Identification of the histidine protein kinase KinB in Pseudomonas aeruginosa and its phosphorylation of the alginate regulator algB.

Sheng Ma,Dennis E Ohman,Daniel J Wozniak

doi:10.1074/jbc.272.29.17952

Abstract

The exopolysaccharide alginate is an important virulence factor in chronic lung infections caused by the bacterium Pseudomonas aeruginosa. Two positive activators for alginate synthesis, algB and algR, are members of a superfamily of response regulators of the two-component regulatory system. AlgB belongs to the NtrC subfamily of response regulators and is required for high-level production of alginate. In this study, an open reading frame encoding a polypeptide of 66 kDa, designated kinB, was identified immediately downstream of algB. The sequence of KinB is homologous to the histidine protein kinase members of two-component regulatory systems. Western blot analysis of a P. aeruginosa strain carrying a kinB-lacZ protein fusion and studies of kinB-phoA fusions indicate that KinB localizes to the inner membrane and has a NH2-terminal periplasmic domain. A KinB derivative containing the COOH terminus of KinB was generated and purified. In the presence of [gamma-32P]ATP, the purified COOH-terminal KinB protein was observed to undergo progressive autophosphorylation in vitro. Moreover, the phosphoryl label of KinB could be rapidly transferred to purified AlgB. Substitutions of the residues conserved among histidine protein kinases abolished KinB autophosphorylation. These results provide evidence that kinB encodes the AlgB cognate histidine protein kinase.

Highlights

The exopolysaccharide alginate is an important virulence factor in chronic lung infections caused by the bacterium Pseudomonas aeruginosa
Cloning and Identification of kinB—We examined whether a gene encoding a sensor kinase was closely linked to a known gene encoding a response regulator that controls the alginate biosynthetic operon in P. aeruginosa
Two positive regulators of alginate production, AlgB and AlgR, affect transcriptional activation of the alginate biosynthetic operon at algD, and both have sequence similarity to the family of response regulators of two-component systems [9, 10]. This suggested that the production of alginate by P. aeruginosa is influenced by environmental factors, some of which may be found in the unique environment of the cystic fibrosis (CF) lung [17]

Summary

Introduction

The exopolysaccharide alginate is an important virulence factor in chronic lung infections caused by the bacterium Pseudomonas aeruginosa. The histidine protein kinase senses a specific environmental stimulus and undergoes autophosphorylation at a histidine residue present in a highly conserved carboxyl-terminal domain of the protein. This phosphate group is subsequently transferred to an aspartate residue in the amino terminus of the response regulator, resulting in a change in the activity of the response regulator that leads to an adaptive response [11, 12]. In this study we identified a gene called kinB, located immediately downstream of algB, that encodes a protein with high similarity to typical histidine protein kinases of two component systems.

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Results

Conclusion