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Journal of Bacteriology, November 2008, p. 7335-7345, Vol. 190, No. 22
0021-9193/08/$08.00+0     doi:10.1128/JB.00360-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Role of the Histone-Like Nucleoid Structuring Protein in the Regulation of rpoS and RpoS-Dependent Genes in Vibrio cholerae ^,

Anisia J. Silva,^1* Syed Zafar Sultan,¹ Weili Liang,² and Jorge A. Benitez¹

Department of Microbiology, Biochemistry, and Immunology, Morehouse School of Medicine, 720 Westview Dr., SW, Atlanta, Georgia 30310,¹ State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changping, Beijing 102206, China²

Received 11 March 2008/ Accepted 27 August 2008

Production of the Zn-metalloprotease hemagglutinin (HA)/protease by Vibrio cholerae has been reported to enhance enterotoxicity in rabbit ileal loops and the reactogenicity of live cholera vaccine candidates. Expression of HA/protease requires the alternate sigma factor ^S (RpoS), encoded by rpoS. The histone-like nucleoid structuring protein (H-NS) has been shown to repress rpoS expression in Escherichia coli. In V. cholerae strains of the classical biotype, H-NS has been reported to silence virulence gene expression. In this study we examined the role of H-NS in the expression of HA/protease and motility in an El Tor biotype strain by constructing a hns mutant. The hns mutant exhibited multiple phenotypes, such as production of cholera toxin in nonpermissive LB medium, reduced resistance to high osmolarity, enhanced resistance to low pH and hydrogen peroxide, and reduced motility. Depletion of H-NS by overexpression of a dominant-negative allele or by deletion of hns resulted in diminished expression of HA/protease. Epistasis analysis of HA/protease expression in hns, rpoS, and hns rpoS mutants, analysis of RpoS reporter fusions, quantitative reverse transcription-PCR measurements, and ectopic expression of RpoS in rpoS and rpoS hns mutants showed that H-NS posttranscriptionally enhances RpoS expression. The hns mutant exhibited a lower degree of motility and lower levels of expression of flaA, flaC, cheR-2, and motX mRNAs than the wild type. Comparison of the mRNA abundances of these genes in wild-type, hns, rpoS, and hns rpoS strains revealed that deletion of rpoS had a more severe negative effect on their expression. Interestingly, deletion of hns in the rpoS background resulted in higher expression levels of flaA, flaC, and motX, suggesting that H-NS represses the expression of these genes in the absence of ^S. Finally, we show that the cyclic AMP receptor protein and H-NS act along the same pathway to positively affect RpoS expression.

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* Corresponding author. Mailing address: Department of Microbiology, Biochemistry, and Immunology, Morehouse School of Medicine, 720 Westview Dr., SW, Atlanta, GA 30310. Phone: (404) 756-6660. Fax: (404) 752-1179. E-mail: asilva-benitez{at}msm.edu

Published ahead of print on 12 September 2008.

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

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Journal of Bacteriology, November 2008, p. 7335-7345, Vol. 190, No. 22
0021-9193/08/$08.00+0     doi:10.1128/JB.00360-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.