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

Abstract

Overproduction of the capsular polysaccharide alginate appears to confer a selective advantage for Pseudomonas aeruginosa in the lungs of cystic fibrosis patients. The regulators AlgB and AlgR, which are both required as positive activators in alginate overproduction, have homology with the regulator class of two-component environmental responsive proteins which coordinate gene expression through signal transduction mechanisms. Signal transduction in this class of proteins generally occurs via autophosphorylation of the sensor kinase protein and phosphotransfer from the sensor to a conserved aspartate residue, which is present in the amino terminus of the response regulator. Recently,kinB was identified downstream of algB and was shown to encode the cognate histidine protein kinase that efficiently phosphorylates AlgB. However, we show here that a null mutation inkinB in a mucoid cystic fibrosis isolate, P. aeruginosa FRD1, did not block alginate production. The role of the conserved aspartate residue in the phosphorylation of AlgB was examined. The predicted phosphorylation site of AlgB (D59) was mutated to asparagine (N), and a derivative of an AlgB lacking the entire amino-terminal phosphorylation domain (AlgBΔ1-145) was constructed. A hexahistidine tag was included at the amino terminus of the wild-type (H-AlgB), H-AlgBΔ1-145, and mutant (H-AlgB.59N) AlgB proteins. These derivatives were purified by Ni2+affinity chromatography and examined for in vitro phosphorylation by the purified sensor kinase protein, KinB. The results indicated that while KinB efficiently phosphorylated H-AlgB, no phosphorylation of H-AlgBΔ1-145 or H-AlgB.D59N was apparent. An allelic exchange system was developed to transfer mutantalgB alleles onto the chromosome of a P. aeruginosa algB mutant to examine the effect on alginate production. Despite the defect in AlgB phosphorylation,P. aeruginosa strains expressing AlgB.D59N or H-AlgBΔ1-145 remained mucoid. The roles of the conserved aspartate residues in the phosphorylation of AlgR were also examined. As seen with AlgB, mutations in the predicted phosphorylation site of AlgR (AlgR.D54N and AlgR.D85N) did not affect alginate production. These results indicate that in vivo phosphorylation of AlgB and AlgR are not required for their roles in alginate production. Thus, the mechanism by which these response regulators activate alginate genes in mucoid P. aeruginosa appears not to be mediated by conventional phosphorylation-dependent signal transduction.

To ∼P or Not to ∼P? Non‐canonical activation by two‐component response regulators

“…AlgRP seems to be required to bind the fim and hcn promoters. On the other hand, transcriptional activation of the alg operon, which is required for the synthesis of the capsular polysaccharide, alginate, is directly mediated by two unphosphorylated response regulators, AlgB and AlgR (Ma et al, 1998;Leech et al, 2008). Pseudomonas strains carrying the D59N AlgB substitution and the D54N AlgR substitution retained the ability to form alginate-rich mucoid colonies.…”

Evidence that WbpD is an N‐acetyltransferase belonging to the hexapeptide acyltransferase superfamily and an important protein for O‐antigen biosynthesis in Pseudomonas aeruginosa PAO1

“…P. aeruginosa and Escherichia coli strains were routinely propagated in Luria-Bertani Broth (LB; Invitrogen), or on Luria-Bertani Agar (LA) plates (LB containing 1.5% Bacto Agar). Pseudomonas Isolation Agar (PIA) was used in selection of transconjugants following mating experiments, and resolving media (5% sucrose, 1.2% tryptone, 0.5% yeast extract; Ma et al, 1998) supplemented with 1.5% Bacto Agar was used in gene replacement experiments. Davis Minimal Media was used for the preparation of electrocompetent P. aeruginosa cells.…”

Post‐transcriptional regulation of the virulence‐associated enzyme AlgC by the σ22‐dependent small RNA ErsA of Pseudomonas aeruginosa

“…σ 22 controls the algD promoter (PalgD), which drives the expression of the 12-gene alginate biosynthetic operon (algD-alg8-alg44-algKEGXLIJFA) (Sumita and Ohman, 2004). Three additional positive regulatory proteins, AlgB, AlgR and AmrZ, are also under σ 22 control and required for PalgD expression (Wozniak and Ohman, 1994;Ma et al, 1998;Baynham et al, 2006;Leech et al, 2008). Located distant from the alginate biosynthetic operon, another alg gene, algC, encodes a bifunctional enzyme with phosphomannomutase (PMM) and phosphoglucomutase (PGM) activities; this enzyme provides the sugar precursors for the alginate biosynthetic pathway and for the synthesis of other P. aeruginosa polysaccharide exoproducts such as Psl, Pel, lipopolysaccharide (LPS) and rhamnolipids (Deretic et al, 1995;Olvera et al, 1999;Ma et al, 2012).…”