CcpA represses the divergent cit operon of Enterococcus faecalis through multiple cre sites

BLANCATO, VICTOR; SUÁREZ C.; PINCET, S; DEUTSCHER, J; MAGNI, CHRISTIAN

Puerto Madryn

Congreso; XLVI Reunión Anual de SAIB; 2010

Sociedad Argentina de Investigación en Bioquímica y Bilogía Molecular

In E. faecalis the genes encoding the enzymes involved in citrate metabolism are organized in two divergent operons, citHO and oadHDB-citCDEFX-oadA-citMG (citCL). Both operons are specifically activated by addition of citrate to culture medium. We observed that the citHO and citCL promoters are repressed in the presence of sugars transported by the PTS, strongly suggesting a Carbon Catabolic Repression (CCR). Moreover, our results showed that repression was relieved in a ccpA-deficient E. faecalis strain indicating that the pleiotropic transcriptional factor CcpA was involved in the regulation. Sequence analysis of the proximal intergenic region of the cit operons revealed the presence of three putative catabolite responsive elements (cre). In this work, we analyzed the role of CcpA and cre sites in the regulation of citrate metabolism. By gel mobility shift assays (EMSA) we confirmed that E. faecalis CcpA is capable of binding to each cre site. Furthermore, we analyzed the effect of its cofactor, HPr. The results indicated that unphosphorylated HPr showed a minor increase in CcpA affinity for the cre sites, whereas serine-phosphorylated HPr (P-Ser-HPr) produced a significant increase in affinity. Thus, in E. faecalis the binding of the P-Ser-HPr-CcpA complex to the cre sites repressed the expression of the activator CitO and also inhibited the expression of the catabolic genes.