CitO regulates the citrate degradation gene cluster of Enterococcus faecalis

SUÁREZ C. A.; BLANCATO V. S.; REPIZO G. D.; GUIBERT L. M.; MAGNI C.

Carlos Paz

Congreso; XLIV Reunión Anual de SAIB; 2008

SAIB

We demonstrated that CitO, a member of the GntR family of transcriptional regulators, is a novel positive regulator involved in the expression of the cit operons in Enterococcus faecalis. The transcriptional analysis of the cit clusters revealed two divergent operons, citHO and oadHDBcitCDEFXoadAcitMG, which were activated by specific addition of citrate to the medium. In this work, we showed by means of DNAseI footprinting and EMSA assays that CitO binds to the cis-acting sequences O1 and O2, present on the citHO – oadH intergenic region. Its affinity for the binding sites is increased in the presence of citrate allowing in this way the induction of both cit promoters. After that, to demonstrate that CitO specifically recognizes the intergenic region in vivo, we used a plasmid containing the O1 and O2 sites (pO1O2). Transformation with the empty vector resulted in normal transcriptional induction, while transformation with plasmid pO1O2 reduced the activity of the promoters. To define the role of CitO binding regions in the mechanism of transcription regulation, a set of DNA fragments covering different regions of the cit promoters were fused to a promoterless lacZ reporter gene. Altogether, the results presented indicate that the two CitO binding sites are very likely to be the fundamental cis-acting elements for the regulation of the cit promoters mediated by CitO in vivo.