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 vib riobactin outer membrane receptor, viuA, and the vibriobactin utilization g ene, viuB, was recovered from a Sau3A lambda library of O395 chromosomal DN A. By analogy with the genetic organization of the Escherichia coli enterob actin gene cluster, in which the enterobactin biosynthetic and transport ge nes lie adjacent to the enterobactin outer membrane receptor, fepA, and the utilization gene,fes, the cloned DNA was examined for the ability to resto re siderophore synthesis to E. coli ent mutants. Cross-feeding studies demo nstrated that an E. coli enfF mutant complemented with the cloned DNA regai ned the ability to synthesize enterobactin and to grow in low-iron medium. Sequence analysis of the cloned chromosomal DNA revealed an open reading fr ame downstream of viuB which encoded a deduced protein of greater than 2,15 8 amino acids, homologous to Yersinia sp. HMWP2, Vibrio anguillarum AngR, a nd E. call EntF. A mutant with an in-frame deletion of this gene, named vib F, was created with classical V. cholerae strain O395 by in vivo marker exc hange. In cross-feeding studies, this mutant was unable to synthesize ferri c vibriobactin but was able to utilize exogenous siderophore. Complementati on of the mutant with a cloned vibF fragment restored vibriobactin synthesi s 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 o f vibF was located by primer extension and was found to contain a dyad symm etric nucleotide sequence highly homologous to the E. coli Fur binding cons ensus sequence. A footprint of purified V. cholerae Fur on the vibF promote r, overlapping the Fur binding consensus sequence, was observed using DNase I footprinting. The protein product of vibF is homologous to the multifunc tional nonribosomal protein synthetases and is necessary for the biosynthes is of vibriobactin.