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Volume 272, Number 29, Issue of July 18, 1997 pp. 17952-17960
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of the Histidine Protein Kinase KinB in Pseudomonas aeruginosa and Its Phosphorylation of the Alginate Regulator AlgB

(Received for publication, February 24, 1997, and in revised form, April 30, 1997)

Sheng Ma , Daniel J. Wozniak ^§ and Dennis E. Ohman

From the  Department of Microbiology and Immunology, University of Tennessee and the Veterans Administration Medical Center, Memphis, Tennessee 38163 and the ^§ Department of Microbiology and Immunology, Bowman Gray School of Medicine at Wake Forest University, Winston-Salem, North Carolina 27157-1064

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 [-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.

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