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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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Binding of CcpA to DNA fragments containing different cre sites. A) Nucleotide sequence of the citH-oadH intergenic regions. Locations of transcription start sites are indicated (+1); -10 and -35; regions are shown underlined. Arrows indicate direction of transcription and translation. CitO binding sequences are displayed in dotted boxes and putative cre sites in grey boxes. B, C and D) Images of gel shift assays performed with different amplicons (A, B and C respectively) covering each cre site or mutated cre site amplicons (Bm and Cm), increasing concentrations of CcpA and fixed concentrations of HPr or P-Ser-HPr.
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Figure 4: Binding of CcpA to DNA fragments containing different cre sites. A) Nucleotide sequence of the citH-oadH intergenic regions. Locations of transcription start sites are indicated (+1); -10 and -35; regions are shown underlined. Arrows indicate direction of transcription and translation. CitO binding sequences are displayed in dotted boxes and putative cre sites in grey boxes. B, C and D) Images of gel shift assays performed with different amplicons (A, B and C respectively) covering each cre site or mutated cre site amplicons (Bm and Cm), increasing concentrations of CcpA and fixed concentrations of HPr or P-Ser-HPr.

Mentions: The results presented up to this point show that PTS sugars repress the citrate fermentation pathway through the action of CcpA. A bioinformatic search in the divergent promoter region revealed the presence of three putative catabolite responsive elements (cre sites) highly homologous to the E. faecalis consensus cre site [TG(T/A)NANCGNTN(T/A)CA] [27] and [(T/A)TG(T/A)AA(A/G)CG(C/T)(T/A)(T/A) (T/A)C(T/A)] [29]. cre1 (C1) and cre2 (C2) are located downstream from PcitHO; C1 is located in the coding region of citH and C2 in the untranslated region at 207-bp and 94-bp, respectively, downstream from the transcriptional start site (TSS) of the citHO operon (distances are indicated relative to the center of symmetry). cre3 (C3) is located 97-bp downstream from the citCL TSS within the coding region of oadH (Figure 4).


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)

Binding of CcpA to DNA fragments containing different cre sites. A) Nucleotide sequence of the citH-oadH intergenic regions. Locations of transcription start sites are indicated (+1); -10 and -35; regions are shown underlined. Arrows indicate direction of transcription and translation. CitO binding sequences are displayed in dotted boxes and putative cre sites in grey boxes. B, C and D) Images of gel shift assays performed with different amplicons (A, B and C respectively) covering each cre site or mutated cre site amplicons (Bm and Cm), increasing concentrations of CcpA and fixed concentrations of HPr or P-Ser-HPr.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Binding of CcpA to DNA fragments containing different cre sites. A) Nucleotide sequence of the citH-oadH intergenic regions. Locations of transcription start sites are indicated (+1); -10 and -35; regions are shown underlined. Arrows indicate direction of transcription and translation. CitO binding sequences are displayed in dotted boxes and putative cre sites in grey boxes. B, C and D) Images of gel shift assays performed with different amplicons (A, B and C respectively) covering each cre site or mutated cre site amplicons (Bm and Cm), increasing concentrations of CcpA and fixed concentrations of HPr or P-Ser-HPr.
Mentions: The results presented up to this point show that PTS sugars repress the citrate fermentation pathway through the action of CcpA. A bioinformatic search in the divergent promoter region revealed the presence of three putative catabolite responsive elements (cre sites) highly homologous to the E. faecalis consensus cre site [TG(T/A)NANCGNTN(T/A)CA] [27] and [(T/A)TG(T/A)AA(A/G)CG(C/T)(T/A)(T/A) (T/A)C(T/A)] [29]. cre1 (C1) and cre2 (C2) are located downstream from PcitHO; C1 is located in the coding region of citH and C2 in the untranslated region at 207-bp and 94-bp, respectively, downstream from the transcriptional start site (TSS) of the citHO operon (distances are indicated relative to the center of symmetry). cre3 (C3) is located 97-bp downstream from the citCL TSS within the coding region of oadH (Figure 4).

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