Identification of siderophore biosynthesis genes essential for growth of Aeromonas salmonicida under iron limitation conditions

Aeromonas salmonicida subsp. salmonicida, the etiological agent of furunculosis in fish, produces a catechol-type siderophore under iron-limiting conditions. In this study, the Fur titration assay (FURTA) was used to identify a cluster of six genes, asbG, asbF, asbD, asbC, asbB, and asbI, encoding proteins similar to components of the siderophore biosynthetic machinery in other bacteria. Reverse transcriptase PCR analyses showed that this cluster consists of four iron-regulated transcriptional units. Mutants with deletions in either asbD (encoding a multidomain nonribosomal peptide synthetase), asbG (encoding a histidine decarboxylase), or asbC (encoding a predicted histamine monooxygenase) did not grow under iron-limiting conditions and did not produce siderophores. Growth of the Delta asbG strain under iron starvation conditions was restored by addition of histamine, suggesting that the siderophore in this species could contain a histamine-derived moiety. None of the mutants could grow in the presence of transferrin, indicating that A. salmonicida uses the catechol-type siderophore for removal of iron from transferrin rather than relying on a receptor for this iron-binding protein. All 18 A. salmonicida strains analyzed by DNA probe hybridization were positive in tests for the presence of the asbD gene, and all of them promoted the growth of asbD, asbG, and asbC mutants, suggesting that this siderophore-mediated iron uptake system is conserved among A. salmonicida isolates. This study provides the first description of siderophore biosynthesis genes in this fish pathogen, and the results demonstrate that the asbD, asbG, and asbC genes are necessary for the production of a catecholate siderophore that is essential for the growth of A. salmonicida under iron limitation conditions.