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Journal of Bacteriology, August 2003, p. 4672-4682, Vol. 185, No. 16
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.16.4672-4682.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Mutation in the relA Gene of Vibrio cholerae Affects In Vitro and In Vivo Expression of Virulence Factors

Shruti Haralalka, Suvobroto Nandi,{dagger} and Rupak K. Bhadra*

Infectious Diseases Division, Indian Institute of Chemical Biology, Kolkata 700 032, India

Received 27 January 2003/ Accepted 16 May 2003

The relA gene product determines the level of (p)ppGpp, the effector nucleotides of the bacterial stringent response that are also involved in the regulation of other functions, like antibiotic production and quorum sensing. In order to explore the possible involvement of relA in the regulation of virulence of Vibrio cholerae, a relA homolog from the organism (relAVCH) was cloned and sequenced. The relAVCH gene encodes a 738-amino-acid protein having functions similar to those of other gram-negative bacteria, including Escherichia coli. A {Delta}relA::kan allele was generated by replacing ~31% of the open reading frame of wild-type relA of V. cholerae El Tor strain C6709 with a kanamycin resistance gene. The V. cholerae relA mutant strain thus generated, SHK17, failed to accumulate (p)ppGpp upon amino acid deprivation. Interestingly, compared to the wild type, C6709, the mutant strain SHK17 exhibited significantly reduced in vitro production of two principal virulence factors, cholera toxin (CT) and toxin-coregulated pilus (TCP), under virulence gene-inducing conditions. In vivo experiments carried out in rabbit ileal loop and suckling mouse models also confirmed our in vitro results. The data suggest that (p)ppGpp is essential for maximal expression of CT and TCP during in vitro growth, as well as during intestinal infection by virulent V. cholerae. Northern blot and reverse transcriptase PCR analyses indicated significant reduction in the transcript levels of both virulence factors in the relA mutant strain SHK17. Such marked alteration of virulence phenotypes in SHK17 appears most likely to be due to down regulation of transcript levels of toxR and toxT, the two most important virulence regulatory genes of V. cholerae. In SHK17, the altered expression of the two outer membrane porin proteins, OmpU and OmpT, indicated that the relA mutation most likely affects the ToxR-dependent virulence regulatory pathway, because it had been shown earlier that ToxR directly regulates their expression independently of ToxT.

* Corresponding author. Mailing address: Infectious Diseases Division, Indian Institute of Chemical Biology, Kolkata 700 032, India. Phone: 91-33-2473-0350 or -3491. Fax: 91-33-2473-5197. E-mail: rupakbhadra{at}iicb.res.in.

{dagger} Present address: Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Chicago, Illinois 60208.

Journal of Bacteriology, August 2003, p. 4672-4682, Vol. 185, No. 16
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.16.4672-4682.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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