SummaryThe two TonB systems in Vibrio cholerae were found to have unique as well as common functions. Both systems can mediate transport of haemin and the siderophores vibriobactin and ferrichrome. However, TonB1 specifically mediates utilization of the siderophore schizokinen, whereas TonB2 is required for utilization of enterobactin by V. cholerae. Although either TonB system was sufficient for the use of haemin as an iron source, in vitro competition between TonB1 and TonB2 system mutants indicates a preferential role for TonB1 in haemin utilization. This was most pronounced in conditions of high osmolarity, in which TonB1 system mutants were unable to grow with haemin as the sole iron source. Sequence analysis predicted that the two TonB proteins differ in both amino acid sequence and protein size. An internal deletion in TonB1 was constructed in order to generate a protein of approximately the same size as TonB2. A strain expressing the TonB1 deletion protein, and no other TonB, used haemin as the iron source in low-osmolarity medium, but could not use haemin in high osmolarity. This is the same phenotype as a strain expressing only TonB2 and suggests that TonB1, but not TonB2, can span the increased periplasmic space in high osmolarity and thus mediate haemin transport. Mouse colonization assays indicated a role for both TonB systems, and mutations in either system resulted in reduced ability to compete with the wild type in vivo.

Vibrio cholerae uses the catechol siderophore vibriobactin for iron transport under iron-limiting conditions. We have identified genes for vibriobactin transport and mapped them within the vibriobactin biosynthetic gene cluster. Within this genetic region we have identified four genes, viuP, viuD,viuG and viuC, whose protein products have homology to the periplasmic binding protein, the two integral cytoplasmic membrane proteins, and the ATPase component, respectively, of other iron transport systems. The amino-terminal region of ViuP has homology to a lipoprotein signal sequence, and ViuP could be labeled with [3H]palmitic acid. This suggests that ViuP is a membrane lipoprotein. The ViuPDGC system transports both vibriobactin and enterobactin in Escherichia coli. In the same assay, the E. coli enterobactin transport system, FepBDGC, allowed the utilization of enterobactin but not vibriobactin. Although the entire viuPDGC system could complement mutations infepB, fepD, fepG, orfepC, only viuC was able to independently complement the corresponding fep mutation. This indicates that these proteins usually function as a complex. V. cholerae strains carrying a mutation in viuP or inviuG were constructed by marker exchange. These mutations reduced, but did not completely eliminate, vibriobactin utilization. This suggests that V. cholerae contains genes in addition to viuPDGC that function in the transport of catechol siderophores.

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