Identification, cloning, and sequencing of a gene required for ferric vibriobactin utilization by Vibrio cholerae

Abstract

Chromosomal DNA downstream of the Vibrio cholerae ferric vibriobactin receptor gene, viuA, was cloned and sequenced, revealing an 813-bp open reading frame encoding a deduced protein of 271 amino acids. In vitro transcription-translation of this DNA confirmed expression of a protein of the expected size. A deletion mutation of this gene, viuB, was created in the classical V. cholerae strain O395 by in vivo marker exchange. By cross-feeding studies, this mutant was unable to utilize exogenous ferric vibriobactin but synthesized the siderophore normally; synthesis of siderophore by the mutant was also confirmed by the Arnow assay. Complementation of the mutant with a plasmid encoding only viuB restored ferric vibriobactin utilization to normal. Unexpectedly, hydropathicity analysis of ViuB did not reveal a signal sequence or transmembrane domain, suggesting that ViuB is not a periplasmic or membrane protein but may be a cytoplasmic protein involved in ferric vibriobactin uptake and processing, perhaps analogous to the Escherichia coli protein Fes. ViuB was not, however, homologous to Fes or to other proteins in the database. Complementation studies revealed that the cloned V. cholerae viuB gene could complement an E. coli fes mutant but that the cloned E. coli fes gene could not complement a V. cholerae viuB mutant. Northern (RNA) blot analysis of RNA from wild-type V. cholerae grown in high- and low-iron media revealed a monocistronic viuB message that was negatively regulated by iron at the transcriptional level. The promoter of viuB was located by primer extension and contained a nucleotide sequence highly homologous to the E. coli Fur binding consensus sequence, suggesting that expression of viuB is under the control of the V. cholerae fur gene.