Transcriptional Analysis of a Dehalococcoides-Containing Microbial Consortium Reveals Prophage Activation

Abstract

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. C hlorinated solvents are among the most prevalent groundwater contaminants in industrialized countries, and their presence poses risks to human and environmental health. KB-1 is an anaerobic microbial community dominated by Dehalococcoides organisms that efficiently dechlorinates tetrachloroethene (PCE) and trichloroethene (TCE) to ethene (7,8,38). Dehalococcoides grows slowly in isolation and often has complex nutritional requirements, whereas Dehalococcoides growing in mixed communities grows and dechlorinates more rapidly, producing very robust cultures. This suggests beneficial relationships between nondechlorinating organisms and Dehalococcoides. It is therefore important to identify Dehalococcoides and non-Dehalococcoides genes within these communities that are expressed during dechlorination.There have been a few published microarray studies involving Dehalococcoides. In the first, Johnson et al. (14) examined transcription in Dehalococcoides strain 195 along a timeline from exponential to stationary phase in an effort to understand factors that limit the growth of this slow-growing isolate. They also used the arrays to investigate transc...

Sustainable Growth of Dehalococcoides mccartyi 195 by Corrinoid Salvaging and Remodeling in Defined Lactate-Fermenting Consortia

“…A previous study revealed that a number of phage-related genes in Dhc195 are significantly upregulated when the growth phase transits from late exponential phase to early stationary phase (33). Similarly, a D. mccartyi phage was induced in the dechlorinating KB-1 culture when TCE was omitted (50). This suggests that the phage-related genes in D. mccartyi might be induced under environmental stress.…”

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

“…Early transcriptional studies had detected genes encoding BvcA and VcrA in KB-1 cultures dechlorinating TCE, cis-DCE, VC, and 1,2-DCA and had also found that BvcA gene transcripts were more abundant with the substrate cDCE (17). Subsequent microarray data that found genes for VcrA most highly transcribed in KB-1 cultures in general, with vcrA being the only RDase gene transcribed in KB-1 cultures grown on VC (41). These microarray data also revealed transcription of the Geobacter RDase gene in a TCE-in- duced KB-1 culture (41).…”

“…The KB-1 metagenome (Joint Genome Institute IMG Taxon Object ID 2013843002), including all known curated KB-1 dehalogenase sequences from multiple studies (6,21,41) (final database of 40,772 entries) was used as a sequence database to compare to mass spectra. All MS/MS samples were analyzed using Mascot (version 2.3.02; Matrix Science, London, United Kingdom).…”

Genomic insights into organohalide respiration

“…Low cellular yields in the Dehalococcoidetes and Dehalobacter, oxygen sensitivity of RDases, and failure to produce functional heterologous RDases to date are key hurdles to purification and characterization of an RDase's substrate range. In addition, even when fed a single substrate Dehalococcoides strains have been shown to express multiple RDases simultaneously -further confounding purification of individual RDases from OHRB cultures [47,48,56]. Recent work with heterologous expression of PceA from D. restrictus [49 ] employed removal of the twin arginine ('RR') leader sequence, and addition of two fusions before cloning (streptavidin, to help with recovery, and PceT trigger factor).…”