Posttranslational control of the algT (algU)-encoded sigma22 for expression of the alginate regulon in Pseudomonas aeruginosa and localization of its antagonist proteins MucA and MucB (AlgN)

Abstract

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 ( 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 22. The goal of this study was to better understand the functional interactions between 22 and its antagonist regulators during alginate production. Nonmucoid strain PAO1 was made to overproduce alginate (indicating high algD promoter activity) through increasing 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 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 showed that 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 22 in order to control certain 22 -dependent promoters like algD. By Western blotting and phoA fusion analyses, the MucB antagonist of 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-22 interact via an inner membrane complex that controls the stability of 22 protein in order to control alginate biosynthesis.

Calcium-Induced Virulence Factors Associated with the Extracellular Matrix of MucoidPseudomonas aeruginosaBiofilms

“…Alginate biosynthesis is regulated by a complex hierarchy of proteins (71), including the stress response sigma factor e (11,15,41). In nonmucoid strains, isolated from environmental sources other than chronic CF pulmonary infections, this sigma factor is sequestered in the membrane by the antisigma factor MucAB (40,43). Nonmucoid strains show no evidence of alginate production under any environmental conditions described, including in biofilms (72).…”

“…Nonmucoid strains show no evidence of alginate production under any environmental conditions described, including in biofilms (72). Alginate-producing strains isolated from the pulmonary fluid of CF patients often have a mutation in mucA (40), causing release of e from membrane sequestration and subsequent expression of the e -controlled regulon, including the alginate biosynthetic gene cluster (43). Dehydration and high osmolarity have been shown to play roles in alg gene expression in a mucoid strain (3,10).…”

Sequence diversity of the mucABD locus in Pseudomonas aeruginosa isolates from patients with cystic fibrosis

“…The algU gene, which is essential for alginate production, encodes the alternative sigma factor s 22 . MucA is an anti-sigma factor that sequesters s 22 (Mathee et al, 1997;Schurr et al, 1996), and MucB is a periplasmic negative regulator (Martin et al, 1993b). MucD is a homologue of the Escherichia coli periplasmic serine protease HtrA, and is apparently both a chaperone and a regulator of alginate production (Wood & Ohman, 2006).…”

The Pseudomonas aeruginosa Two-Component Regulator AlgR Directly Activates rsmA Expression in a Phosphorylation-Independent Manner

“…In chronic P. aeruginosa infections, the bacteria frequently acquire spontaneous mucA mutations that free the alternative sigma factor AlgU (12,14,(24)(25)(26). AlgU and AlgR regulate the alginate biosynthesis genes producing mucoid colonies (12).…”