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Appl. Environ. Microbiol. doi:10.1128/AEM.02728-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

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

Department of Microbiology and Parasitology, Institute of Aquaculture and Faculty of Biology, University of Santiago de Compostela, Santiago de Compostela, Galicia, Spain

^* To whom correspondence should be addressed. Email: crosorio{at}usc.es.

	Abstract

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 6 genes, asbG, asbF, asbD, asbC, asbB and asbI encoding proteins similar to components of the siderophore biosynthetic machinery in other bacteria. RT-PCR analyses showed that this cluster consists of four iron-regulated transcriptional units. Deletion mutants in either asbD (encoding a multidomain nonribosomal peptide synthetase), asbG (encoding a histidine decarboxylase) or asbC (encoding a predicted histamine monooxygenase) failed to grow under iron-limiting conditions and did not produce siderophores. Growth of asbG under iron starvation 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. Of 18 A. salmonicida strains analyzed by DNA probe hybridization, all tested positive for the presence of 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 represents the first description of siderophore biosynthesis genes in this fish pathogen, and demonstrates that asbD, asbG, and asbC genes are necessary for the production of a catecholate siderophore that is essential for the growth of A. salmonicida under conditions of iron limitation.