title: Evidence for sigma factor competition in the regulation of alginate production by Pseudomonas aeruginosa.
abstract: 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 (AlgU(A61V)). 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 AlgU(A61V), 2) an overexpression of the stringent starvation protein A (SspA), and 3) a reduced expression of the major sigma factor RpoD (σ(70)). Induction of AlgU(A61V) in trans caused conversion to mucoidy in CF149 and PAO1DalgU, suggesting that AlgU(A61V) is functional in activating alginate production. Furthermore, the level of AlgU(A61V) was increased in all three mutants relative to CF149. However, compared to the wild type AlgU, AlgU(A61V) had a reduced activity in promoting alginate production in PAO1ΔalgU. SspA and three other anti-σ(70) 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-σ(70) factors can induce conversion to mucoidy in P. aeruginosa CF149 with algU-suppressor mutation via modulation of RpoD.
authors.affiliation: Department of Biochemistry and Microbiology, Joan C. Edwards School of Medicine at Marshall University, Huntington, West Virginia, USA.
authors.forename: Yeshi
authors.initials: Y
authors.lastname: Yin
authors.forename: T Ryan
authors.initials: TR
authors.lastname: Withers
authors.forename: Xin
authors.initials: X
authors.lastname: Wang
authors.forename: Hongwei D
authors.initials: HD
authors.lastname: Yu
mesh_terms: D000464:Alginates; D015153:Blotting, Western; D003550:Cystic Fibrosis / Q000150:complications; D004251:DNA Transposable Elements; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D020723:Glucuronic Acid / Q000096:biosynthesis; D006603:Hexuronic Acids; D006801:Humans; D009093:Mucus; D011552:Pseudomonas Infections / Q000150:complications / Q000503:physiopathology; D011550:Pseudomonas aeruginosa / Q000254:growth & development / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D012808:Sigma Factor / Q000378:metabolism*
chemical_list: D000464:Alginates; D004251:DNA Transposable Elements; D006603:Hexuronic Acids; D012808:Sigma Factor; D020723:Glucuronic Acid; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases