DsbA and DsbC affect extracellular enzyme formation in Pseudomonas aeruginosa

DsbA and DsbC proteins involved in the periplasmic formation of disulfide b onds in Pseudomonas aeruginosa were identified acid shown to play an import ant role for the formation of extracellular enzymes. Mutants deficient in e ither dsbA or dsbC or both genes were constructed, and extracellular elasta se, alkaline phosphatase, and lipase activities were determined. The dsbA m utant no longer produced these enzymes, whereas the lipase activity was dou bled in the dsbC mutant. Also, extracellar lipase production was severely r educed in a P. aeruginosa dsbA mutant in which an inactive DsbA variant car rying the mutation C34S was expressed. Even when the lipase gene lipA was c onstitutively expressed in trans in a lipA dsbA double mutant, lipase activ ity in cell extracts acid culture supernatants was still reduced to about 2 5%. Interestingly, the presence of dithiothreitol in the growth medium comp letely inhibited the formation of extracellular lipase whereas the addition of dithiothreitol to a cell-free culture supernatant did not affect lipase activity. We conclude that the correct formation of the disulfide bond cat alyzed in vivo by DsbA is necessary to stabilize periplasmic lipase. Such a stabilization is the prerequisite for efficient secretion using the type I I pathway.