Posttranslational control of the algT (algU)-encoded sigma(22) for expression of the alginate regulon in Pseudomonas aeruginosa and localization of its antagonist proteins MucA and MucB (AlgN)

Pseudomonas aeruginosa strains associated with cystic fibrosis are often mucoid due to the copious production of alginate, an exopolysaccharide and virulence factor. Alginate gene expression is transcriptionally controlled by a gene cluster at 68 min on the chromosome: algT (algU)-mucA-mucB (algN)-mucC (algM)-mucD (algY). The algT gene encodes a 22-kDa alternative sigma factor (sigma(22)) that autoregulates its own promoter (PalgT) as well as the promoters of algR, algB, and algD. The other genes in the algT cluster appear to regulate the expression or activity of sigma(22). The goal of this study was to better understand the functional interactions between sigma(22) and its antagonist regulators during alginate production. Nonmucoid strain PAO1 was made to overproduce alginate (indicating high algD promoter activity) through increasing sigma(22) in the cell by introducing a plasmid clone containing algT from mucA22(Def) strain FRD1. However, the bacterial cells remained nonmucoid if the transcriptionally coupled mucB on the clone remained intact. This suggested that a stoichiometric relationship between sigma(22) and MucB may be required to control sigma factor activity. When the transcription and translational initiation of algT were measured with lacZ fusions, alginate production correlated with only about a 1.2- to 1.7-fold increase in algT-lacZ activity, respectively. An algR-lacZ transcriptional fusion showed a 2.8-fold increase in transcription with alginate production under the same conditions. A Western blot analysis of total cell extracts shelved that sigma(22) was approximately 10-fold higher in strains that overproduced alginate, even though algT expression increased less than 2-fold. This suggested that a posttranscriptional mechanism may exist to destabilize sigma(22) in order to control certain sigma(22)-dependent promoters like algD. By Western blotting and phoA fusion analyses, the MucB antagonist of sigma(22) was found to localize to the periplasm of the cell. Similar experiments suggest that MucA localizes to the inner membrane via one transmembrane domain with amino- and carboxy-terminal domains in the cytoplasm and periplasm, respectively. These data were used to propose a model in which MucB-MucA-sigma(22) interact via an inner membrane complex that controls the stability of sigma(22) protein in order to control alginate biosynthesis.