IMPORTANCE During chronic lung infections, such as in cystic fibrosis patients, Pseudomonas aeruginosa produces the exopolysaccharide alginate and forms biofilms that shield the organisms from the immune response and increase resistance to antibiotics. Activation of alginate genes is under the control of an extracytoplasmic stress response system that releases an alternative sigma factor (σ22) in response to cell wall stress and then activates expression of a large regulon. In this study, a mutant analysis of 27 members of the regulon showed that 11 play a role in envelope homeostasis and affect the stress response system itself. Interestingly, some genes demonstrate effects only in either the planktonic (free-swimming) or the sessile (biofilm) mode of growth, which leads to persistence and antibiotic tolerance. The studies presented here provide an important initial step in dissecting the mechanisms that regulate a critical signal transduction pathway that impacts P. aeruginosa pathogenesis.