Alginate-regulating genes are identified in the clinical cystic fibrosis isolate of Pseudomonas aeruginosa PA2192

Cystic fibrosis (CF) is a genetic disorder that leads to a buildup of mucus in the lungs ideal for bacterial colonization. When Pseudomonas aeruginosa enters the CF lung, it undergoes a conversion from nonmucoid to mucoid; colonization by a mucoid strain of P. aeruginosa greatly increases mortality. The mucoid phenotype is due to the production of alginate. The regulator of alginate production is the AlgT/U sigma factor. The observed phenotypic conversion is due to a mutation in the mucA gene coding for an anti-sigma factor, MucA, which sequesters AlgT/U. This mucoid phenotype is unstable when the strains are removed from the lung as they acquire second-site mutations. This in vitro reversion phenomenon is utilized to identify novel genes regulating alginate production. Previously, second-site mutations were mapped to algT/U, algO, and mucP, demonstrating their role in alginate regulation. Most of these studies were performed using a non-CF isolate. It was hypothesized that second site mutations in a clinical strain would be mapped to the same genes. In this study, a clinical, hyper-mucoid P. aeruginosa strain PA2192 was used to study the reversion phenomenon. This study found that PA2192 has a novel mucA mutation which was named them mucA180 allele. Twelve colonies were sub-cultured for two weeks without aeration at room temperature in order to obtain nonmucoid suppressors of alginate production (sap). Only 41 sap mutants were stable for more than 48 hours -- a reversion frequency of 3.9% as compared to ~90% in laboratory strains showing that PA2192 has a stable mucoid phenotype. This phenotype was restored in 28 of the 41 sap mutants when complemented with plasmids harboring algT/U. Four of the sap mutants are complemented by algO. Sequence analyses of the algT/U mutants have found no mutations in the coding region or promoter leading to the hypothesis that there is another, as yet unidentified mechanism of alginate regulation in this clinical strain.