CONICET | Buscador de Institutos y Recursos Humanos

In E. faecalis the genes encoding the enzymes involved in citrate metabolism are organized in two divergent operons: citHO and oadHDB-citCDEFX-oadA-citMG. Both operons are specifically activated by the addition of citrate to the medium. This activation is mediated by binding of CitO, a transcriptional regulator of the GntR family, to the cis-acting sequences located in the cit intergenic region. Early studies indicated that citrate and glucose could not be co-metabolized suggesting some form of catabolite repression. Carbon catabolite repression (CCR) is the general phenomenon to adjust expression of catabolic genes in response to the availability of rapidly metabolizable carbon sources. In Gram-positive bacteria CCR is exerted through binding of the CcpA protein to cis-acting catabolite responsive element (cre). Our previous results indicated that citrate metabolism is repressed by PTS sugars. Detailed sequence analysis of the promoter regions revealed the presence of multiple cre sites, two located downstream of PcitHO (precisely 96 and 208 bp) and one downstream of PcitCL (at 98 bp), strongly suggesting that the pleiotropic transcriptional factor CcpA, could be involved in the repression of the citrate metabolism. In this work, we shown by western blot analysis of cell extracts of E. faecalis grown with different initial concentrations of glucose, that the level of the activator CitO is tightly regulated by the availability of preferential carbon sources. In accordance, cell extracts showed a reduction in the citrate lyase activity. Also, we analyzed the role of the cre sites in the mechanism of transcription regulation. Gel mobility shift assays using DNA fragments corresponding to the individual cre sites, confirmed that the E. faecalis CcpA protein is capable of binding to each of them. To determine if the binding of CcpA to the cre sites downregulates the expression of the operons, a set of DNA fragments covering alternative versions of the cit promoters (cre sites deleted or mutated) were fused to the promoterless lacZ reporter gene of pTCV-lac vector. The level of accumulated β-galactosidase activity of the resulting strains, grown in LB supplemented with glucose and citrate, suggested that the cre sites are indeed involved in the regulation of the cit promoters. Moreover, we found that either of the cre sites found in the PcitHO promoter were able to repress this operon, and all the cre sites worked independently in the conditions assayed. These results confirm the presence of multiple cre sites involved in the CCR, which suggest a more complex mechanism implicated in the fine tuning of the regulation mediated by CcpA.