title: 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.
abstract: Pseudomonas aeruginosa strains that cause chronic pulmonary infections in cystic fibrosis patients typically undergo mucoid conversion. The mucoid phenotype indicates alginate overproduction and is often due to defects in MucA, an antisigma factor that controls the activity of sigma-22 (AlgT [also called AlgU]), which is required for the activation of genes for alginate biosynthesis. In this study we hypothesized that mucoid conversion may be part of a larger response that activates genes other than those for alginate synthesis. To address this, a two-dimensional (2-D) gel analysis was employed to compare total proteins in strain PAO1 to those of its mucA22 derivative, PDO300, in order to identify protein levels enhanced by mucoid conversion. Six proteins that were clearly more abundant in the mucoid strain were observed. The amino termini of such proteins were determined and used to identify the gene products in the genomic database. Proteins involved in alginate biosynthesis were expected among these, and two (AlgA and AlgD) were identified. This result verified that the 2-D gel approach could identify gene products under sigma-22 control and upregulated by mucA mutation. Two other protein spots were also clearly upregulated in the mucA22 background, and these were identified as porin F (an outer membrane protein) and a homologue of DsbA (a disulfide bond isomerase). Single-copy gene fusions were constructed to test whether these proteins were enhanced in the mucoid strain due to increased transcription. The oprF-lacZ fusion showed little difference in levels of expression in the two strains. However, the dsbA-lacZ fusion showed two- to threefold higher expression in PDO300 than in PAO1, suggesting that its promoter was upregulated by the deregulation of sigma-22 activity. 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 sensitivity to dithiothreitol, reduced type IV pilin-mediated twitching motility, and reduced accumulation of extracellular proteases, including elastase. Although efficient secretion of elastase in the dsbA mutant was still demonstrable, the elastase produced appeared to be unstable, possibly as a result of mispaired disulfide bonds. Disruption of dsbA in the mucoid PDO300 background did not affect alginate production. Thus, even though dsbA is coregulated with mucoid conversion, it was not required for alginate production. This suggests that mucA mutation, which deregulates sigma-22, results in a global response that includes other factors in addition to increasing the production of alginate.
authors.affiliation: Department of Microbiology, University of Tennessee, Memphis, Tennessee, USA.
authors.forename: S
authors.initials: S
authors.lastname: Malhotra
authors.forename: L A
authors.initials: LA
authors.lastname: Silo-Suh
authors.forename: K
authors.initials: K
authors.lastname: Mathee
authors.forename: D E
authors.initials: DE
authors.lastname: Ohman
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000464:Alginates / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D003550:Cystic Fibrosis / Q000382:microbiology; D004229:Dithiothreitol / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D049071:Endopeptidase Clp; D004790:Enzyme Induction; D036781:Fimbriae Proteins; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D018272:Porins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism*; D020543:Proteome / Q000032:analysis*; D011552:Pseudomonas Infections / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics / Q000472:pathogenicity* / Q000502:physiology*; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism
chemical_list: D000464:Alginates; D001426:Bacterial Proteins; D008565:Membrane Proteins; C107895:MucA protein, Pseudomonas; D018272:Porins; D020543:Proteome; D036781:Fimbriae Proteins; D000469:Alkaline Phosphatase; D012697:Serine Endopeptidases; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; D019704:Protein Disulfide-Isomerases; D004229:Dithiothreitol