Molecular Biomarker-Based Biokinetic Modeling of a PCE-Dechlorinating and Methanogenic Mixed Culture

Heavner, Gretchen L.; Rowe, Annette R.; Mansfeldt, Cresten; Pan, Ju Khuan; Gossett, James M.; Richardson, Ruth

doi:10.1021/es303517s

Cited by 22 publications

(22 citation statements)

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“…All 100-ml subcultures studied in this experiment were derived from a previously described 6-liter mixed-community culture containing D. mccartyi strain 195 (the D2 culture [27][28][29][30][31][32]) (the sequenced D2 metagenome is freely available at JGI's Integrated Microbial Genomes database under taxon ID 2088090019). The culture contains a D. mccartyi strain 195 population (ϳ5 ϫ 10 8 cells/ml; 60% of the total community population) mixed with methanogens and organisms that ferment organic substrates.…”

Section: Methodsmentioning

confidence: 99%

“…The setup for the 50 continuously fed cultures has been described previously (27,30). In brief, Pressure-Lok syringes (VICI Precision Sampling, Baton Rouge, LA) were loaded onto a Cole Parmer (Vernon Hills, IL) syringe pump, model 74900, to deliver the electron acceptor and donor stocks at the desired rates.…”

Section: Methodsmentioning

confidence: 99%

“…The complete setup for each of these experiments is detailed in the literature (29,30) and in Table S1 in the supplemental material.…”

Section: Methodsmentioning

confidence: 99%

“…Previous experiments investigating continuously fed cultures tied the abundances of preselected transcripts, determined via reverse transcription-quantitative PCR (RT-qPCR) assays, to the respiration rate of D. mccartyi strain 195 (27)(28)(29)(30). Those studies found the strongest correlations with the respiration rate for the tceA and hupL transcripts.…”

mentioning

confidence: 99%

“…Those studies found the strongest correlations with the respiration rate for the tceA and hupL transcripts. In the current study, we used two-color microarrays to monitor the transcriptome of the sole organohalorespiring D. mccartyi strain, strain 195, in the well-characterized D2 mixedcommunity culture (27)(28)(29)(30)(31)(32) (29). By performing meta-analyses of the full transcriptome rather than a limited number of preselected RT-qPCR targets, genome-wide expression responses were noted, suggesting novel connections between annotated proteins.…”

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confidence: 99%

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Meta-Analyses of Dehalococcoides mccartyi Strain 195 Transcriptomic Profiles Identify a Respiration Rate-Related Gene Expression Transition Point and Interoperon Recruitment of a Key Oxidoreductase Subunit

Mansfeldt

Rowe

Heavner

et al. 2014

Appl Environ Microbiol

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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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