In CCR or CCA (carbon catabolite activation) the CcpA/HPr-Ser-P complex regulates transcription through binding to the cre-sites . Most of the differential gene expression observed in our experiments could be ascribed to carbon catabolite regulation via cre-sites. CCR in E. faecalis has been studied by others, but not by transcriptomic analysis. It has been reported that enzymes for degradation of citrate, arginine, serine, galactose and GSK872 nmr glycerol are under control of CCR in E. faecalis [47–50]. This is in agreement with our finding
that these genes are up-regulated and associated with cre-sites. The metabolism of glycerol shows that LY2874455 our mutants were catabolic derepressed. The consensus sequence of the extragenic putative
cre-sites compiled in this study is WTGWAARCGYWWWC, very similar to what has been reported in B. subtilis . Most of the operons affected contain upstream cre-sites, but in several cases the putative cre-site is found within the open reading frames. Interestingly, three of the differentially expressed genes have the putative cre-site positioned in the intergenic region immediately downstream of the genes. Regulation of transcriptional initiation involving a 3′-cre located within the open reading frame but distantly separated from the promoter has been suggested to involve DNA looping . To our knowledge, cres located downstream of the regulated gene have not been reported. Another down-regulated gene with a putative cre-site in its promoter was EF0082, encoding a major facilitator GDC941 family transporter. The gene has also been found to be positively regulated by a PrfA-like regulator, Ers, encoded by EF0074 . Altogether, transcription involving about 90 cre-sites appeared to be affected in E. faecalis by disturbing its mannose PTS. About 65% of the putatively CCR regulated
genes encode proteins involved in uptake and metabolism of alternative energy sources. It is noteworthy that a number of genes showing increased transcription Inositol oxygenase in our mutants encode transcription regulators suggesting that regulatory cascades are involved. Among them were EF1025 and EF1026, encoding the homologs of CcpN and Yqfl which are involved in CcpA independent CCR in B. subtilis . When phosphorylated at His-15 by phosphotransfer from phosphoenolpyruvate via enzyme I, HPr has other regulatory functions. HPr-His-P reaches high levels in cells with a low energy status in response to reduced levels of glycolytic intermediates and ATP, and increased level of Pi and PEP . It can by phosphorylation regulate the activity of PTSs, enzymes such as DhaK and GlpK and transcriptional regulators [13, 48, 54, 55]. Interestingly, not only the spontaneous mutants but also the mptD-inactivated mutant showed a strong reduced transcription of the mpt operon.