Molecular biomarkers could provide critical insight into myriad in situ microbial activities. In this study we explore correlations of both mRNA and protein biomarkers with chloroethene respiration rate in Dehalococcoides. In a series of continuously fed dechlorinating mixed-culture microcosm experiments (n = 26), we varied respiratory substrates, substrate ratios and feeding rates. Transcript levels for most biomarkers were responsive down to 0.01× the culture's maximum respiration rate. The dehalogenase TceA and the Ni−Fe hydrogenase HupL transcripts were positively correlated (Pearson's r of 0.89 and 0.88, respectively) with respiration rates on log−log plots between 1.5 and 280 μeeq/ L-hr for mRNA abundances of 10 7 to 10 10 transcripts/mL (0.07−230 transcripts/genome). These trends were independent of the types of chloroethene or electron donors fed. Other mRNA target levels plateaued or declined at respiration rates above 5 μeeq/L-hr. Using both relative and absolute protein quantification methods, we found that per-genome protein abundances of most targeted biomarkers did not statistically change over the experimental time frames. However, quantified enzyme levels allowed us to calculate in vivo enzyme-specific rate constants (k cat ) for the dehalogenases PceA and TceA: 400 and 22 substrate molecules/enzyme-sec, respectively. Overall, these data support the promise of both mRNA and protein biomarkers for estimating process rates through either empirical (mRNAbased) or kinetic (protein-based) models, but they require follow-up studies in other cultures and at active remediation sites.

A cDNA-microarray was designed and used to monitor the transcriptomic profile of Dehalococcoides mccartyi strain 195 (in a mixed community) respiring various chlorinated organics, including chloroethenes and 2,3-dichlorophenol. The cultures were continuously fed in order to establish steady-state respiration rates and substrate levels. The organization of array data into a clustered heat map revealed two major experimental partitions. This partitioning in the data set was further explored through principal component analysis. The first two principal components separated the experiments into those with slow (1.6 ؎ 0.6 M Cl ؊ /h)-and fast (22.9 ؎ 9.6 M Cl ؊ /h)-respiring cultures. Additionally, the transcripts with the highest loadings in these principal components were identified, suggesting that those transcripts were responsible for the partitioning of the experiments. By analyzing the transcriptomes (n ‫؍‬ 53) across experiments, relationships among transcripts were identified, and hypotheses about the relationships between electron transport chain members were proposed. One hypothesis, that the hydrogenases Hup and Hym and the formate dehydrogenase-like oxidoreductase (DET0186-DET0187) form a complex (as displayed by their tight clustering in the heat map analysis), was explored using a nondenaturing protein separation technique combined with proteomic sequencing. Although these proteins did not migrate as a single complex, DET0112 (an FdhB-like protein encoded in the Hup operon) was found to comigrate with DET0187 rather than with the catalytic Hup subunit DET0110. On closer inspection of the genome annotations of all Dehalococcoides strains, the DET0185-to-DET0187 operon was found to lack a key subunit, an FdhB-like protein. Therefore, on the basis of the transcriptomic, genomic, and proteomic evidence, the place of the missing subunit in the DET0185-to-DET0187 operon is likely filled by recruiting a subunit expressed from the Hup operon (DET0112). 5), which pass an electron to halogenated organics, and hydrogenases (H 2 ases), which oxidize the sole known electron donor of D. mccartyi, hydrogen (15, 16). Although expression has been confirmed at both the mRNA and protein levels for RDases (differing with the strain), H 2 ases (Hup, Hym-1, Hym-2, Vhu, Ech, and Hyc [5,17,18]), and other putative membrane-bound oxidoreductase proteins, the interactions among these proteins are not well described (19). Three of the other notable putative oxidoreductases are DET0187, a predicted (but not biochemically confirmed) molybdopterin-containing protein annotated as a formate dehydrogenase that maintains an atypical serine in its active site rather than the typical cysteine or selenocysteine (15); Nuo, a NADH-ubiquinone oxidoreductase that lacks the NADH-receiving subunits (NuoEFG) in its predicted operon; and Mod, an additional predicted (but not biochemically confirmed) molybdopterin-containing oxidoreductase.The main goal of this study was to observe broad transcriptional expression patterns during the steady-state...

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Bioremediation of chlorinated ethenes via anaerobic reductive dechlorination relies upon the activity of specific microbial populations--most notably Dehalococcoides (DHC) strains. In the lab and field Dehalococcoides grow most robustly in mixed communities which usually contain both fermenters and methanogens. Recently, researchers have been developing quantitative molecular biomarkers to aid in field site diagnostics and it is hoped that these biomarkers could aid in the modeling of anaerobic reductive dechlorination. A comprehensive biokinetic model of a community containing Dehalococcoides mccartyi (formerly D. ethenogenes) was updated to describe continuously fed reactors with specific biomass levels based on quantitative PCR (qPCR)-based population data (DNA and RNA). The model was calibrated and validated with subsets of chemical and molecular biological data from various continuous feed experiments (n = 24) with different loading rates of the electron acceptor (1.5 to 482 μeeq/L-h), types of electron acceptor (PCE, TCE, cis-DCE) and electron donor to electron acceptor ratios. The resulting model predicted the sum of dechlorination products vinyl chloride (VC) and ethene (ETH) well. However, VC alone was under-predicted and ETH was over predicted. Consequently, competitive inhibition among chlorinated ethenes was examined and then added to the model. Additionally, as 16S rRNA gene copy numbers did not provide accurate model fits in all cases, we examined whether an improved fit could be obtained if mRNA levels for key functional enzymes could be used to infer respiration rates. The resulting empirically derived mRNA "adjustment factors" were added to the model for both DHC and the main methanogen in the culture (a Methanosaeta species) to provide a more nuanced prediction of activity. Results of this study suggest that at higher feeding rates competitive inhibition is important and mRNA provides a more accurate indicator of a population's instantaneous activity than 16S rRNA gene copies alone as biomass estimates.