Phosphorylation-Independent Activity of the Response Regulators AlgB and AlgR in Promoting Alginate Biosynthesis in Mucoid Pseudomonas aeruginosa

Cystic fibrosis (CF) is a common, serious, and often fatal genetic disease characterized by oversecretion of pulmonary mucus, bacterial infections, respiratory congestion, and, in many cases, death due to respiratory failure. Although the lungs of CF patients are colonized by several microorganisms, infections by Pseudomonas aeruginosa are the most common, are usually chronic, and are the most serious in terms of clinical prognosis (15, 23). P. aeruginosa isolates from such chronic infections often have a mucoid colony appearance. This phenotype is due to the overproduction and secretion of a capsular polysaccharide called alginate which plays an important role in chronic P. aeruginosa infections in CF patients (for a review, see reference 23). Alginate production is controlled by a complex regulatory hierarchy involving several genes (65). A key element in alginate gene regulation is the alternative sigma factor ς22 (alternatively known as AlgT and AlgU), which is a member of the RpoE family of extracytoplasmic function sigma factors (13, 30). The activity of ς22 appears to be modulated by the mucABCD gene products, which are encoded by the algT gene operon at 68 min on the P. aeruginosa chromosome (23, 34, 41). Many mucoid P. aeruginosa isolates derived from CF patients harbor mutations in mucA (32), and inactivation of mucA or mucB (also referred to as algN) in wild-type nonmucoid P. aeruginosa strains causes induction of alginate synthesis (20, 31, 32). A membrane complex formed by MucA-MucB may be involved in regulating the stability of ς22 in the cell (34). Biochemical data show that MucA has an affinity for ς22 (50, 66). Active ς22 induces the expression of at least four genes or operons which are required for alginate synthesis. These include the algT operon (13, 33), the algD operon encoding most of the genes required for alginate synthesis (8, 11, 65), algR (33, 65), and the algB operon (29, 64, 65). The algB and algR genes encode proteins that have homology to response regulators of the two-component superfamily (44). Both AlgR and AlgB control alginate levels by activating transcription of algD, the first gene of the alginate biosynthetic operon located at 34 min on the P. aeruginosa chromosome (8, 11, 65). AlgR activates algD expression directly by binding to three sites, two of which are located unusually far upstream of the algD transcription start site (25, 37). The mechanism by which AlgB stimulates algD transcription and alginate production is unclear. AlgB shows homology to response regulators of the NtrC subfamily (63). Although AlgR contains a conserved amino-terminal phosphorylation domain typical of response regulators, its output domain does not appear to fall into a known subfamily (10). Response regulators generally have a cognate sensor kinase protein that responds to environmental stimuli and undergoes autophosphorylation at a histidine residue. The phosphate is then transferred to an aspartate residue in the amino-terminal domain of the response regulator. This phosphorylation usually activates the response regulator leading to an adaptive response. This kind of phosphorelay is a general mechanism for the activation of response regulators of two-component regulatory systems (for a review, see reference 43). Recently, a gene downstream of algB, designated kinB, was identified to encode the cognate sensor kinase for AlgB (29). The KinB protein was localized to the membrane, and a purified carboxyl terminus of KinB was able to undergo autophosphorylation and to phosphorylate AlgB (29). Upstream of algR is fimS, a gene involved in type 4 pilus-mediated twitching motility that encodes an atypical sensor protein (62); it has also been termed algZ (68). Despite the evidence that KinB-AlgB and FimS-AlgR are cognate sensor response regulator pairs, it has not been established that phosphorylation of AlgB or AlgR is required for alginate production in vivo. This is a clinically important question, since it has been proposed that inhibitors of two-component signal transduction systems might have therapeutic value for CF patients colonized with P. aeruginosa (46). In the present study, P. aeruginosa strains with mutations in kinB, algB, and algR were constructed to test the role of phosphorylation and signal transduction in alginate synthesis. These studies showed that the response regulators AlgB and AlgR did not require phosphorylation in order to promote alginate production in mucoid P. aeruginosa. This suggests that an alternative and unusual mechanism may be used by these response regulators to activate gene expression.