Absolute quantification of pceABCT gene products and characterization of the membrane protein complex responsible for the reduction of tetrachloroethene in Dehalobacter restrictus

The pceABCT gene cluster from Dehalobacter restrictus is one model system for tetrachloroethene (PCE) respiration. In the present study we aim to decipher the stoichiometry of the pceABCT gene products and the composition of PceA-containing membrane-bound protein complexes in D. restrictus. Quantitative proteomics (qProt), membrane extraction, CN-PAGE, and an in-gel enzymatic assay were developed. A qProt analysis performed on the membrane fraction revealed a 1.5:1 stoichiometric relationship between PceA and PceB, while PceC was about 60-times less abundant than PceA. Data from CN-PAGE and a newly developed in-gel PceA enzymatic assay revealed several forms of active PceA-containing complexes with masses of approximately 140, 180, and 250 kDa, depending on the physiological state of D. restrictus cells.