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CcpA ensures optimal metabolic fitness of Streptococcus pneumoniae.

Carvalho SM, Kloosterman TG, Kuipers OP, Neves AR - PLoS ONE (2011)

Bottom Line: In agreement, CcpA influenced the level of many intracellular metabolites potentially involved in metabolic regulation.Our data strengthen the view that a true understanding of cell physiology demands thorough analyses at different cellular levels.Moreover, integration of transcriptional and metabolic data uncovered a link between CcpA and the association of surface molecules (e.g. capsule) to the cell wall.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.

ABSTRACT
In gram-positive bacteria, the transcriptional regulator CcpA is at the core of catabolite control mechanisms. In the human pathogen Streptococcus pneumoniae, links between CcpA and virulence have been established, but its role as a master regulator in different nutritional environments remains to be elucidated. Thus, we performed whole-transcriptome and metabolic analyses of S. pneumoniae D39 and its isogenic ccpA mutant during growth on glucose or galactose, rapidly and slowly metabolized carbohydrates presumably encountered by the bacterium in different host niches. CcpA affected the expression of up to 19% of the genome covering multiple cellular processes, including virulence, regulatory networks and central metabolism. Its prevalent function as a repressor was observed on glucose, but unexpectedly also on galactose. Carbohydrate-dependent CcpA regulation was also observed, as for the tagatose 6-phosphate pathway genes, which were activated by galactose and repressed by glucose. Metabolite analyses revealed that two pathways for galactose catabolism are functionally active, despite repression of the Leloir genes by CcpA. Surprisingly, galactose-induced mixed-acid fermentation apparently required CcpA, since genes involved in this type of metabolism were mostly under CcpA-repression. These findings indicate that the role of CcpA extends beyond transcriptional regulation, which seemingly is overlaid by other regulatory mechanisms. In agreement, CcpA influenced the level of many intracellular metabolites potentially involved in metabolic regulation. Our data strengthen the view that a true understanding of cell physiology demands thorough analyses at different cellular levels. Moreover, integration of transcriptional and metabolic data uncovered a link between CcpA and the association of surface molecules (e.g. capsule) to the cell wall. Hence, CcpA may play a key role in mediating the interaction of S. pneumoniae with its host. Overall, our results support the hypothesis that S. pneumoniae optimizes basic metabolic processes, likely enhancing in vivo fitness, in a CcpA-mediated manner.

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Fermentation profiles of D39 and D39ΔccpA on Glc and Gal.Growth curves, substrate consumption and end-products formed by the D39 (A and C) and D39ΔccpA (B and D) strains growing on Glc (A and B) or Gal (C and D). Culture supernatant samples for substrate and end product analysis by HPLC and/or 1H-NMR were harvested for each of the conditions in the mid-exponential, transition-to-stationary and growth arrest (maximal biomass) time points of the respective growth curves (bars in the plots). To calculate end-product concentrations, values of at least two independent experiments were averaged and the error was below 7% for major products (>2 mM) and 25% for minor products (<2 mM). Initial concentrations of Glc and Gal were 56±1 mM and 57±1 mM, respectively. Time points in substrate consumption curves are averages of at least two independent experiments and the error is below 5%. Symbols: (○), substrate consumption; (▪), growth curve; white bars, lactate; hatched bars, formate; black bars, acetate; stripped bars, ethanol. Growth curves as in Fig. S1, except that OD600 scale (y-axis) is decimal.
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pone-0026707-g003: Fermentation profiles of D39 and D39ΔccpA on Glc and Gal.Growth curves, substrate consumption and end-products formed by the D39 (A and C) and D39ΔccpA (B and D) strains growing on Glc (A and B) or Gal (C and D). Culture supernatant samples for substrate and end product analysis by HPLC and/or 1H-NMR were harvested for each of the conditions in the mid-exponential, transition-to-stationary and growth arrest (maximal biomass) time points of the respective growth curves (bars in the plots). To calculate end-product concentrations, values of at least two independent experiments were averaged and the error was below 7% for major products (>2 mM) and 25% for minor products (<2 mM). Initial concentrations of Glc and Gal were 56±1 mM and 57±1 mM, respectively. Time points in substrate consumption curves are averages of at least two independent experiments and the error is below 5%. Symbols: (○), substrate consumption; (▪), growth curve; white bars, lactate; hatched bars, formate; black bars, acetate; stripped bars, ethanol. Growth curves as in Fig. S1, except that OD600 scale (y-axis) is decimal.

