Identification of Genes in the σ 22 Regulon of Pseudomonas aeruginosa Required for Cell Envelope Homeostasis in Either the Planktonic or the Sessile Mode of Growth

Abstract

ABSTRACTThe Pseudomonas aeruginosa extracytoplasmic functioning (ECF) sigma factor σ22 is encoded by algT/algU and is inhibited by anti-sigma factor MucA. σ22 was originally discovered for its essential role in the expression of the exopolysaccharide alginate by mucoid strains associated with chronic pulmonary infection. However, σ22 is now known to also have a large regulon associated with the response to cell wall stress. Our recent transcriptome analysis identified 293 open reading frames (ORFs) in the σ22 stress stimulon that include genes for outer envelope biogenesis and remodeling, although most of the genes have undefined functions. To better understand the σ22-dependent stress response, mutants affected in 27 genes of the σ22 stimulon were examined and expression was studied with lacZ fusions. Mutants constructed in the 27 genes showed no major change in response to cell wall-acting antibiotics or growth at elevated temperatures nor in alginate production. The mutants were examined for their effects on the expression of the σ22-dependent promoter of the alginate biosynthetic operon (PalgD) as a measure of σ22 derepression from MucA. By testing PalgD expression under both planktonic and sessile growth conditions, 11 genes were found to play a role in the stress response that activates σ22. Some mutations caused an increase or a decrease in the response to cell wall stress. Interestingly, mutations in 7 of the 11 genes caused constitutive PalgD expression under nonstressed conditions and thus showed that these genes are involved in maintaining envelope homeostasis. Mutations in PA0062 and PA1324 showed constitutive PalgD expression during both the planktonic and the sessile modes of growth. However, the PA5178 mutation caused constitutive PalgD expression only during planktonic growth. In contrast, mutations in PA2717, PA0567, PA3040, and PA0920 caused constitutive PalgD expression only in the sessile/biofilm mode of growth. This provides evidence that the σ22 stimulon for cell envelope homeostasis overlaps with biofilm control mechanisms.