures in medium without ammonium while providing VC as the sole electron acceptor was hypothesized to select for a mutant strain of D. mccartyi that could both fix nitrogen and respire VC. However, after over 4 years of incubation, no evidence for horizontal gene transfer of the vcrA-GI was found. Rather, we observed VC-dechlorinating activity attributed to the TCE reductase, TceA, in the recipient strain. We also observed that D. mccartyi can grow by scavenging low concentrations of fixed nitrogen sources. During this experiment we identified two additional D. mccartyi strains in the KB-1 TCE-enriched culture that could fix nitrogen. The presence of multiple strains of D. mccartyi with distinct phenotypes may enhance bioaugmentation success, but here it may have undermined attempts to force horizontal gene transfer of the vcrA-GI.IMPORTANCEDehalococcoides mccartyi are a powerful bioremediation tool for the degradation of chlorinated solvent contamination in soil and groundwater. Only a few D. mccartyi strains have the ability to dechlorinate toxic chlorinated compounds like vinyl chloride. Interestingly, the genetic ability to dechlorinate vinyl chloride is theorized to be shared among D. mccartyi strains. In this study we attempted to promote the genetic transfer of vinyl chloride degrading ability from one D. mccartyi strain to another. Although we did not observe this exchange, our findings suggest there may be restrictions of genetic transfer between specific clades or sub-groups of D. mccartyi strains. Developing our understanding of genetic transfer among D. mccartyi strains could allow for enhanced degradation of chlorinated solvent contamination in situ.

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Chlorinated electron acceptor availability selects for specificDehalococcoidespopulations in dechlorinating enrichment cultures and in groundwater

Cited by 3 publications

References 66 publications

Biomarkers’ Responses to Reductive Dechlorination Rates and Oxygen Stress in Bioaugmentation Culture KB-1TM

Biomarkers’ Responses to Reductive Dechlorination Rates and Oxygen Stress in Bioaugmentation Culture KB-1TM

Detection of Organohalide-Respiring Enzyme Biomarkers at a Bioaugmented TCE-Contaminated Field Site

Long term survival ofDehalococcoides mccartyistrains in mixed cultures under electron acceptor and ammonium limitation

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