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CcpA represses the expression of the divergent cit operons of Enterococcus faecalis through multiple cre sites.

Suárez CA, Blancato VS, Poncet S, Deutscher J, Magni C - BMC Microbiol. (2011)

Bottom Line: We found that they are all active and able to bind the CcpA/P-Ser-HPr complex, which downregulates the expression of the cit operons.Systematic mutation of the CcpA/P-Ser-HPr binding sites revealed that cre1 and cre2 contribute to citHO repression, while cre3 is involved in CCR of citCL.In conclusion, our study establishes that expression of the cit operons in E. faecalis is controlled by CCR via CcpA-dependent and -independent mechanisms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET), Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, (S2002LRK) Rosario, Argentina.

ABSTRACT

Background: In Enterococcus faecalis the genes encoding the enzymes involved in citrate metabolism are organized in two divergent operons, citHO and oadHDB-citCDEFX-oadA-citMG (citCL locus). Expression of both operons is specifically activated by adding citrate to the medium. This activation is mediated by binding of the GntR-like transcriptional regulator (CitO) 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, however the molecular mechanism remained unknown.

Results: In this study, we observed that the citHO promoter is repressed in the presence of sugars transported by the Phosphoenolpyruvate:carbohydrate Phosphotranserase System (PTS sugars). This result strongly suggested that Carbon Catabolic Repression (CCR) impedes the expression of the activator CitO and the subsequent induction of the cit pathway. In fact, we demonstrate that CCR is acting on both promoters. It is partially relieved in a ccpA-deficient E. faecalis strain indicating that a CcpA-independent mechanism is also involved in regulation of the two operons. Furthermore, sequence analysis of the citH/oadH intergenic region revealed the presence of three putative catabolite responsive elements (cre). We found that they are all active and able to bind the CcpA/P-Ser-HPr complex, which downregulates the expression of the cit operons. Systematic mutation of the CcpA/P-Ser-HPr binding sites revealed that cre1 and cre2 contribute to citHO repression, while cre3 is involved in CCR of citCL.

Conclusion: In conclusion, our study establishes that expression of the cit operons in E. faecalis is controlled by CCR via CcpA-dependent and -independent mechanisms.

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Schematic representation of the pTCV-lac derived plasmids. Promoter regions of the citHO and citCL operons are shown. The different cre sites are indicated by boxes (C1, C2, C3 and M for mutated cre sites). The glucose repression index represents the ratio of accumulated β-galactosidase activity between cell extracts from cultures grown in LBC and LBCG medium (MULBC/MULBGC) for 7 hours.
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Figure 5: Schematic representation of the pTCV-lac derived plasmids. Promoter regions of the citHO and citCL operons are shown. The different cre sites are indicated by boxes (C1, C2, C3 and M for mutated cre sites). The glucose repression index represents the ratio of accumulated β-galactosidase activity between cell extracts from cultures grown in LBC and LBCG medium (MULBC/MULBGC) for 7 hours.

Mentions: In order to test the role of these sites in the transcription regulation mechanism mediated by CcpA, a set of DNA fragments corresponding to altered cit promoter regions (i.e. cre sites deleted or mutated) were fused to the promoterless lacZ reporter gene of the pTCV-lac vector (Figure 5). Plasmids harboring the Pcit-lacZ transcriptional fusions were electroporated into the E. faecalis JHB11 strain.


CcpA represses the expression of the divergent cit operons of Enterococcus faecalis through multiple cre sites.

Suárez CA, Blancato VS, Poncet S, Deutscher J, Magni C - BMC Microbiol. (2011)

Schematic representation of the pTCV-lac derived plasmids. Promoter regions of the citHO and citCL operons are shown. The different cre sites are indicated by boxes (C1, C2, C3 and M for mutated cre sites). The glucose repression index represents the ratio of accumulated β-galactosidase activity between cell extracts from cultures grown in LBC and LBCG medium (MULBC/MULBGC) for 7 hours.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198936&req=5

Figure 5: Schematic representation of the pTCV-lac derived plasmids. Promoter regions of the citHO and citCL operons are shown. The different cre sites are indicated by boxes (C1, C2, C3 and M for mutated cre sites). The glucose repression index represents the ratio of accumulated β-galactosidase activity between cell extracts from cultures grown in LBC and LBCG medium (MULBC/MULBGC) for 7 hours.
Mentions: In order to test the role of these sites in the transcription regulation mechanism mediated by CcpA, a set of DNA fragments corresponding to altered cit promoter regions (i.e. cre sites deleted or mutated) were fused to the promoterless lacZ reporter gene of the pTCV-lac vector (Figure 5). Plasmids harboring the Pcit-lacZ transcriptional fusions were electroporated into the E. faecalis JHB11 strain.

Bottom Line: We found that they are all active and able to bind the CcpA/P-Ser-HPr complex, which downregulates the expression of the cit operons.Systematic mutation of the CcpA/P-Ser-HPr binding sites revealed that cre1 and cre2 contribute to citHO repression, while cre3 is involved in CCR of citCL.In conclusion, our study establishes that expression of the cit operons in E. faecalis is controlled by CCR via CcpA-dependent and -independent mechanisms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET), Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, (S2002LRK) Rosario, Argentina.

ABSTRACT

Background: In Enterococcus faecalis the genes encoding the enzymes involved in citrate metabolism are organized in two divergent operons, citHO and oadHDB-citCDEFX-oadA-citMG (citCL locus). Expression of both operons is specifically activated by adding citrate to the medium. This activation is mediated by binding of the GntR-like transcriptional regulator (CitO) 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, however the molecular mechanism remained unknown.

Results: In this study, we observed that the citHO promoter is repressed in the presence of sugars transported by the Phosphoenolpyruvate:carbohydrate Phosphotranserase System (PTS sugars). This result strongly suggested that Carbon Catabolic Repression (CCR) impedes the expression of the activator CitO and the subsequent induction of the cit pathway. In fact, we demonstrate that CCR is acting on both promoters. It is partially relieved in a ccpA-deficient E. faecalis strain indicating that a CcpA-independent mechanism is also involved in regulation of the two operons. Furthermore, sequence analysis of the citH/oadH intergenic region revealed the presence of three putative catabolite responsive elements (cre). We found that they are all active and able to bind the CcpA/P-Ser-HPr complex, which downregulates the expression of the cit operons. Systematic mutation of the CcpA/P-Ser-HPr binding sites revealed that cre1 and cre2 contribute to citHO repression, while cre3 is involved in CCR of citCL.

Conclusion: In conclusion, our study establishes that expression of the cit operons in E. faecalis is controlled by CCR via CcpA-dependent and -independent mechanisms.

Show MeSH
Related in: MedlinePlus