Aeromonas salmonicida is the etiological agent of furunculosis in salmonid fish. The disease is responsible for severe economic losses in intensively cultured salmon and trout. Bacterin vaccines provide inadequate protection against infection. We have constructed an aromatic-dependent mutant of A. salmonicida in order to investigate the possibility of an effective live-attenuated vaccine. The aroA gene of A. salmonicida was cloned in Escherichia coli, and the nucleotide sequence was determined. The codon usage pattern of aroA was found to be quite distinct from that of the vapA gene coding for the surface array protein layer (A layer). The aroA gene was inactivated by inserting a fragment expressing kanamycin resistance within the coding sequence. The aroA::Kar mutation was introduced into the chromosome of virulent A. salmonicida 644Rb and 640V2 by allele replacement by using a suicide plasmid delivery system. The aroA mutation did not revert at a detectable frequency (< 10(-11). The mutation resulted in attenuation when bacteria were injected intramuscularly into Atlantic salmon (Salmo salar L.). Introduction of the wild-type aroA gene into the A. salmonicida mutants on a broad-host-range plasmid restored virulence. A. salmonicida mutant 644Rb aroA::Kar persisted in the kidney of brown trout (Salmo trutta L.) for 12 days at 10 degrees C. Vaccination of brown trout with 10(7) CFU of A. salmonicida 644Rb aroA by intraperitoneal injection resulted in a 253-fold increase in the 50% lethal dose (LD50) compared with unvaccinated controls challenged with a virulent clinical isolate 9 weeks later. A second vaccination after 6 weeks increased the LD50 by a further 16-fold.