Functional analysis of paralogous thiol-disulfide oxidoreductases in Bacillus subtilis

The in vivo formation of disulfide bonds, which is critical for the stabili ty and/or activity of many proteins, is catalyzed by thiol-disulfide oxidor eductases. In the present studies, we show that the Gram-positive eubacteri um Bacillus subtilis contains three genes, denoted bdbA, bdbB, and bdbC, fo r thiol-disulfide oxidoreductases, Escherichia coil alkaline phosphatase, c ontaining two disulfide bonds, was unstable when secreted by B. subtilis ce lls lacking BdbB or BdbC, and notably, the expression levels of bdbB and bd bC appeared to set a limit for the secretion of active alkaline phosphatase , Cells lacking BdbC also showed decreased stability of cell-associated for ms of E. coli TEM-beta-lactamase, containing one disulfide bond. In contras t, BdbA was not required for the stability of alkaline phosphatase or beta- lactamase. Because BdbB and BdbC are typical membrane proteins, our finding s suggest that they promote protein folding at the membrane-cell wall inter face. Interestingly, pre-beta-lactamase processing to its mature form was s timulated in cells lacking BdbC, suggesting that the unfolded form of this precursor is a preferred substrate for signal peptidase. Surprisingly, cell s lacking BdbC did not develop competence for DNA uptake, indicating the in volvement of disulfide bond-containing proteins in this process. Unlike E. coli and yeast, none of the thiol-disulfide oxidoreductases of B, subtilis was required for growth in the presence of reducing agents. In conclusion, our observations indicate that BdbB and BdbC have a general role in disulfi de bond formation, whereas BdbA may be dedicated to a specific process.