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.

Degenerate primers were used to amplify 14 distinct reductive-dehalogenase-homologous (RDH) genes from the Dehalococcoides-containing mixed culture KB1. Most of the corresponding predicted proteins were highly similar (97 to >99% amino acid identity) to previously reported Dehalococcoides reductive dehalogenases. To examine the differential transcription of these RDH genes, KB1 was split into five subcultures amended with either trichloroethene, cis-1,2-dichloroethene, vinyl chloride, 1,2-dichlorethane, or no chlorinated electron acceptor. Total RNA was extracted following the onset of reductive dechlorination, and RDH transcripts were reverse transcribed and amplified using degenerate primers. The results indicate that the transcription of RDH genes requires the presence of a chlorinated electron acceptor, and for all treatments, multiple RDH genes were simultaneously transcribed, with transcripts of two of the genes being present under all four electron-accepting conditions. Two of the transcribed sequences were highly similar to reported vinyl chloride reductase genes, namely, vcrA from Dehalococcoides sp. strain VS and bvcA from Dehalococcoides sp. strain BAV1. These findings suggest that multiple RDH genes are induced by a single chlorinated substrate and that multiple reductive dehalogenases contribute to chloroethene degradation in KB1.