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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 glucose concentrations on the expression of cit operons, CitO levels and citrate lyase activity. A and B) JHB2 (JH2-2/pTCV-PcitHO), JHB6 (JH2-2/pTCV-PcitCL), CL1 (CL14/pTCV-PcitHO) and CL2 strains (CL14/pTCV-PcitCL) were grown in LBC (circle) or LBC supplemented with different initial concentrations of glucose: 0.25% (square), 0.5% (up-pointing triangle) and 1% (down-pointing triangle). The corresponding open symbols indicate the remaining glucose concentration in the culture medium (right axis). Levels of accumulated β-galactosidase activity were measured at different times as indicated in the figure. C and D) E. faecalis strains were grown in the same conditions of panels A and B, and cells extracts were obtained 7 h after inoculation, C) Western blot analysis was performed with polyclonal antibodies raised against CitO. D) Citrate lyase activity was determined as described previously [5]. Error bars represent standard deviation of triplicate measurements.
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Figure 2: Effect of glucose concentrations on the expression of cit operons, CitO levels and citrate lyase activity. A and B) JHB2 (JH2-2/pTCV-PcitHO), JHB6 (JH2-2/pTCV-PcitCL), CL1 (CL14/pTCV-PcitHO) and CL2 strains (CL14/pTCV-PcitCL) were grown in LBC (circle) or LBC supplemented with different initial concentrations of glucose: 0.25% (square), 0.5% (up-pointing triangle) and 1% (down-pointing triangle). The corresponding open symbols indicate the remaining glucose concentration in the culture medium (right axis). Levels of accumulated β-galactosidase activity were measured at different times as indicated in the figure. C and D) E. faecalis strains were grown in the same conditions of panels A and B, and cells extracts were obtained 7 h after inoculation, C) Western blot analysis was performed with polyclonal antibodies raised against CitO. D) Citrate lyase activity was determined as described previously [5]. Error bars represent standard deviation of triplicate measurements.

Mentions: Subsequently, we tested whether expression of the cit operons depends on the glucose concentration. Hence, we measured the β-galactosidase activity in wild-type and ccpA mutant strains carrying either one of the two transcriptional cit promoter-lacZ fusions. In the wild-type-derived strains (JHB2 and JHB6) β-galactosidase activity decreased when the initial concentration of glucose was raised from 0.25 to 1% (Figure 2A). On the other hand, in the CcpA-deficient strains (CL1 and CL2) activity of the cit promoters was independent of the glucose concentration (Figure 2B). These results suggest that the activity of the cit promoters is tightly regulated by the availability of glucose and that the pleiotropic transcriptional factor CcpA is involved in this process.


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 glucose concentrations on the expression of cit operons, CitO levels and citrate lyase activity. A and B) JHB2 (JH2-2/pTCV-PcitHO), JHB6 (JH2-2/pTCV-PcitCL), CL1 (CL14/pTCV-PcitHO) and CL2 strains (CL14/pTCV-PcitCL) were grown in LBC (circle) or LBC supplemented with different initial concentrations of glucose: 0.25% (square), 0.5% (up-pointing triangle) and 1% (down-pointing triangle). The corresponding open symbols indicate the remaining glucose concentration in the culture medium (right axis). Levels of accumulated β-galactosidase activity were measured at different times as indicated in the figure. C and D) E. faecalis strains were grown in the same conditions of panels A and B, and cells extracts were obtained 7 h after inoculation, C) Western blot analysis was performed with polyclonal antibodies raised against CitO. D) Citrate lyase activity was determined as described previously [5]. 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 2: Effect of glucose concentrations on the expression of cit operons, CitO levels and citrate lyase activity. A and B) JHB2 (JH2-2/pTCV-PcitHO), JHB6 (JH2-2/pTCV-PcitCL), CL1 (CL14/pTCV-PcitHO) and CL2 strains (CL14/pTCV-PcitCL) were grown in LBC (circle) or LBC supplemented with different initial concentrations of glucose: 0.25% (square), 0.5% (up-pointing triangle) and 1% (down-pointing triangle). The corresponding open symbols indicate the remaining glucose concentration in the culture medium (right axis). Levels of accumulated β-galactosidase activity were measured at different times as indicated in the figure. C and D) E. faecalis strains were grown in the same conditions of panels A and B, and cells extracts were obtained 7 h after inoculation, C) Western blot analysis was performed with polyclonal antibodies raised against CitO. D) Citrate lyase activity was determined as described previously [5]. Error bars represent standard deviation of triplicate measurements.
Mentions: Subsequently, we tested whether expression of the cit operons depends on the glucose concentration. Hence, we measured the β-galactosidase activity in wild-type and ccpA mutant strains carrying either one of the two transcriptional cit promoter-lacZ fusions. In the wild-type-derived strains (JHB2 and JHB6) β-galactosidase activity decreased when the initial concentration of glucose was raised from 0.25 to 1% (Figure 2A). On the other hand, in the CcpA-deficient strains (CL1 and CL2) activity of the cit promoters was independent of the glucose concentration (Figure 2B). These results suggest that the activity of the cit promoters is tightly regulated by the availability of glucose and that the pleiotropic transcriptional factor CcpA is involved in this process.

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