Alginate overproduction, or mucoidy, plays an important role in the pathogenesis of P. aeruginosa lung infection in cystic fibrosis (CF). Mucoid strains with mucA mutations predominantly populate in chronically-infected patients. However, the mucoid strains can revert to nonmucoidy in vitro through suppressor mutations. We screened a mariner transposon library using CF149, a non-mucoid clinical isolate with a misssense mutation in algU(AlgUA61V). The wild type AlgU is a stress-related sigma factor that activates transcription of alginate biosynthesis. Three mucoid mutants were identified with transposon insertions that caused 1) an overexpression of AlgUA61V, 2) an overexpression of the stringent starvation protein A (SspA), and 3) a reduced expression of the major sigma factor RpoD (σ70). Induction of AlgUA61V in trans caused conversion to mucoidy in CF149 and PAO1DalgU, suggesting that AlgUA61V is functional in activating alginate production. Furthermore, the level of AlgUA61V was increased in all three mutants relative to CF149. However, compared to the wild type AlgU, AlgUA61V had a reduced activity in promoting alginate production in PAO1ΔalgU. SspA and three other anti-σ70 orthologues, P. aeruginosa AlgQ, E. coli Rsd, and T4 phage AsiA, all induced mucoidy, suggesting that reducing activity of RpoD is linked to mucoid conversion in CF149. Conversely, RpoD overexpression resulted in suppression of mucoidy in all mucoid strains tested, indicating that sigma factor competition can regulate mucoidy. Additionally, an RpoD-dependent promoter (PssrA) was more active in non-mucoid strains than in isogenic mucoid variants. Altogether, our results indicate that the anti-σ70 factors can induce conversion to mucoidy in P. aeruginosa CF149 with algU-suppressor mutation via modulation of RpoD.
Evidence for Sigma Factor Competition in the Regulation of Alginate Production by Pseudomonas aeruginosaBiochemistry and Microbiology
Citation InformationYin Y, Withers TR, Wang X, Yu HD (2013) Evidence for Sigma Factor Competition in the Regulation of Alginate Production by Pseudomonas aeruginosa. PLoS ONE 8(8): e72329. doi:10.1371/journal.pone.0072329