Loop-Mediated Isothermal Amplification (LAMP) for Rapid Detection and Quantification of Dehalococcoides Biomarker Genes in Commercial Reductive Dechlorinating Cultures KB-1 and SDC-9

ABSTRACTReal-time quantitative PCR (qPCR) protocols specific to the reductive dehalogenase (RDase) genesvcrA,bvcA, andtceAare commonly used to quantifyDehalococcoidesspp. in groundwater from chlorinated solvent-contaminated sites. In this study, loop-mediated isothermal amplification (LAMP) was developed as an alternative approach for the quantification of these genes. LAMP does not require a real-time thermal cycler (i.e., amplification is isothermal), allowing the method to be performed using less-expensive and potentially field-deployable detection devices. Six LAMP primers were designed for each of three RDase genes (vcrA,bvcA, andtceA) using Primer Explorer V4. The LAMP assays were compared to conventional qPCR approaches using plasmid standards, two commercially available bioaugmentation cultures, KB-1 and SDC-9 (both containDehalococcoidesspecies). DNA was extracted over a growth cycle from KB-1 and SDC-9 cultures amended with trichloroethene and vinyl chloride, respectively. All three genes were quantified for KB-1, whereas onlyvcrAwas quantified for SDC-9. A comparison of LAMP and qPCR using standard plasmids indicated that quantification results were similar over a large range of gene concentrations. In addition, the quantitative increase in gene concentrations over one growth cycle of KB-1 and SDC-9 using LAMP was comparable to that of qPCR. The developed LAMP assays forvcrAandtceAgenes were validated by comparing quantification on the Gene-Z handheld platform and a real-time thermal cycler using DNA isolated from eight groundwater samples obtained from an SDC-9-bioaugmented site (Tulsa, OK). These assays will be particularly useful at sites subject to bioaugmentation with these two commonly usedDehalococcoidesspecies-containing cultures.

A comparative genomics and reductive dehalogenase gene transcription study of two chloroethene-respiring bacteria, Dehalococcoides mccartyi strains MB and 11a

Genomes of two trichloroethene (TCE)-respiring Dehalococcoides (Dhc) mccartyi, strains MB and 11a, were sequenced to identify reductive dehalogenases (RDase) responsible for oraganohalide respiration. Transcription analyses were conducted to verify the roles of RDase subunit A genes (rdhA) in chloroethene respiration. Some interesting features of the strain MB draft genome include a large genome size, two CRISPR-cas type I systems and 38 rdhA genes. Strain 11a has a stream-lined genome with 11 rdhA genes, of which nine are distinct. Quantitative real-time PCR transcription analysis of RDase gene transcripts showed that a single RDase gene, designated mbrA, was up-regulated upon exposure to TCE and no other RDase genes were considerably expressed in strain MB. A single RDase gene, designated vcrA, was up-regulated upon exposure to TCE and expressed at a steady level until all chloroethenes were completely dechlorinated to ethene at 147 h in strain 11a. Overall, this study reports the genomes of two distinct Dhc strains; both contain numerous uncharacterized RDase genes, but in each strain only one such gene was expressed highly during organohalide respiration.

Functional Characterization of Reductive Dehalogenases by Using Blue Native Polyacrylamide Gel Electrophoresis

ABSTRACTDehalococcoides mccartyistrains are obligate organohalide-respiring bacteria harboring multiple distinct reductive dehalogenase (RDase) genes within their genomes. A major challenge is to identify substrates for the enzymes encoded by these RDase genes. We demonstrate an approach that involves blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by enzyme activity assays with gel slices and subsequent identification of proteins in gel slices using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). RDase expression was investigated in cultures ofDehalococcoides mccartyistrain BAV1 and in the KB-1 consortium growing on chlorinated ethenes and 1,2-dichloroethane. In cultures of strain BAV1, BvcA was the only RDase detected, revealing that this enzyme catalyzes the dechlorination not only of vinyl chloride, but also of all dichloroethene isomers and 1,2-dichloroethane. In cultures of consortium KB-1, five distinctDehalococcoidesRDases and oneGeobacterRDase were expressed under the conditions tested. Three of the five RDases included orthologs to the previously identified chlorinated ethene-dechlorinating enzymes VcrA, BvcA, and TceA. This study revealed substrate promiscuity for these three enzymes and provides a path forward to further explore the largely unknown RDase protein family.

