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Journal of Bacteriology, July 2008, p. 4822-4830, Vol. 190, No. 14
0021-9193/08/$08.00+0     doi:10.1128/JB.00231-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Transcription of the ehx Enterohemolysin Gene Is Positively Regulated by GrlA, a Global Regulator Encoded within the Locus of Enterocyte Effacement in Enterohemorrhagic Escherichia coli ^,

Takehito Saitoh,¹ Sunao Iyoda,^2* Shouji Yamamoto,² Yan Lu,² Ken Shimuta,² Makoto Ohnishi,² Jun Terajima,² and Haruo Watanabe²

Infectious Disease Surveillance Center,¹ Department of Bacteriology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan²

Received 15 February 2008/ Accepted 8 May 2008

The pathogenicity island termed locus of enterocyte effacement (LEE) encodes a type 3 protein secretion system, whose function is required for full virulence of enterohemorrhagic Escherichia coli (EHEC). GrlR and GrlA are LEE-encoded negative and positive regulators, respectively, for controlling transcription of the ler gene, which encodes a central activator of LEE gene expression. We previously reported that the GrlR-GrlA regulatory system controls not only the LEE genes but also flagellar gene expression in EHEC (S. Iyoda et al., J. Bacteriol. 188:5682-5692, 2006). In order to further explore virulence-related genes under the control of the GrlR-GrlA regulatory system, we characterized a grlR-deleted EHEC O157 strain, which was found to have high and low levels of expression of LEE and flagellar genes, respectively. We report here that the grlR deletion significantly induced enterohemolysin (Ehx) activity of EHEC O157 on plates containing defibrinated sheep erythrocytes. Ehx levels were not induced in the grlR grlA double mutant strain but increased markedly by overexpression of GrlA even in the ler mutant, indicating that GrlA is responsible for this regulation. Ehx of the EHEC O157 Sakai strain is encoded by the ehxCABD genes, which are carried on the large plasmid pO157. The expression of ehxC fused with FLAG tag or a promoterless lacZ gene on pO157 was significantly induced under conditions in which GrlA was overproduced. These results together suggest that GrlA acts as a positive regulator for the ehx transcription in EHEC.

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* Corresponding author. Mailing address: Department of Bacteriology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan. Phone: 81-3-5285-1111. Fax: 81-3-5285-1163. E-mail: siyoda{at}nih.go.jp

Published ahead of print on 16 May 2008.

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

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Journal of Bacteriology, July 2008, p. 4822-4830, Vol. 190, No. 14
0021-9193/08/$08.00+0     doi:10.1128/JB.00231-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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