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Journal of Bacteriology, July 2006, p. 5089-5100, Vol. 188, No. 14
0021-9193/06/$08.00+0     doi:10.1128/JB.01929-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Molecular Characterization of Pantoea stewartii subsp. stewartii HrpY, a Conserved Response Regulator of the Hrp Type III Secretion System, and its Interaction with the hrpS Promoter{dagger}

Massimo Merighi,1,{ddagger} Doris R. Majerczak,1 Michael Zianni,2 Kimberly Tessanne,2 and David L. Coplin1*

Department of Plant Pathology and the Plant Molecular Biology and Biotechnology Program,1 Plant-Microbe Genomic Facility, The Ohio State University, Columbus, Ohio 432102

Received 16 December 2005/ Accepted 27 April 2006

Pantoea stewartii subsp. stewartii is a bacterial pathogen of corn. Its pathogenicity depends on the translocation of effector proteins into host cells by the Hrp type III secretion system. We previously showed by genetic analysis that the HrpX sensor kinase and the HrpY response regulator are at the head of a complex cascade of regulators controlling hrp/hrc secretion and wts effector genes. This cascade also includes the HrpS response regulator and the HrpL alternative sigma factor. These regulators are shared among many important plant pathogens in the genera Pantoea, Erwinia, and Pseudomonas. In this study, we dissect the regulatory elements in the hrpS promoter region, using genetic and biochemical approaches, and show how it integrates various environmental signals, only some of which are dependent on phosphorylation of HrpY. Primer extension located the transcriptional start site of hrpS at a {sigma}70 promoter 601 bp upstream of the open reading frame. Electrophoretic mobility shift assays and DNase I footprinting analysis demonstrated that HrpY binds to conserved regulatory elements immediately adjacent to this promoter, and its binding affinity was increased by phosphorylation at D57. A consensus sequence for the two direct repeats bound by HrpY is proposed. Deletion analysis of the promoter region revealed that both the HrpY binding site and additional sequences farther upstream, including a putative integration host factor binding site, are required for hrpS expression. This finding suggests that other unknown regulatory proteins may act cooperatively with HrpY.

* Corresponding author. Mailing address: Department of Plant Pathology, The Ohio State University, 201 Kottman Hall, 2021 Coffey Rd., Columbus, OH 43210-1087. Phone: (614) 292-8503. Fax: (614) 292-4455. E-mail: coplin.3{at}osu.edu.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

{ddagger} Present address: Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115.

Journal of Bacteriology, July 2006, p. 5089-5100, Vol. 188, No. 14
0021-9193/06/$08.00+0     doi:10.1128/JB.01929-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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