Isolation and Characterization of “Dehalococcoides” sp. Strain MB, Which Dechlorinates Tetrachloroethene to trans-1,2-Dichloroethene

ABSTRACT In an attempt to understand the microorganisms involved in the generation of trans-1,2-dichloroethene (trans-DCE), pure-culture “Dehalococcoides” sp. strain MB was isolated from environmental sediments. In contrast to currently known tetrachloroethene (PCE)- or trichloroethene (TCE)-dechlorinating pure cultures, which generate cis-DCE as the predominant product, Dehalococcoides sp. strain MB reductively dechlorinates PCE to trans-DCE and cis-DCE at a ratio of 7.3 (±0.4):1. It utilizes H2 as the sole electron donor and PCE or TCE as the electron acceptor during anaerobic respiration. Strain MB is a disc-shaped, nonmotile bacterium. Under an atomic force microscope, the cells appear singly or in pairs and are 1.0 μm in diameter and ∼150 nm in depth. The purity was confirmed by culture-based approaches and 16S rRNA gene-based analysis and was corroborated further by putative reductive dehalogenase (RDase) gene-based, quantitative real-time PCR. Although strain MB shares 100% 16S rRNA gene sequence identity with Dehalococcoides ethenogenes strain 195, these two strains possess different dechlorinating pathways. Microarray analysis revealed that 10 putative RDase genes present in strain 195 were also detected in strain MB. Successful cultivation of strain MB indicates that the biotic process could contribute significantly to the generation of trans-DCE in chloroethene-contaminated sites. It also enhances our understanding of the evolution of this unusual microbial group, Dehalococcoides species.

Monitoring Abundance and Expression of “Dehalococcoides” Species Chloroethene-Reductive Dehalogenases in a Tetrachloroethene-Dechlorinating Flow Column

ABSTRACT We investigated the distribution and activity of chloroethene-degrading microorganisms and associated functional genes during reductive dehalogenation of tetrachloroethene to ethene in a laboratory continuous-flow column. Using real-time PCR, we quantified “Dehalococcoides” species 16S rRNA and chloroethene-reductive dehalogenase (RDase) genes (pceA, tceA, vcrA, and bvcA) in nucleic acid extracts from different sections of the column. Dehalococcoides 16S rRNA gene copies were highest at the inflow port [(3.6 ± 0.6) × 106 (mean ± standard deviation) per gram soil] where the electron donor and acceptor were introduced into the column. The highest transcript numbers for tceA, vcrA, and bvcA were detected 5 to 10 cm from the column inflow. bvcA was the most highly expressed of all RDase genes and the only vinyl chloride reductase-encoding transcript detectable close to the column outflow. Interestingly, no expression of pceA was detected in the column, despite the presence of the genes in the microbial community throughout the column. By comparing the 16S rRNA gene copy numbers to the sum of all four RDase genes, we found that 50% of the Dehalococcoides population in the first part of the column did not contain either one of the known chloroethene RDase genes. Analysis of 16S rRNA gene clone libraries from both ends of the flow column revealed a microbial community dominated by members of Firmicutes and Actinobacteria. Higher clone sequence diversity was observed near the column outflow. The results presented have implications for our understanding of the ecophysiology of reductively dehalogenating Dehalococcoides spp. and their role in bioremediation of chloroethenes.

Quantifying Genes and Transcripts To Assess the In Situ Physiology of “Dehalococcoides” spp. in a Trichloroethene-Contaminated Groundwater Site

ABSTRACT Quantitative PCR (qPCR) was coupled with reverse transcription (RT) to analyze both gene copy numbers and transcripts of the 16S rRNA gene and three reductive dehalogenase (RDase) genes (tceA, vcrA, and bvcA) as biomarkers of “Dehalococcoides” spp. in the groundwater of a trichloroethene-dense nonaqueous-phase liquid site at Fort Lewis, WA, that was sequentially subjected to biostimulation and bioaugmentation. Dehalococcoides cells carrying the tceA, vcrA, and bvcA genes were indigenous to the site. The sum of the three identified RDase gene copy numbers closely correlated to 16S rRNA gene copy numbers throughout the biostimulation and bioaugmentation activity, suggesting that these RDase genes represented the major Dehalococcoides metabolic functions at this site. Biomarker quantification revealed an overall increase of more than 3 orders of magnitude in the total Dehalococcoides population through the 1-year monitoring period (spanning biostimulation and bioaugmentation), and measurement of the respective RDase gene concentrations indicated different growth dynamics among Dehalococcoides cells. The Dehalococcoides cells containing the tceA gene consistently lagged behind other Dehalococcoides cells in population numbers and made up less than 5% of the total Dehalococcoides population, whereas the vcrA- and bvcA-containing cells represented the dominant fractions. Quantification of transcripts in groundwater samples verified that the 16S rRNA gene and the bvcA and vcrA genes were consistently highly expressed in all samples examined, while the tceA transcripts were detected inconsistently, suggesting a less active physiological state of the cells with this gene. The production of vinyl chloride and ethene toward the end of treatment supported the physiological activity of the bvcA- and vcrA-carrying cells. A clone library of the expressed RDase genes in field samples produced with degenerate primers revealed the expression of two putative RDase genes that were not previously monitored with RT-qPCR. The level of abundance of one of the putative RDase genes (FtL-RDase-1638) identified in the cDNA clone library tracked closely in field samples with abundance of the bvcA gene, suggesting that the FtL-RDase-1638 gene was likely colocated in genomes containing the bvcA gene. Overall, results from this study demonstrate that quantification of biomarker dynamics at field sites can provide useful information about the in situ physiology of Dehalococcoides strains and their associated activity.

