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Journal of Bacteriology, May 2008, p. 3444-3455, Vol. 190, No. 10
0021-9193/08/$08.00+0     doi:10.1128/JB.00141-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

An Ortholog of OxyR in Legionella pneumophila Is Expressed Postexponentially and Negatively Regulates the Alkyl Hydroperoxide Reductase (ahpC2D) Operon{triangledown}

Jason J. LeBlanc,1,2 Ann Karen C. Brassinga,4 Fanny Ewann,4 Ross J. Davidson,1,2,3 and Paul S. Hoffman2,3,4,5*

Department of Pathology and Laboratory Medicine, Queen Elizabeth II Health Sciences Center, Halifax, Nova Scotia, Canada,1 Department of Microbiology and Immunology,2 Medicine, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada,3 Department of Medicine, Division of Infectious Diseases and International Health,4 Department of Microbiology, University of Virginia School of Medicine, Charlottesville, Virginia 22908-13405

Received 28 January 2008/ Accepted 11 March 2008

Legionella pneumophila expresses two peroxide-scavenging alkyl hydroperoxide reductase systems (AhpC1 and AhpC2D) that are expressed differentially during the bacterial growth cycle. Functional loss of the postexponentially expressed AhpC1 system is compensated for by increased expression of the exponentially expressed AhpC2D system. In this study, we used an acrylamide capture of DNA-bound complexes (ACDC) technique and mass spectrometry to identify proteins that bind to the promoter region of the ahpC2D operon. The major protein captured was an ortholog of OxyR (OxyRLp). Genetic studies indicated that oxyRLp was an essential gene expressed postexponentially and only partially complemented an Escherichia coli oxyR mutant (GS077). Gel shift assays confirmed specific binding of OxyRLp to ahpC2D promoter sequences, but not to promoters of ahpC1 or oxyRLp; however, OxyRLp weakly bound to E. coli OxyR-regulated promoters (katG, oxyR, and ahpCF). DNase I protection studies showed that the OxyRLp binding motif spanned the promoter and transcriptional start sequences of ahpC2 and that the protected region was unchanged by treatments with reducing agents or hydrogen peroxide (H2O2). Moreover, the OxyRLp (pBADLpoxyR)-mediated repression of an ahpC2-gfp reporter construct in E. coli GS077 (the oxyR mutant) was not reversed by H2O2 challenge. Alignments with other OxyR proteins revealed several amino acid substitutions predicted to ablate thiol oxidation or conformational changes required for activation. We suggest these mutations have locked OxyRLp in an active DNA-binding conformation, which has permitted a divergence of function from a regulator of oxidative stress to a cell cycle regulator, perhaps controlling gene expression during postexponential differentiation.

* Corresponding author. Mailing address: Division of Infectious Diseases and International Health, Room 2146, MR-4 Bldg., 409 Lane Road, University of Virginia Health Systems, Charlottesville, VA 22908-1340. Phone: (434) 924-2893. Fax: (434) 924-0075. E-mail: psh2n{at}virginia.edu

{triangledown} Published ahead of print on 21 March 2008.

Journal of Bacteriology, May 2008, p. 3444-3455, Vol. 190, No. 10
0021-9193/08/$08.00+0     doi:10.1128/JB.00141-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Morash, M. G., Brassinga, A. K. C., Warthan, M., Gourabathini, P., Garduno, R. A., Goodman, S. D., Hoffman, P. S. (2009). Reciprocal Expression of Integration Host Factor and HU in the Developmental Cycle and Infectivity of Legionella pneumophila. Appl. Environ. Microbiol. 75: 1826-1837 [Abstract] [Full Text]  


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