Bioindicators of Reductive Dechlorination in a Dehalococcoides ethenogenes-Containing Mixed Culture: Transcriptional Trends With Respect to Substrate Type, Substrate Concentration, and Culture Operation
Expression levels of potentially important gene transcripts were examined in a Dehalococcoides ethenogenes strain 195 (DET)-containing mixed culture (D2) capable of reductively dechlorinating tetrachloroethene (PCE) successively to trichloroethene (TCE), cis-1,2-dichloroethene (cDCE), vinyl chloride (VC), and non-toxic ethene using H2 as electron donor. Gene transcripts associated with general cell activity, including those corresponding to 16S rRNA and subunits of RNA polymerase (RpoB) and an ATP synthase, and also multiple reductive dehalogenase (RDase), hydrogenase (H2ase) and other oxidoreductase enzymes were selected for study as potential bioindicators of reductive dechlorination in DET. Quantitative reverse-transcriptase PCR (qRT-PCR) data on selected gene transcripts in batch PCE-fed microcosms indicated that a subset of targets, including RDases TceA, PceA, DET1559 and DET1545, the H2ase Hup, and a gene annotated as formate dehydrogenase (Fdh), were highly up-regulated within 1 to 12 h after PCE feeding. Transcription profiles over time suggested that genes belonging to similar functional groups were regulated in similar ways. Expression studies in cultures fed either electron donor or acceptor showed that neither PCE nor H2 alone was sufficient to signal up-regulation of chosen bioindicators. Experiments in microcosms continuously fed medium containing PCE showed that pseudo-steady-state mRNA levels were achieved and that increases in PCE loading rate led to corresponding increases in chloroethene respiration rate. Regulation of steady-state transcript levels of most bioindicators was sensitive to chloroethene respiration rate and/or concentration. Within a limited range of respiration rates (1.5 ? 4.8 ?mol/L/hr), Fdh, Hup, TceA, PceA and DET1559 transcript levels displayed positive correlation with respiration rate, and could be well-fit with linear regression models (R2 between 0.95 - 0.97). At high PCE respiration rates, however, most bioindicator levels reached a plateau or decreased, the reasons for which remain unknown. At very low PCE respiration rates, RDases DET1559 and DET1545 were the only potential bioindicators up-regulated above time-0 levels, suggesting they play key roles in reductive dechlorination when substrate concentrations are low. Fdh, Hup and TceA transcript abundances were also high in microcosms continuously fed medium containing TCE or cDCE. Experiments using these alternate electron acceptors indicated that while some RDase bioindicators could reflect substrate utilization, the H2ase Hup was a more accurate and sensitive indicator of cellular respiration rate.
Reductive Dechlorination; Chlorinated Ethenes; Dehalococcoides; Transcription
dissertation or thesis