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Atlantic salmon (Salmo salar) were challenged with Vibrio anguillarum strains M93Sm and NB10 and empA null mutants M99 and NB12. Both wild types were virulent when administered by intraperitoneal (i.p.) injection or anal intubation. NB12 was avirulent via either route of infection. M99 virulence was attenuated when delivered by intubation, but fully virulent by i.p. injection. Northern blot analysis revealed empA expression in M93Sm and NB10 cells incubated in mucus, while incubation in Luria-Bertani broth plus 2% NaCl (LB20) induced empA expression only in NB10. Nucleotide differences between M93Sm and NB10 empA sequences were found in regions located 207 and 229 bp upstream of the empA translational start. Reverse transcription-PCR and 5′ rapid amplification of cDNA ends revealed the empA transcriptional start site 85 bp upstream of the translational start for both strains. A putative σS-dependent promoter was identified upstream of the transcriptional start in both strains. Site-directed mutagenesis was used to create rpoS mutants of M93Sm and NB10. Neither rpoS mutant exhibited protease activity. Since empA is expressed during stationary phase, the effects of conditioned medium on protease activity were examined. M99 conditioned LB20 supernatants stimulated protease activity in NB10 while allowing M93Sm to produce protease in LB20. Neither acyl homoserine lactones nor AI-2 induced protease activity. Conditioned LB20 supernatant from a V. anguillarum luxS mutant caused a more rapid induction of protease activity in wild-type cells. Our data show that expression of empA is differentially regulated in V. anguillarum strains NB10 and M93Sm and requires σS, quorum-sensing molecules, and gastrointestinal mucus.