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Effect of a glucose impulse on the CcpA regulon in Staphylococcus aureus.

Seidl K, Müller S, François P, Kriebitzsch C, Schrenzel J, Engelmann S, Bischoff M, Berger-Bächi B - BMC Microbiol. (2009)

Bottom Line: Phosphotransacetylase and acetate kinase, converting acetyl-CoA to acetate with a concomitant substrate-level phosphorylation, were neither regulated by glucose nor by CcpA.Interestingly, the expression of a larger number of genes was found to be affected by ccpA inactivation in the absence of glucose than after glucose addition, suggesting that glucose-independent effects due to CcpA may have a particular impact in S. aureus.In the presence of glucose, CcpA was found to regulate the expression of genes involved in metabolism, but also that of genes coding for virulence determinants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland. katiseidl@gmail.com

ABSTRACT

Background: The catabolite control protein A (CcpA) is a member of the LacI/GalR family of transcriptional regulators controlling carbon-metabolism pathways in low-GC Gram-positive bacteria. It functions as a catabolite repressor or activator, allowing the bacteria to utilize the preferred carbon source over secondary carbon sources. This study is the first CcpA-dependent transcriptome and proteome analysis in Staphylococcus aureus, focussing on short-time effects of glucose under stable pH conditions.

Results: The addition of glucose to exponentially growing S. aureus increased the expression of genes and enzymes of the glycolytic pathway, while genes and proteins of the tricarboxylic acid (TCA) cycle, required for the complete oxidation of glucose, were repressed via CcpA. Phosphotransacetylase and acetate kinase, converting acetyl-CoA to acetate with a concomitant substrate-level phosphorylation, were neither regulated by glucose nor by CcpA. CcpA directly repressed genes involved in utilization of amino acids as secondary carbon sources. Interestingly, the expression of a larger number of genes was found to be affected by ccpA inactivation in the absence of glucose than after glucose addition, suggesting that glucose-independent effects due to CcpA may have a particular impact in S. aureus. In the presence of glucose, CcpA was found to regulate the expression of genes involved in metabolism, but also that of genes coding for virulence determinants.

Conclusion: This study describes the CcpA regulon of exponentially growing S. aureus cells. As in other bacteria, CcpA of S. aureus seems to control a large regulon that comprises metabolic genes as well as virulence determinants that are affected in their expression by CcpA in a glucose-dependent as well as -independent manner.

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Growth, glucose consumption and acetate build-up. Growth, glucose consumption and acetate formation in strain Newman (wt) and its isogenic ΔccpA mutant (ΔccpA). Cells were grown to an OD600 of 1, cultures were split and 10 mM glucose was added to one half of the culture (squares), while the other half remained without glucose (triangles). Cells were sampled at an OD600 of 1 and 30 min after glucose addition for RNA isolation (indicated by arrows). Experiments shown are representative for three independent experiments.
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Figure 1: Growth, glucose consumption and acetate build-up. Growth, glucose consumption and acetate formation in strain Newman (wt) and its isogenic ΔccpA mutant (ΔccpA). Cells were grown to an OD600 of 1, cultures were split and 10 mM glucose was added to one half of the culture (squares), while the other half remained without glucose (triangles). Cells were sampled at an OD600 of 1 and 30 min after glucose addition for RNA isolation (indicated by arrows). Experiments shown are representative for three independent experiments.

Mentions: The transcriptomes of strain Newman and its isogenic ΔccpA mutant MST14 were analyzed in LB, a complex medium essentially free of glucose and other rapidly catabolizable sugars [25], under controlled pH conditions in exponential growth (OD600 of 1), and 30 min after the addition of 10 mM glucose. In the absence of glucose, the wild-type had a slightly lower doubling time than the mutant (25.7 ± 1.29 min versus 31.8 ± 1.29 min, P < 0.01). The addition of 10 mM glucose at OD600 of 1 increased the growth rate of the wild-type but had only a minor effect on that of the mutant (Fig. 1). 60 min after glucose addition, glucose was depleted from the medium down to 0.3 mM by the wild-type, while still 3 mM of glucose were left in the culture of the mutant (Fig. 1). Despite increased growth and glucose consumption rates in the wild-type culture, acetate production was only slightly enhanced compared to the mutant, in line with previous findings [24]. No lactate was excreted under these conditions at any time point sampled, confirming the aerobic growth conditions. Acidification of the medium upon glucose metabolism was prevented by HEPES-buffering, which allowed maintaining the pH of the growth media at 7.5 for both strains and under both growth conditions for at least 2 h past glucose addition.