Mentions: The pneumococcal strains were grown as above. Substrate consumption and concentrations of end-products are shown in Fig. 3. During growth on Glc strain D39 showed a typical homolactic behaviour, the major end-product being lactate, which reached a concentration of 52±1 mM at the time of growth arrest (maximal biomass), accounting for 93% of the Glc consumed (Table 2); formate was also formed in minor amounts (Fig. 3A and Table 2). Loss of CcpA caused a shift towards a more mixed acid fermentation: a 5-fold increase in the yields of formate, as well as formation of acetate and ethanol, were accompanied by a decrease on lactate production, which accounted for 85% of the Glc consumed as compared to 93% in D39 (Fig. 3B and Table 2). Hydrogen peroxide (H2O2), a known minor end-product of streptococcal aerobic metabolism, was also detected. In the wild-type, the maximal concentration reached was about 0.04±0.002 mM, while in the mutant this concentration was circa 7-fold enhanced. The low concentrations measured most likely reflect a limitation in oxygen availability due to the nearly anaerobic conditions used for growth. At the time point of growth arrest, the ccpA mutant had consumed 8% less Glc, and the maximal consumption rate was about 30% of the wild-type level (Table 2). The observed decrease in Glc consumption was paralleled by the reduction in growth rate, which was 53% lower in D39ΔccpA.


CcpA ensures optimal metabolic fitness of Streptococcus pneumoniae.

Carvalho SM, Kloosterman TG, Kuipers OP, Neves AR - PLoS ONE (2011)

Fermentation profiles of D39 and D39ΔccpA on Glc and Gal.Growth curves, substrate consumption and end-products formed by the D39 (A and C) and D39ΔccpA (B and D) strains growing on Glc (A and B) or Gal (C and D). Culture supernatant samples for substrate and end product analysis by HPLC and/or 1H-NMR were harvested for each of the conditions in the mid-exponential, transition-to-stationary and growth arrest (maximal biomass) time points of the respective growth curves (bars in the plots). To calculate end-product concentrations, values of at least two independent experiments were averaged and the error was below 7% for major products (>2 mM) and 25% for minor products (<2 mM). Initial concentrations of Glc and Gal were 56±1 mM and 57±1 mM, respectively. Time points in substrate consumption curves are averages of at least two independent experiments and the error is below 5%. Symbols: (○), substrate consumption; (▪), growth curve; white bars, lactate; hatched bars, formate; black bars, acetate; stripped bars, ethanol. Growth curves as in Fig. S1, except that OD600 scale (y-axis) is decimal.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198803&req=5

