Vibrio cholerae VibF is required for vibriobactin synthesis and is a member of the family of nonribosomal peptide synthetases

Abstract

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.

FIG. 1

Partial restriction map of O395 chromosomal DNA with relevant restriction enzyme sites, locations of the cloned fragments in pJRB20 and pMHC4, and position of the internal deletion of vibF in JRB18, JRB19, and JRB20. The extents of the coding regions of viuA, viuB, and vibF are indicated by solid arrows. The numbering of sites corresponds to that in reference .

FIG. 2

Organization of domains and core sequence motifs within the peptide synthetase module identified from the deduced amino acid sequence of VibF. The relative locations of the core sequences (cores 1 to 6) and spacer motif, their amino acid sequences in one-letter code, and their putative functions are indicated. The locations of the activation, acyl carrier protein (ACP), and elongation domains are marked.

FIG. 3

Primer extension analysis of RNA from strain O395 grown in low-iron medium using a 21-mer oligonucleotide at an annealing temperature of 55°C. Lanes A, C, G, and T are the corresponding lanes of the DNA sequencing ladder. Lane 1 is the control without added primer, and lane 2 is the primer extension reaction. The identified transcriptional start site (arrow) is shown with an asterisk in Fig. 4.

FIG. 4

Nucleotide sequence of the putative promoter region of vibF. The deduced amino acid sequence of VibF is shown in single-letter code beneath the DNA sequence. The likely transcriptional start site (∗), −10 box (−10), and −35 box (−35) are indicated above the sequence. Three potential starting methionine codons are shown. An inverted repeat homologous to the Fur binding consensus sequence of E. coli is underlined. The binding site for Fur deduced from DNase I protection (see Fig. 5) is boxed.

FIG. 5

Various amounts of purified Fur were incubated with a DNA fragment containing the viuB-vibF intergenic region (10 nM concentration) and subjected to DNase I digestion. Lanes: 1, DNA fragment alone; 2, DNA fragment incubated with 10 nM Fur; 3, DNA fragment incubated with 50 nM Fur. The DNase I footprint is indicated, as is the location of the Fur binding consensus sequence shown in Fig. 4.