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Background  The induction of metalloprotease encoded by  empA  in  Vibrio anguillarum  occurs at high cell density in salmon intestinal mucus. Previously we have shown that there are significant differences in  empA  expression in two strains of  V. anguillarum , M93Sm and NB10. It is hypothesized that differences in  empA  regulation are due to differences in binding of regulatory elements.    Results  Two strains of  V. anguillarum , M93Sm and NB10, were examined and compared for the presence of DNA regulatory proteins that bind to and control the  empA  promoter region. Gel mobility shift assays, using a digoxigenin (DIG)-labeled oligomer containing a  lux  box-like element and the promoter for  empA , were done to demonstrate the presence of a DNA-binding protein. Protein extracts from NB10 cells incubated in Luria Bertani broth + 2% NaCl (LB20), nine salts solution + 200 μg/ml mucus (NSSM), 3M (marine minimal medium), or NSS resulted in a gel mobility shift. No gel mobility shift was seen when protein extracts from either LB20- or NSSM-grown M93Sm cells were mixed with the DIG-labeled  empA  oligomer. The azocasein assay detected protease activity in all incubation conditions for NB10 culture supernatants. In contrast, protease activity was detected in M93Sm culture supernatants only when incubated in NSSM. Since the  luxR  homologue in  V. anguillarum ,  vanT , has been cloned, sequenced, and shown to be required for protease activity, we wanted to determine if  vanT  mutants of NB10 exhibit the same gel shift observed in the wild-type. Site-directed mutagenesis was used to create  vanT  mutants in  V. anguillarum  M93Sm and NB10 to test whether VanT is involved with the gel mobility shift. Both  vanT  mutants, M02 and NB02, did not produce protease activity in any conditions. However, protein extracts from NB02 incubated in each condition still exhibited a gel shift when mixed with the DIG-labeled  empA  oligomer.    Conclusions  The data demonstrate that protein extracts of  V. anguillarum  NB10 cells contain a protein that binds to a 50 bp oligomer containing the  empA  promoter- lux  box-like region. NB10 cells express  empA  during stationary phase in all growth conditions. The DNA binding protein is not present in M93Sm extracts. M93Sm cells express protease activity only when incubated at high cell density in fish gastrointestinal mucus. The gel shift observed with NB10 cells is not due to VanT binding. The data also suggest that the DNA binding protein is responsible for the less restrictive expression of  empA  in NB10 compared to M93Sm.

Gel shift analysis of the empA promoter region in Vibrio anguillarum.