Gel shift analysis of the <it>empA </it>promoter region in <it>Vibrio anguillarum</it>

• Main Authors: Denkin Steven M, Sekaric Pedja, Nelson David R
• Format: Article
• Language: English
• Published: BMC 2004-10-01
• Series: BMC Microbiology
• Online Access: http://www.biomedcentral.com/1471-2180/4/42

<p>Abstract</p> <p>Background</p> <p>The induction of metalloprotease encoded by <it>empA </it>in <it>Vibrio anguillarum </it>occurs at high cell density in salmon intestinal mucus. Previously we have shown that there are significant differences in <it>empA </it>expression in two strains of <it>V. anguillarum</it>, M93Sm and NB10. It is hypothesized that differences in <it>empA </it>regulation are due to differences in binding of regulatory elements.</p> <p>Results</p> <p>Two strains of <it>V. anguillarum</it>, M93Sm and NB10, were examined and compared for the presence of DNA regulatory proteins that bind to and control the <it>empA </it>promoter region. Gel mobility shift assays, using a digoxigenin (DIG)-labeled oligomer containing a <it>lux </it>box-like element and the promoter for <it>empA</it>, 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 <it>empA </it>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 <it>luxR </it>homologue in <it>V. anguillarum</it>, <it>vanT</it>, has been cloned, sequenced, and shown to be required for protease activity, we wanted to determine if <it>vanT </it>mutants of NB10 exhibit the same gel shift observed in the wild-type. Site-directed mutagenesis was used to create <it>vanT </it>mutants in <it>V. anguillarum </it>M93Sm and NB10 to test whether VanT is involved with the gel mobility shift. Both <it>vanT </it>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 <it>empA </it>oligomer.</p> <p>Conclusions</p> <p>The data demonstrate that protein extracts of <it>V. anguillarum </it>NB10 cells contain a protein that binds to a 50 bp oligomer containing the <it>empA </it>promoter-<it>lux </it>box-like region. NB10 cells express <it>empA </it>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 <it>empA </it>in NB10 compared to M93Sm.</p>