Alginate overproduction by Pseudomonas aeruginosa, which causes a mucoid phenotype, is a major virulence factor associated with chronic pulmonary infections in cystic fibrosis patients. Expression of the algD operon for alginate biosynthesis requires three major regulators in association with the ECF sigma factor, σ22, in mucoid strains that are typically defective in anti-sigma factor, MucA. One such algD regulator is AlgB, a member of the NtrC family of two-component systems, which typically utilize σ54. However, neither σ54 nor the cognate sensor kinase (KinB) of AlgB are required for algD expression in such mucoid strains. I hypothesized that KinB-phosphorylated AlgB must play some role in gene regulation, and so I sought to construct a constitutively active AlgB that simulated kinase-phosphorylation. I took a predictive approach and genetically introduced substitutions in AlgB that had been shown to activate DctD, a close homologue of AlgB in Rhizobium (52). When one such substitution, AlgBE125K, was transferred to a nonmucoid P. aeruginosa PAO ΔalgB-kinB (JK159) strain, alginate overproduction was observed. Interestingly, introduction of an algT mutation to remove σ22 did not block alginate production induced by AlgBE125K; although, it did stimulate the production of alginate in the presence of AlgBwt in trans to similar levels induced by the constitutive mutant. In contrast, introduction of an rpoN mutation showed that alginate production mediated by AlgBwt and AlgBE125K was σ54 dependent. The increase in expression of alginate by AlgBwt in the presence of σ54 and the absence of σ22 suggested a competition between the sigma factors for binding to PalgD. Biochemical assays were conducted to assess the constitutive property of AlgBE125K. For the ATPase assay, an equivalent amount of ATP hydrolysis was observed between the mutant and the wild type AlgB proteins. Slight differences seen for the EMSA data suggested possible higher order complex formation for AlgBE125K compared to AlgBwt. Collectively, these results suggested that in wild-type (MucA+) P. aeruginosa, expression of the algD operon is dependent on the phosphorylation of AlgB by KinB in a typical two-component fashion that is triggered by some as yet unknown environmental stimulus.