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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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Effect of different sugars on expression of the cit operons. A) Genetic organization of E. faecalis cit metabolic operons. PcitHO, promoter of the citHO operon composed of CitH (Me2+-citrate transporter) and CitO (GntR transcriptional regulator); PcitCL promoter of the citCL operon composed of OadHDBA (oxaloacetate decarboxylase membrane complex), CitCDEFXG (citrate lyase and accessory proteins) and CitM (soluble oxaloacetate decarboxylase). O1 and O2 binding sites of the activator CitO. B and C) Influence of diverse PTS and non-PTS sugars on the expression of PcitHO-lacZ and PcitCL-lacZ fusions. JHB2 (JH2-2/pTCV-PcitHO), JHB6 (JH2-2/pTCV-PcitCL), CL1 (CL14/pTCV-PcitHO) and CL2 (CL14/pTCV-PcitCL) were grown in LBC and LBC supplemented with 30 mM initial concentration of different sugars. Levels of accumulated β-galactosidase activity were measured 7 h after inoculation. Error bars represent standard deviation of triplicate measurements.
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Figure 1: Effect of different sugars on expression of the cit operons. A) Genetic organization of E. faecalis cit metabolic operons. PcitHO, promoter of the citHO operon composed of CitH (Me2+-citrate transporter) and CitO (GntR transcriptional regulator); PcitCL promoter of the citCL operon composed of OadHDBA (oxaloacetate decarboxylase membrane complex), CitCDEFXG (citrate lyase and accessory proteins) and CitM (soluble oxaloacetate decarboxylase). O1 and O2 binding sites of the activator CitO. B and C) Influence of diverse PTS and non-PTS sugars on the expression of PcitHO-lacZ and PcitCL-lacZ fusions. JHB2 (JH2-2/pTCV-PcitHO), JHB6 (JH2-2/pTCV-PcitCL), CL1 (CL14/pTCV-PcitHO) and CL2 (CL14/pTCV-PcitCL) were grown in LBC and LBC supplemented with 30 mM initial concentration of different sugars. Levels of accumulated β-galactosidase activity were measured 7 h after inoculation. Error bars represent standard deviation of triplicate measurements.

Mentions: We recently described that the molecular mechanism underlying activation of the cit operons (citHO and citCL) in E. faecalis requires the transcriptional factor CitO [6]. Rea and Cogan had previously suggested that glucose represses citrate metabolism in this bacterium [15]. We therefore studied whether different carbon sources might affect transcription of the genes involved in citrate utilization. To accomplish this task, we measured the activity of the cit promoters (PcitHO and PcitCL, Figure 1A) by fusing them to the promoterless lacZ gene in the vector pTCV-lac [26]. β-Galactosidase activity was determined in cell extracts of E. faecalis JH2-2 harboring plasmid pTCV-PcitHO or pTCV-PcitCL, constructed in a previous work by Blancato et al., 2008 (strains JHB2 and JHB6, Table 1) [6].


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)

Effect of different sugars on expression of the cit operons. A) Genetic organization of E. faecalis cit metabolic operons. PcitHO, promoter of the citHO operon composed of CitH (Me2+-citrate transporter) and CitO (GntR transcriptional regulator); PcitCL promoter of the citCL operon composed of OadHDBA (oxaloacetate decarboxylase membrane complex), CitCDEFXG (citrate lyase and accessory proteins) and CitM (soluble oxaloacetate decarboxylase). O1 and O2 binding sites of the activator CitO. B and C) Influence of diverse PTS and non-PTS sugars on the expression of PcitHO-lacZ and PcitCL-lacZ fusions. JHB2 (JH2-2/pTCV-PcitHO), JHB6 (JH2-2/pTCV-PcitCL), CL1 (CL14/pTCV-PcitHO) and CL2 (CL14/pTCV-PcitCL) were grown in LBC and LBC supplemented with 30 mM initial concentration of different sugars. Levels of accumulated β-galactosidase activity were measured 7 h after inoculation. Error bars represent standard deviation of triplicate measurements.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Effect of different sugars on expression of the cit operons. A) Genetic organization of E. faecalis cit metabolic operons. PcitHO, promoter of the citHO operon composed of CitH (Me2+-citrate transporter) and CitO (GntR transcriptional regulator); PcitCL promoter of the citCL operon composed of OadHDBA (oxaloacetate decarboxylase membrane complex), CitCDEFXG (citrate lyase and accessory proteins) and CitM (soluble oxaloacetate decarboxylase). O1 and O2 binding sites of the activator CitO. B and C) Influence of diverse PTS and non-PTS sugars on the expression of PcitHO-lacZ and PcitCL-lacZ fusions. JHB2 (JH2-2/pTCV-PcitHO), JHB6 (JH2-2/pTCV-PcitCL), CL1 (CL14/pTCV-PcitHO) and CL2 (CL14/pTCV-PcitCL) were grown in LBC and LBC supplemented with 30 mM initial concentration of different sugars. Levels of accumulated β-galactosidase activity were measured 7 h after inoculation. Error bars represent standard deviation of triplicate measurements.
Mentions: We recently described that the molecular mechanism underlying activation of the cit operons (citHO and citCL) in E. faecalis requires the transcriptional factor CitO [6]. Rea and Cogan had previously suggested that glucose represses citrate metabolism in this bacterium [15]. We therefore studied whether different carbon sources might affect transcription of the genes involved in citrate utilization. To accomplish this task, we measured the activity of the cit promoters (PcitHO and PcitCL, Figure 1A) by fusing them to the promoterless lacZ gene in the vector pTCV-lac [26]. β-Galactosidase activity was determined in cell extracts of E. faecalis JH2-2 harboring plasmid pTCV-PcitHO or pTCV-PcitCL, constructed in a previous work by Blancato et al., 2008 (strains JHB2 and JHB6, Table 1) [6].

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