Proteome analysis of the effect of mucoid conversion on global protein expression in Pseudomonas aeruginosa strain PAO1 shows induction of the disulfide bond isomerase, DsbA

Pseudomonas aeruginosa strains that cause chronic pulmonary infections in c ystic fibrosis patients typically undergo mucoid conversion, The mucoid phe notype indicates alginate overproduction and is often due to defects in Muc A, an antisigma factor that controls the activity of sigma-22 (AlgT [also c alled AlgU]), which is required for the activation of genes for alginate bi osynthesis, In this study we hypothesized that mucoid conversion may be par t of a larger response that activates genes other than those for alginate s ynthesis. To address this, a two-dimensional (2-D) gel analysis was employe d to compare total proteins in strain PAO1 to those of its mucA22 derivativ e, PDO300, in order to identify protein levels enhanced by mucoid conversio n. Six proteins that were clearly more abundant in the mucoid strain were o bserved. The amino termini of such proteins were determined and used to ide ntify the gene products in the genomic database. Proteins involved in algin ate biosynthesis were expected among these, and two (AlgA and AlgD) were id entified. This result verified that the 2-D gel approach could identify gen e products under sigma-22 control and upregulated by mucA mutation. Two oth er protein spots were also clearly upregulated in the mucA22 background, an d these were identified as porin F (an outer membrane protein) and a homolo gue of DsbA (a disulfide bond isomerase), Single-copy gene fusions were con structed to test whether these proteins were enhanced in the mucoid strain due to increased transcription. The oprF-lacZ fusion showed little differen ce in levels of expression in the two strains. However, the dsbA-lacZ fusio n showed two- to threefold higher expression in PDO300 than in PAO1, sugges ting that its promoter was upregulated by the deregulation of sigma-22 acti vity. A dsbA-null mutant was constructed in PAO1 and shown to have defects predicted for a cell with reduced disulfide bond isomerase activity, namely , reduction in periplasmic alkaline phosphatase activity, increased sensiti vity to dithiothreitol, reduced type IV pilin-mediated twitching motility, and reduced accumulation of extracellular proteases, including elastase, Al though efficient secretion of elastase in the dsbA mutant was still demonst rable, the elastase produced appeared to be unstable, possibly as a result of mispaired disulfide bonds. Disruption of dsbA in the mucoid PDO300 backg round did not affect alginate production. Thus, even though dsbA is coregul ated with mucoid conversion, it was not required for alginate production. T his suggests that mucA mutation, which deregulates sigma-22, results in a g lobal response that includes other factors in addition to increasing the pr oduction of alginate.