Effect of a glucose impulse on the CcpA regulon in Staphylococcus aureus.

Seidl K, Müller S, François P, Kriebitzsch C, Schrenzel J, Engelmann S, Bischoff M, Berger-Bächi B - BMC Microbiol. (2009)

Growth, glucose consumption and acetate build-up. Growth, glucose consumption and acetate formation in strain Newman (wt) and its isogenic ΔccpA mutant (ΔccpA). Cells were grown to an OD600 of 1, cultures were split and 10 mM glucose was added to one half of the culture (squares), while the other half remained without glucose (triangles). Cells were sampled at an OD600 of 1 and 30 min after glucose addition for RNA isolation (indicated by arrows). Experiments shown are representative for three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2697999&req=5

Figure 1: Growth, glucose consumption and acetate build-up. Growth, glucose consumption and acetate formation in strain Newman (wt) and its isogenic ΔccpA mutant (ΔccpA). Cells were grown to an OD600 of 1, cultures were split and 10 mM glucose was added to one half of the culture (squares), while the other half remained without glucose (triangles). Cells were sampled at an OD600 of 1 and 30 min after glucose addition for RNA isolation (indicated by arrows). Experiments shown are representative for three independent experiments.
Mentions: The transcriptomes of strain Newman and its isogenic ΔccpA mutant MST14 were analyzed in LB, a complex medium essentially free of glucose and other rapidly catabolizable sugars [25], under controlled pH conditions in exponential growth (OD600 of 1), and 30 min after the addition of 10 mM glucose. In the absence of glucose, the wild-type had a slightly lower doubling time than the mutant (25.7 ± 1.29 min versus 31.8 ± 1.29 min, P < 0.01). The addition of 10 mM glucose at OD600 of 1 increased the growth rate of the wild-type but had only a minor effect on that of the mutant (Fig. 1). 60 min after glucose addition, glucose was depleted from the medium down to 0.3 mM by the wild-type, while still 3 mM of glucose were left in the culture of the mutant (Fig. 1). Despite increased growth and glucose consumption rates in the wild-type culture, acetate production was only slightly enhanced compared to the mutant, in line with previous findings [24]. No lactate was excreted under these conditions at any time point sampled, confirming the aerobic growth conditions. Acidification of the medium upon glucose metabolism was prevented by HEPES-buffering, which allowed maintaining the pH of the growth media at 7.5 for both strains and under both growth conditions for at least 2 h past glucose addition.

Bottom Line: Phosphotransacetylase and acetate kinase, converting acetyl-CoA to acetate with a concomitant substrate-level phosphorylation, were neither regulated by glucose nor by CcpA.Interestingly, the expression of a larger number of genes was found to be affected by ccpA inactivation in the absence of glucose than after glucose addition, suggesting that glucose-independent effects due to CcpA may have a particular impact in S. aureus.In the presence of glucose, CcpA was found to regulate the expression of genes involved in metabolism, but also that of genes coding for virulence determinants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland. katiseidl@gmail.com

ABSTRACT

Background: The catabolite control protein A (CcpA) is a member of the LacI/GalR family of transcriptional regulators controlling carbon-metabolism pathways in low-GC Gram-positive bacteria. It functions as a catabolite repressor or activator, allowing the bacteria to utilize the preferred carbon source over secondary carbon sources. This study is the first CcpA-dependent transcriptome and proteome analysis in Staphylococcus aureus, focussing on short-time effects of glucose under stable pH conditions.

Results: The addition of glucose to exponentially growing S. aureus increased the expression of genes and enzymes of the glycolytic pathway, while genes and proteins of the tricarboxylic acid (TCA) cycle, required for the complete oxidation of glucose, were repressed via CcpA. Phosphotransacetylase and acetate kinase, converting acetyl-CoA to acetate with a concomitant substrate-level phosphorylation, were neither regulated by glucose nor by CcpA. CcpA directly repressed genes involved in utilization of amino acids as secondary carbon sources. Interestingly, the expression of a larger number of genes was found to be affected by ccpA inactivation in the absence of glucose than after glucose addition, suggesting that glucose-independent effects due to CcpA may have a particular impact in S. aureus. In the presence of glucose, CcpA was found to regulate the expression of genes involved in metabolism, but also that of genes coding for virulence determinants.

Conclusion: This study describes the CcpA regulon of exponentially growing S. aureus cells. As in other bacteria, CcpA of S. aureus seems to control a large regulon that comprises metabolic genes as well as virulence determinants that are affected in their expression by CcpA in a glucose-dependent as well as -independent manner.

Show MeSH
Related in: MedlinePlus