Novel role of the lipopolysaccharide O1 side chain in ferric siderophore transport and virulence of Vibrio anguillarum

From a library of approximately 20,000 transposon mutants, we have identified mutants affected in chromosomal genes involved in synthesis of the siderophore anguibactin, as well as in ferric anguibactin utilization. Genetic and sequence analyses of one such transport-defective mutant revealed that the transposon insertion occurred in an open reading frame (ORF) with homology to rmlC, a dTDP-rhamnose biosynthetic gene. This ORF resides within a cluster of four ORFs, all of which are predicted to function in the biosynthesis of this O side chain precursor. The same phenotype was seen in a mutant obtained by allelic exchange in rmlD, another ORF in this dTDP-rhamnose biosynthetic cluster. This mutation could be complemented with the wild-type rmlD gene, restoring both production of the O1 antigen side chain and ferric anguibactin transport. Presence of the O1 side chain was crucial for the resistance of Vibrio anguillarum to the bactericidal action of nonimmune serum from the fish host. Surprisingly, further analysis demonstrated that these mutations were pleiotropic, leading to a dramatic decrease in the levels of FatA, the outer membrane protein receptor for ferric anguibactin transport, and a concomitant reduction in iron transport. Thus, our results in this work demonstrate that the lipopolysaccharide O1 side chain is required for the operation of two critical virulence factors in V. anguillarum: serum resistance and anguibactin-mediated iron transport. These factors allow V. anguillarum to survive in serum and multiply in the iron-limiting milieu of the host vertebrate.