Patricia A. Carroll

Phase variation in tcpH modulates expression of the ToxR regulon in Vibrio cholerae

We evaluated a spontaneous mutant of Vibrio cholerae, which was avirulent in an infant mouse and had reduced expression of cholera toxin and TcpA in response to environmental signals. The toxR, toxS and toxT genes in the mutant were normal, but transcription of toxT was absent. A plasmid expressing wild‐type tcpP and tcpH complemented the mutant. The mutation resulted from a frameshift in a string of nine G residues within tcpH; similar slipped‐strand mutations in tcpH arose at a frequency of 10−4 during overnight growth and in the majority of colonies by the end of 5 days of growth in ToxR‐inducing conditions. Transcription of tcpPH was regulated by temperature and pH independently of ToxR or ToxT. These results suggest that TcpH couples environmental signals (temperature and pH) to expression of the ToxR regulon, and provide a model for phase variation in the co‐ordinate expression of cholera virulence factors.

Vibrio cholerae VibF Is Required for Vibriobactin Synthesis and Is a Member of the Family of Nonribosomal Peptide Synthetases

A 7.5-kbp fragment of chromosomal DNA downstream of the Vibrio cholerae vibriobactin outer membrane receptor, viuA, and the vibriobactin utilization gene, viuB, was recovered from a Sau3A lambda library of O395 chromosomal DNA. By analogy with the genetic organization of the Escherichia coli enterobactin gene cluster, in which the enterobactin biosynthetic and transport genes lie adjacent to the enterobactin outer membrane receptor, fepA, and the utilization gene, fes, the cloned DNA was examined for the ability to restore siderophore synthesis to E. coli ent mutants. Cross-feeding studies demonstrated that an E. coli entF mutant complemented with the cloned DNA regained the ability to synthesize enterobactin and to grow in low-iron medium. Sequence analysis of the cloned chromosomal DNA revealed an open reading frame downstream of viuB which encoded a deduced protein of greater than 2,158 amino acids, homologous to Yersinia sp. HMWP2, Vibrio anguillarum AngR, and E. coli EntF. A mutant with an in-frame deletion of this gene, named vibF, was created with classical V. cholerae strain O395 by in vivo marker exchange. In cross-feeding studies, this mutant was unable to synthesize ferric vibriobactin but was able to utilize exogenous siderophore. Complementation of the mutant with a cloned vibF fragment restored vibriobactin synthesis to normal. The expression of the vibF promoter was found to be negatively regulated by iron at the transcriptional level, under the control of the V. cholerae fur gene. Expression of vibF was not autoregulatory and neither affected nor was affected by the expression of irgA or viuA. The promoter of vibF was located by primer extension and was found to contain a dyad symmetric nucleotide sequence highly homologous to the E. coli Fur binding consensus sequence. A footprint of purified V. cholerae Fur on the vibF promoter, overlapping the Fur binding consensus sequence, was observed using DNase I footprinting. The protein product of vibF is homologous to the multifunctional nonribosomal protein synthetases and is necessary for the biosynthesis of vibriobactin.