IDENTIFICATION, CLONING, AND SEQUENCING OF A GENE REQUIRED FOR FERRICVIBRIOBACTIN UTILIZATION BY VIBRIO-CHOLERAE

Chromosomal DNA downstream of the Vibrio cholerae ferric vibriobactin receptor gene, viuA, was cloned and sequenced, revealing an 813-bp ope n reading frame encoding a deduced protein of 271 amino acids. In vitr o transcription-translation of this DNA confirmed expression of a prot ein of the expected size. A deletion mutation of this gene, viuB, was created in the classical V. cholerae strain O395 by in vivo marker exc hange. By cross-feeding studies, this mutant was unable to utilize exo genous ferric vibriobactin but synthesized the siderophore normally; s ynthesis 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, hyd ropathicity analysis of ViuB did not reveal a signal sequence or trans membrane domain, suggesting that ViuB is not a periplasmic or membrane protein but may be a cytoplasmic protein involved in ferric vibriobac tin uptake and processing, perhaps analogous to the Escherichia coli p rotein Fes. ViuB was not, however, homologous to Fes or to other prote ins 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 mu tant; Northern (RNA) blot analysis of RNA from wild-type V. cholerae g rown in high- and low-iron media revealed a monocistronic viuB message that was negatively regulated by iron at the transcriptional level. T he promoter of viuB was located by primer extension and contained a nu cleotide sequence highly homologous to the E. coli Fur binding consens us sequence, suggesting that expression of viuB is under the control o f the V. cholerae fur gene.