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Journal of Bacteriology, November 1999, p. 6615-6622, Vol. 181, No. 21
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Characterization of a Hank's Type Serine/Threonine Kinase and Serine/Threonine Phosphoprotein Phosphatase in Pseudomonas aeruginosa

Subhendu Mukhopadhyay, Vinayak Kapatral, Wenbin Xu, and A. M. Chakrabarty*

Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, Illinois 60612

Received 19 May 1999/Accepted 18 August 1999

Pseudomonas aeruginosa is an opportunistic pathogen that causes infections in eye, urinary tract, burn, and immunocompromised patients. We have cloned and characterized a serine/threonine (Ser/Thr) kinase and its cognate phosphoprotein phosphatase. By using oligonucleotides from the conserved regions of Ser/Thr kinases of mycobacteria, an 800-bp probe was used to screen P. aeruginosa PAO1 genomic library. A 20-kb cosmid clone was isolated, from which a 4.5-kb DNA with two open reading frames (ORFs) were subcloned. ORF1 was shown to encode Ser/Thr phosphatase (Stp1), which belongs to the PP2C family of phosphatases. Overlapping with the stp1 ORF, an ORF encoding Hank's type Ser/Thr kinase was identified. Both ORFs were cloned in pGEX-4T1 and expressed in Escherichia coli. The overexpressed proteins were purified by glutathione-Sepharose 4B affinity chromatography and were biochemically characterized. The Stk1 kinase is 39 kDa and undergoes autophosphorylation and can phosphorylate eukaryotic histone H1. A site-directed Stk1 (K86A) mutant was shown to be incapable of autophosphorylation. A two-dimensional phosphoamino acid analysis of Stk1 revealed strong phosphorylation at a threonine residue and weak phosphorylation at a serine residue. The Stp1 phosphatase is 27 kDa and is an Mn2+-, but not a Ca2+- or a Mg2+-, dependent Ser/Thr phosphatase. Its activity is inhibited by EDTA and NaF, but not by okadaic acid, and is similar to that of PP2C phosphatase.

* Corresponding author. Mailing address: Department of Microbiology and Immunology (M/C 790), University of Illinois at Chicago College of Medicine, 835 S. Wolcott Ave., Chicago, IL 60612. Phone: (312) 996-4586. Fax: (312) 996-6415. E-mail: Ananda.Chakrabarty{at}uic.edu.

Journal of Bacteriology, November 1999, p. 6615-6622, Vol. 181, No. 21
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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