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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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Related in: MedlinePlus

Effect of ccpA deletion on intracellular concentrations of phosphorylated metabolites during growth on Glc or Gal.Phosphorylated metabolites were measured by 31P-NMR in ethanol extracts of S. pneumoniae D39 and ΔccpA strains grown to mid-exponential (M, white bars, OD600 of 0.35±0.02) or transition-to-stationary phases (TS, black bars, OD600 of 1.3±0.1) of growth in CDM supplemented with 56±1 mM Glc (white background) or 57±1 mM Gal (light grey background). Phosphorylated metabolites measured in extracts comprised glycolytic metabolites, phosphorylated carbohydrate-specific metabolites, UDP-activated metabolites, and co-factors. The values are the mean of three independent experiments ± SD.
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pone-0026707-g004: Effect of ccpA deletion on intracellular concentrations of phosphorylated metabolites during growth on Glc or Gal.Phosphorylated metabolites were measured by 31P-NMR in ethanol extracts of S. pneumoniae D39 and ΔccpA strains grown to mid-exponential (M, white bars, OD600 of 0.35±0.02) or transition-to-stationary phases (TS, black bars, OD600 of 1.3±0.1) of growth in CDM supplemented with 56±1 mM Glc (white background) or 57±1 mM Gal (light grey background). Phosphorylated metabolites measured in extracts comprised glycolytic metabolites, phosphorylated carbohydrate-specific metabolites, UDP-activated metabolites, and co-factors. The values are the mean of three independent experiments ± SD.

Mentions: Inactivation of CcpA renders a strain with altered growth and fermentation profiles both on Glc and on Gal. In particular, the disparate substrate consumption rates and the fermentation end-product patterns, led us to surmise that levels of intracellular metabolites are also under the influence of CcpA. Intracellular metabolites were determined in ethanol extracts obtained as for transcriptome analyses (M and TS phases of growth) by targeted metabolomics using 31P-NMR (Fig. 4). In this way we were able to quantify intracellular concentrations of metabolites in central carbon utilization pathways (carbohydrate-specific pathways, glycolysis), catabolic precursors in the synthesis of surface structures (cell wall, capsule), and co-factors (Fig. 5), detailed below.


CcpA ensures optimal metabolic fitness of Streptococcus pneumoniae.

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

Effect of ccpA deletion on intracellular concentrations of phosphorylated metabolites during growth on Glc or Gal.Phosphorylated metabolites were measured by 31P-NMR in ethanol extracts of S. pneumoniae D39 and ΔccpA strains grown to mid-exponential (M, white bars, OD600 of 0.35±0.02) or transition-to-stationary phases (TS, black bars, OD600 of 1.3±0.1) of growth in CDM supplemented with 56±1 mM Glc (white background) or 57±1 mM Gal (light grey background). Phosphorylated metabolites measured in extracts comprised glycolytic metabolites, phosphorylated carbohydrate-specific metabolites, UDP-activated metabolites, and co-factors. The values are the mean of three independent experiments ± SD.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198803&req=5

pone-0026707-g004: Effect of ccpA deletion on intracellular concentrations of phosphorylated metabolites during growth on Glc or Gal.Phosphorylated metabolites were measured by 31P-NMR in ethanol extracts of S. pneumoniae D39 and ΔccpA strains grown to mid-exponential (M, white bars, OD600 of 0.35±0.02) or transition-to-stationary phases (TS, black bars, OD600 of 1.3±0.1) of growth in CDM supplemented with 56±1 mM Glc (white background) or 57±1 mM Gal (light grey background). Phosphorylated metabolites measured in extracts comprised glycolytic metabolites, phosphorylated carbohydrate-specific metabolites, UDP-activated metabolites, and co-factors. The values are the mean of three independent experiments ± SD.
Mentions: Inactivation of CcpA renders a strain with altered growth and fermentation profiles both on Glc and on Gal. In particular, the disparate substrate consumption rates and the fermentation end-product patterns, led us to surmise that levels of intracellular metabolites are also under the influence of CcpA. Intracellular metabolites were determined in ethanol extracts obtained as for transcriptome analyses (M and TS phases of growth) by targeted metabolomics using 31P-NMR (Fig. 4). In this way we were able to quantify intracellular concentrations of metabolites in central carbon utilization pathways (carbohydrate-specific pathways, glycolysis), catabolic precursors in the synthesis of surface structures (cell wall, capsule), and co-factors (Fig. 5), detailed below.

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