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Journal of Bacteriology, November 2007, p. 7549-7555, Vol. 189, No. 21
0021-9193/07/$08.00+0     doi:10.1128/JB.00893-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Chlamydophila pneumoniae PknD Exhibits Dual Amino Acid Specificity and Phosphorylates Cpn0712, a Putative Type III Secretion YscD Homolog{triangledown}

Dustin L. Johnson and James B. Mahony*

Faculty of Health Sciences and Department of Pathology and Molecular Medicine, McMaster University, and Father Sean O'Sullivan Research Centre, St. Joseph's Healthcare, Hamilton, Ontario, Canada

Received 7 June 2007/ Accepted 17 August 2007

Chlamydophila pneumoniae is an obligate intracellular bacterium that causes bronchitis, pharyngitis, and pneumonia and may be involved in atherogenesis and Alzheimer's disease. Genome sequencing has identified three eukaryote-type serine/threonine protein kinases, Pkn1, Pkn5, and PknD, that may be important signaling molecules in Chlamydia. Full-length PknD was cloned and expressed as a histidine-tagged protein in Escherichia coli. Differential centrifugation followed by sodium carbonate treatment of E. coli membranes demonstrated that His-PknD is an integral membrane protein. Fusions of overlapping PknD fragments to alkaline phosphatase revealed that PknD contains a single transmembrane domain and that the kinase domain is in the cytoplasm. To facilitate solubility, the kinase domain was cloned and expressed as a glutathione S-transferase (GST) fusion protein in E. coli. Purified GST-PknD kinase domain autophosphorylated, and catalytic mutants (K33G, D156G, and K33G-D156G mutants) and activation loop mutants (T185A and T193A) were inactive. PknD phosphorylated recombinant Cpn0712, a type III secretion YscD homolog that has two forkhead-associated domains. Thin-layer chromatography revealed that the PknD kinase domain autophosphorylated on threonine and tyrosine and phosphorylated the FHA-2 domain of Cpn0712 on serine and tyrosine. To our knowledge, this is the first demonstration of a bacterial protein kinase with amino acid specificity for both serine/threonine and tyrosine residues and this is the first study to show phosphorylation of a predicted type III secretion structural protein.

* Corresponding author. Mailing address: Regional Virology and Chlamydiology Laboratory, St. Joseph's Hospital, 50 Charlton Avenue East, Hamilton, Ontario, Canada L8N 4A6. Phone: (905) 521-6021. Fax: (905) 521-6083. E-mail: mahonyj{at}mcmaster.ca

{triangledown} Published ahead of print on 31 August 2007.

Journal of Bacteriology, November 2007, p. 7549-7555, Vol. 189, No. 21
0021-9193/07/$08.00+0     doi:10.1128/JB.00893-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Stone, C. B., Johnson, D. L., Bulir, D. C., Gilchrist, J. D., Mahony, J. B. (2008). Characterization of the Putative Type III Secretion ATPase CdsN (Cpn0707) of Chlamydophila pneumoniae. J. Bacteriol. 190: 6580-6588 [Abstract] [Full Text]  
  • Johnson, D. L., Stone, C. B., Mahony, J. B. (2008). Interactions between CdsD, CdsQ, and CdsL, Three Putative Chlamydophila pneumoniae Type III Secretion Proteins. J. Bacteriol. 190: 2972-2980 [Abstract] [Full Text]  


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