DsbA and DsbC Affect Extracellular Enzyme Formation in Pseudomonas aeruginosa

Abstract

DsbA and DsbC proteins involved in the periplasmic formation of disulfide bonds in Pseudomonas aeruginosawere 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.Disulfide bonds are important for the structure and stability of numerous proteins. For Escherichia coli it is now well established that the formation of disulfide bonds is an assisted process which occurs in the periplasm and is catalyzed by the thiol-disulfide oxidoreductase DsbA (7, 34). DsbA acts as a donor of disulfides to newly synthesized periplasmic proteins (23) and is reoxidized by DsbB, a second protein located in the inner membrane (5, 6). DsbA and DsbB are members of the Dsb system (the system for disulfide bond formation) which consists of at least six redox proteins belonging to the thioredoxin superfamily. These proteins contain a canonical C-X-X-C motif in the dithiol active site and seem to be conserved throughout the gram-negative bacteria (49). DsbC is another member which is suggested to act as a disulfide isomerase (56, 67). DsbD (39) keeps DsbC in a reduced state (57). More recently, DsbG was described as a novel member of the Dsb family in E. coli which oxidizes so far unknown substrates (4,8).Undoubtedly, the process of protein secretion in gram-negative bacteria is related to the function of the Dsb system. However, the results obtained so far are contradictory. Four major secretion pathways exist in gram-negative bacteria to direct proteins into the extracellular medium (37). In type I and type III pathways the secreted proteins directly pass both the inner and the outer membrane using a machinery formed by either three or more than 20 different proteins, respectively. In the type II pathway, which is also called the general secretory pathway, secretion occurs in two consecutive steps (48), with an intermediate state in the periplasm where the formation of disulfide bonds can take place. Since E. coli does not secrete exoprotein...

The bdbDC Operon of Bacillus subtilisEncodes Thiol-disulfide Oxidoreductases Required for Competence Development

“…2) 2 and with the DsbG protein of Chlamydia trachomatis (22) (55 and 53% identical residues and conservative replacements in regions of 171 and 176 amino acids, respectively). In addition, we observed sequence similarity, albeit more limited, with DsbG of E. coli as well as the DsbA proteins of Haemophilus influenzae (23), Neisseria meningitidis (24), and Pseudomonas aeruginosa (25). A further characteristic of several thiol-disulfide oxidoreductases (but not of DsbA of S. aureus), namely a conserved Phe residue at position Ϫ5 relative to the CXXC motif (8,26), is also present in the predicted BdbD protein.…”

Characterization of a Pseudomonas putida ABC transporter (AatJMQP) required for acidic amino acid uptake: biochemical properties and regulation by the Aau two-component system

“…role in the maturation of extracellular enzymes (Urban et al, 2001), while ppsA (PP2082) encodes phosphoenolpyruvate synthase (PpsA), a key enzyme in gluconeogenesis from aketo acids (Chao et al, 1993).…”

Disulfide Bond in Pseudomonas aeruginosa Lipase Stabilizes the Structure but Is Not Required for Interaction with Its Foldase

“…The enzyme contains two cysteine residues which form a disulfide bond (20). Recently, it has been shown that this lipase is not secreted in a P. aeruginosa mutant strain devoid of the oxidoreductase DsbA, indicating the importance of the disulfide bond-forming system to the secretion of this enzyme (41). Here, we show by construction and characterization of cysteine-to-serine variants that the primary function of the disulfide bond in P. aeruginosa lipase is to stabilize the protein during and after translocation over the outer membrane.…”