pone-0026707-g003: Fermentation profiles of D39 and D39ΔccpA on Glc and Gal.Growth curves, substrate consumption and end-products formed by the D39 (A and C) and D39ΔccpA (B and D) strains growing on Glc (A and B) or Gal (C and D). Culture supernatant samples for substrate and end product analysis by HPLC and/or 1H-NMR were harvested for each of the conditions in the mid-exponential, transition-to-stationary and growth arrest (maximal biomass) time points of the respective growth curves (bars in the plots). To calculate end-product concentrations, values of at least two independent experiments were averaged and the error was below 7% for major products (>2 mM) and 25% for minor products (<2 mM). Initial concentrations of Glc and Gal were 56±1 mM and 57±1 mM, respectively. Time points in substrate consumption curves are averages of at least two independent experiments and the error is below 5%. Symbols: (○), substrate consumption; (▪), growth curve; white bars, lactate; hatched bars, formate; black bars, acetate; stripped bars, ethanol. Growth curves as in Fig. S1, except that OD600 scale (y-axis) is decimal.
Mentions: The pneumococcal strains were grown as above. Substrate consumption and concentrations of end-products are shown in Fig. 3. During growth on Glc strain D39 showed a typical homolactic behaviour, the major end-product being lactate, which reached a concentration of 52±1 mM at the time of growth arrest (maximal biomass), accounting for 93% of the Glc consumed (Table 2); formate was also formed in minor amounts (Fig. 3A and Table 2). Loss of CcpA caused a shift towards a more mixed acid fermentation: a 5-fold increase in the yields of formate, as well as formation of acetate and ethanol, were accompanied by a decrease on lactate production, which accounted for 85% of the Glc consumed as compared to 93% in D39 (Fig. 3B and Table 2). Hydrogen peroxide (H2O2), a known minor end-product of streptococcal aerobic metabolism, was also detected. In the wild-type, the maximal concentration reached was about 0.04±0.002 mM, while in the mutant this concentration was circa 7-fold enhanced. The low concentrations measured most likely reflect a limitation in oxygen availability due to the nearly anaerobic conditions used for growth. At the time point of growth arrest, the ccpA mutant had consumed 8% less Glc, and the maximal consumption rate was about 30% of the wild-type level (Table 2). The observed decrease in Glc consumption was paralleled by the reduction in growth rate, which was 53% lower in D39ΔccpA.

Bottom Line: In agreement, CcpA influenced the level of many intracellular metabolites potentially involved in metabolic regulation.Our data strengthen the view that a true understanding of cell physiology demands thorough analyses at different cellular levels.Moreover, integration of transcriptional and metabolic data uncovered a link between CcpA and the association of surface molecules (e.g. capsule) to the cell wall.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.

ABSTRACT
In gram-positive bacteria, the transcriptional regulator CcpA is at the core of catabolite control mechanisms. In the human pathogen Streptococcus pneumoniae, links between CcpA and virulence have been established, but its role as a master regulator in different nutritional environments remains to be elucidated. Thus, we performed whole-transcriptome and metabolic analyses of S. pneumoniae D39 and its isogenic ccpA mutant during growth on glucose or galactose, rapidly and slowly metabolized carbohydrates presumably encountered by the bacterium in different host niches. CcpA affected the expression of up to 19% of the genome covering multiple cellular processes, including virulence, regulatory networks and central metabolism. Its prevalent function as a repressor was observed on glucose, but unexpectedly also on galactose. Carbohydrate-dependent CcpA regulation was also observed, as for the tagatose 6-phosphate pathway genes, which were activated by galactose and repressed by glucose. Metabolite analyses revealed that two pathways for galactose catabolism are functionally active, despite repression of the Leloir genes by CcpA. Surprisingly, galactose-induced mixed-acid fermentation apparently required CcpA, since genes involved in this type of metabolism were mostly under CcpA-repression. These findings indicate that the role of CcpA extends beyond transcriptional regulation, which seemingly is overlaid by other regulatory mechanisms. In agreement, CcpA influenced the level of many intracellular metabolites potentially involved in metabolic regulation. Our data strengthen the view that a true understanding of cell physiology demands thorough analyses at different cellular levels. Moreover, integration of transcriptional and metabolic data uncovered a link between CcpA and the association of surface molecules (e.g. capsule) to the cell wall. Hence, CcpA may play a key role in mediating the interaction of S. pneumoniae with its host. Overall, our results support the hypothesis that S. pneumoniae optimizes basic metabolic processes, likely enhancing in vivo fitness, in a CcpA-mediated manner.

Show MeSH
Related in: MedlinePlus