DsbA and DsbC affect extracellular enzyme formation in Pseudomonas aeruginosa.
DsbA and DsbC proteins involved in the periplasmic formation of disulfide bonds in Pseudomonas aeruginosa were identified and shown to play an important role for the formation of extracellular enzymes. Mutants deficient in either dsbA or dsbC or both genes were constructed, and extracellular elastase, alkaline phosphatase, and lipase activities were determined. The dsbA mutant no longer produced these enzymes, whereas the lipase activity was doubled in the dsbC mutant. Also, extracellar lipase production was severely reduced in a P. aeruginosa dsbA mutant in which an inactive DsbA variant carrying the mutation C34S was expressed. Even when the lipase gene lipA was constitutively expressed in trans in a lipA dsbA double mutant, lipase activity in cell extracts and culture supernatants was still reduced to about 25%. Interestingly, the presence of dithiothreitol in the growth medium completely 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 catalyzed in vivo by DsbA is necessary to stabilize periplasmic lipase. Such a stabilization is the prerequisite for efficient secretion using the type II pathway.
Urban
A
A
Lehrstuhl für Biologie der Mikroorganismen, Ruhr-Universität Bochum, D-44780 Bochum, Germany.
Leipelt
M
M
Eggert
T
T
Jaeger
K E
KE
D000469:Alkaline Phosphatase / Q000378:metabolism*; D001426:Bacterial Proteins; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005816:Genetic Complementation Test; D008049:Lipase / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009068:Movement; D009154:Mutation; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits; D011550:Pseudomonas aeruginosa / Q000201:enzymology*
D001426:Bacterial Proteins; D021122:Protein Subunits; D008049:Lipase; D000469:Alkaline Phosphatase; D008666:Metalloendopeptidases; C043859:pseudolysin, Pseudomonas aeruginosa; D019704:Protein Disulfide-Isomerases