Biochemistry of respiration in Dehalococcoides strain CBDB1

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Strain CBDB1 will be grown on several different electron acceptors. A native gel electrophoresis approach in combination with mass spectrometry will be applied to identify reductive dehalogenases in the organism. Special emphasis will be focused on differentiation of the enzyme sets synthesized with comparable substrates such as 1,2,3- vs. 1,2,4-trichlorobenzene or 2,3,4-trichlorobiphenyl vs. 2,4,5-trichlorobiphenyl. Native gel electrophoresis, possibly after treatment with crosslinking agents in combination with mass spectrometry of digestion peptides, will be used to analyze interactions with other proteins of the respiratory chain. Biochemical tests (activity test with viologens, inhibition studies) will characterize in more detail identified dehalogenases with respect to cofactors, substrate spectra, orientation in the membrane and the structure and role of the B-protein. As in S. multivorans the B-protein is a small transmembrane protein that is always encoded in reductive dehalogenase operons and is thought to be a membrane anchor. In cooperation with subproject 4, which will study hydrogenases and the annotated formate dehydrogenase in strain CBDB1, we will try to identify membrane-bound electron mediators that shuttle electrons between hydrogenase(s) and the reductive dehalogenase(s). For the identification of membrane-bound redox mediators we will use GC-MS, electrochemical reactions and predictions deduced from current comparative genome annotations.