Characterization of the angR gene of Vibrio anguillarum: Essential role invirulence

The ability to utilize the iron bound by high-affinity iron-binding protein s in the vertebrate host is an important virulence factor for the marine fi sh pathogen Vibrio anguillarum. Virulence in septicemic infections is due t o the presence of a highly efficient plasmid-encoded iron transport system, AngR a 110-kDa protein component of this system, appears to play a role in both regulation of the expression of the iron transport genes fatDCBA and the production of the siderophore anguibactin. Therefore, study of the expr ession of the angR gene and the properties of its product, the AngR protein ? may contribute to the understanding of the mechanisms of virulence of thi s pathogen, In this work, we present genetic and molecular evidence from tr ansposition mutagenesis experiments and RNA analysis that angR, which maps immediately downstream of the fatA gene, is part of a polycistronic transcr ipt that also includes the iron transport genes fatDCBA and angT, a gene lo cated downstream of angR which showed domain homology to certain thioestera ses involved in nonribosomal peptide synthesis of siderophores and antibiot ics. In order to dissect the specific domains of AngR associated with regul ation of iron transport gene expression, anguibactin production, and virule nce, we also generated a panel of site-directed angR mutants, as well as de letion derivatives. Both virulence and anguibactin production were dramatic ally affected by each one of the angR modifications, In contrast to the nee d for an intact AngR molecule for anguibactin production and virulence, the regulation of iron transport gene expression does not require the entire A ngR molecule, since truncation of the carboxy terminus carrying the nonribo somal peptide synthetase cores, as well as the site-directed mutations, res ulted in derivatives that retained their ability to regulate gene expressio n which aas only abolished after truncation of amino-terminal sequences con taining helix-turn-helix and leucine zipper motifs and a specialized hetero cyclization and condensation domain found in certain nonribosomal peptide s ynthetases, The evidence, while not rigorously eliminating the possibility that a separate regulatory polypeptide exists and is encoded somewhere with in the 5'-end region of the angR gene, strongly supports the idea that AngR is a bifunctional protein and that it plays an essential role in the virul ence mechanisms of V. anguillarum. We also show in this study that the angT gene, found downstream of angR, intervenes in the mechanism of anguibactin production but is not essential for virulence or iron transport gene expre ssion.