Project name: Dehalococcoides mccartyi 195

Description: 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 throughprincipal component analysis. The first two principal components separated the experiments into those with slow (1.6 plus or minus 0.6 M Cl- per h) and fast (22.9 plus or minus 9.6 M Cl- per 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 Hupand 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).Overall design: 53 arrays total: 3 biological replicates of PCE High, 2 biological replicates of PCE High Inhibited, 3 biological replicates of PCE Low, 3 biological replicates of PCE Half Butyrate, 2 biological replicates of PCE Lactate, 2 biological replicates of PCE Butyrate, 2 replicates of hydrogen control, 2 biological replicates of PCE hydrogen, 2 biological replicates of PCE + no donor, 2 biological replicates of PCE + yeast extract, 2 biological replicates of PCE + fermented yeast extract, 2 biological replicates of each 3 feed rates for PCE (6 total), 2 biological replicates of each 3 feed rates for TCE (6 total), 2 biological replicates of each 3 feed rates for dCE (6 total), 3 biological replicates for dCE 10, 2 biological replicates for 2 feed rates for Chlorophenols (4 total), and 3 decay samples.

Data type: Transcriptome or Gene expression

Sample scope: Multiisolate

Relevance: Other

Last updated: 2010-12-22