Identification of heme uptake genes in the fish pathogen Aeromonas salmonicida subsp. salmonicida

Aeromonas salmonicida subsp. salmonicida, the causative agent of furunculosis in fish, can use heme as the sole iron source. We applied the Fur Titration Assay to isolate a cluster including six genes hutAZXBCD that showed similarity to heme uptake genes of other Gram-negative bacteria, and three genes orf123 of unknown function. The spatial organization of these nine genes, arranged in five transcriptional units, was similar to that of a homologous cluster in A. hydrophila. When a TonB system was provided, this cluster allowed Escherichia coli 101ESD (an ent mutant, unable to synthesize enterobactin) to utilize hemin and hemoglobin as iron sources. Mutation of hutB, a gene that encodes a predicted periplasmic hemin-binding protein, caused a drastic defect in the ability of A. salmonicida to grow with hemin as unique source of iron. Interestingly, a mutant for hutA gene (encoding the outer membrane hemin receptor) showed initially a reduced ability to grow with hemin as sole iron source, but after 24 h it achieved growth levels similar to parental strain. Thus mutation of hutA could not abolish the growth with hemin as iron source, suggesting that redundant outer membrane heme transport functions might be encoded in the A. salmonicida genome.