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

Abstract

Multiple reductive dehalogenase (RDase), hydrogenase (H 2 ase), 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 H 2 ases, including a periplasmic [Ni/Fe] H 2 ase (Hup) and a cytoplasmic [Fe] H 2 ase (Vhu). mRNA transcripts extracted from tetrachloroethene-dechlorinating mixed cultures corresponding to Fdh, the H 2 ase 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). H 2 ase 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.Chlorinated ethenes are common groundwater contaminants that are found at more than half of the sites on the EPA's National Priorities List (1). A mixed enrichment culture capable of reductively dechlorinating tetrachloroethene (PCE) successively to trichloroethene (TCE), cis-1,2-dichloroethene (cDCE), vinyl chloride (VC), and the harmless compound ethene was developed to study this microbially mediated process (4). This enrichment culture (D2) has been maintained for more than a decade and is currently the source of an extract required to grow pure cultures of Dehalococcoides ethenogenes strain 195, a member of the Chloroflexi phylum capable of complete anaerobic reductive dechlorination of PCE. D. ethenogenes uses H 2 as its sole electron donor; in the D2 enrichment culture, H 2 is formed from the anaerobic fermentation of butyric acid to acetate and H 2 (3, 6). This organism, which was isolated from an earlier generation of the D2 enrichment culture, has been well characterized and its genome has been sequenced (14,16,18,20). Characterization of additional Dehalococcoides strains indicates that although all representatives have multiple reductive dehalogenase (RDase) genes, both the number of RDase genes and the corresponding substrate ranges can vary by strain (8,11,22).Of the 19 putat...

Identification and transcriptional analysis of trans-DCE-producing reductive dehalogenases in Dehalococcoides species

“…The dceA7 gene (100% identity to DET0088) is truncated, and the 3 0 anchoring protein is replaced by a hypothetical protein. Nevertheless, recent studies have reported that the expression of DET0088 was upregulated during PCE dechlorination in Alameda Naval Air Station enrichments and mixed cultures containing strain 195 (Rahm et al, 2006;West et al, 2008). Also, the dceA7 gene was identified using the newly designed primer pairs, RDH F1C and RDH R1C, revealing inadequacies of the previous degenerate primer pair.…”

Regulation of reductive dehalogenase gene transcription in Dehalococcoides mccartyi

“…Studies with trichloroethene (TCE)-and tetrachloroethene (PCE)-grown cells of strain 195 indicated that the genes encoding the PCE and TCE reductive dehalogenases (PceA, TceA) were transcribed to the highest levels among all 17 rdhA genes [25], and the corresponding proteins were identified with a high coverage in the proteome of the respective cells [26]. Furthermore, rdhA gene expression levels depended on the growth phase and the PCE respiration rate [27][28][29]. Consequently, they were used as biomarkers for active PCE or TCE dechlorination in technical and groundwater systems [30,31].…”

The restricted metabolism of the obligate organohalide respiring bacterium Dehalobacter restrictus: lessons from tiered functional genomics

“…This genus, although phylogenetically distant to Dehalobacter, inhabits similar ecological niches, and is exclusively dependent on OHR metabolism with H 2 as electron donor. These studies have used both transcriptomics using full genome microarrays and proteomics to identify key components of the metabolism of OHRB under different growth conditions or growth phases [23][24][25][26][27][28][29].…”