Unusual Codon Bias in Vinyl Chloride Reductase Genes of Dehalococcoides Species

ABSTRACT Vinyl chloride reductases (VC-RDase) are the key enzymes for complete microbial reductive dehalogenation of chloroethenes, including the groundwater pollutants tetrachloroethene and trichloroethene. Analysis of the codon usage of the VC-RDase genes vcrA and bvcA showed that these genes are highly unusual and are characterized by a low G+C fraction at the third position. The third position of codons in VC-RDase genes is biased toward the nucleotide T, even though available Dehalococcoides genome sequences indicate the absence of any tRNAs matching codons that end in T. The comparatively high level of abnormality in the codon usage of VC-RDase genes suggests an evolutionary history that is different from that of most other Dehalococcoides genes.

Comparative Proteomics of Dehalococcoides spp. Reveals Strain-Specific Peptides Associated with Activity

ABSTRACT Anaerobic reductive dehalogenation by Dehalococcoides spp. is an ideal system for studying functional diversity of closely related strains of bacteria. In Dehalococcoides spp., reductive dehalogenases (RDases) are key respiratory enzymes involved in the anaerobic detoxification of halogenated compounds at contaminated sites globally. Although housekeeping genes sequenced from Dehalococcoides spp. are >85% identical at the amino acid level, different strains are capable of dehalogenating diverse ranges of compounds, depending largely on the suite of RDase genes that each strain harbors and expresses. We identified RDase proteins that corresponded to known functions in four characterized cultures and predicted functions in an uncharacterized Dehalococcoides-containing mixed culture. Homologues within RDase subclusters containing PceA, TceA, and VcrA were among the most frequently identified proteins. Several additional proteins, including a formate dehydrogenase-like protein (Fdh), had high coverage in all strains and under all growth conditions.

Temporal Expression of Respiratory Genes in an Enrichment Culture Containing Dehalococcoides ethenogenes

ABSTRACT Multiple reductive dehalogenase (RDase), hydrogenase (H2ase), and other respiration-associated (RA) oxidoreductase genes have been identified in cultured representatives of Dehalococcoides. Although their products are likely to play key roles in the environmentally important process of reductive dechlorination, very little information is available about their regulation and specific functions. Here we show increased expression and temporal variability in the expression of five RDase genes and in the expression of genes for a putative formate dehydrogenase (Fdh) and two H2ases, including a periplasmic [Ni/Fe] H2ase (Hup) and a cytoplasmic [Fe] H2ase (Vhu). mRNA transcripts extracted from tetrachloroethene-dechlorinating mixed cultures corresponding to Fdh, the H2ase Hup, and the RDase targets TceA and DET0162 were expressed most highly, with average levels 34 (± 7.5)-, 23 (± 6.7)-, 16 (± 3.3)-, and 13 (± 3.3)-fold higher, respectively, than that for RNA polymerase (RpoB). H2ase and RA transcripts reached their respective expression maxima within the first 2 h after feeding. RDase transcripts, however, were most highly expressed after 3 h and exhibited greater temporal variability than other transcripts. Comparison with D. ethenogenes strain 195 pure culture expression levels indicated that RDase DET1545 was more highly expressed in mixed cultures, where, on average, its transcript level was sixfold higher than that of RpoB. While the specific functions of several of these gene products remain elusive, the high expression levels and temporal variability reported here suggest that these groups of enzymes are metabolically important for the respiration of chlorinated ethenes in mixed cultures containing Dehalococcoides.

Quantitative PCR Targeting 16S rRNA and Reductive Dehalogenase Genes Simultaneously Monitors Multiple Dehalococcoides Strains

ABSTRACT The 16S rRNA gene provides insufficient information to infer the range of chloroorganic electron acceptors used by different Dehalococcoides organisms. To overcome this limitation and provide enhanced diagnostic tools for growth measurements, site assessment, and bioremediation monitoring, a quantitative real-time PCR (qPCR) approach targeting 16S rRNA genes and three Dehalococcoides reductive dehalogenase (RDase) genes with assigned function (i.e., tceA, bvcA, and vcrA) was designed and evaluated. qPCR standard curves generated for the RDase genes by use of genomic DNA from Dehalococcoides pure cultures correlated with standard curves obtained for both Bacteria- and Dehalococcoides-targeted 16S rRNA genes, suggesting that the RDase genes are useful targets for quantitative assessment of Dehalococcoides organisms. RDase gene probe/primer pairs were specific for the Dehalococcoides strains known to carry the diagnostic RDase gene sequences, and the qPCR method allowed the detection of as few as 1 to 20 and quantification of as few as 50 to 100 tceA, bvcA, or vcrA gene targets per PCR volume. The qPCR approach was applied to dechlorinating enrichment cultures, microcosms, and samples from a contaminated site. In characterized enrichment cultures where known Dehalococcoides strains were enumerated, the sum of the three RDase genes equaled the total Dehalococcoides cell numbers. In site samples and chloroethane-dechlorinating microcosms, the sum of the three RDase genes was much less than that predicted by Dehalococcoides-targeted qPCR, totaling 10 to 30% of the total Dehalococcoides cell numbers. Hence, a large number of Dehalococcoides spp. contain as-yet-unidentified RDase genes, indicating that our current understanding of the dechlorinating Dehalococcoides community is incomplete.