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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

Schematic representation of central metabolic pathways in S. pneumoniae D39.Glc is oxidized to pyruvate via the Embden-Meyerhof-Parnas pathway (Glycolysis, orange box). Homolactic fermentation reduces pyruvate into lactate, whereas mixed-acid fermentation leads to other products, such as formate, acetate and ethanol (Pyruvate metabolism, yellow box). Gal is converted to G6P by the Leloir pathway (light pink box) and to dihydroxyacetone phosphate or glyceraldehyde 3-phosphate by the tagatose 6-phosphate pathway (light orange box). Pathways for capsule, peptidoglycan and phosphorylcholine biosynthesis are shown in light blue, light purple and light green boxes, respectively. Putative and functional characterized genes encoding depicted metabolic steps are shown in white boxes. Proposed pathways were reconstructed based on genome information (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi), literature and database surveys (KEGG, MetaCyc). Gene annotation downloaded from NCBI: galM, aldose 1-epimerase; galK, galactokinase; galE-1, UDP-glucose 4-epimerase; galT-2, galactose 1-phosphate uridylyltransferase; pgm, phosphoglucomutase/phosphomannomutase family protein; galU, UTP-glucose 1-phosphate uridylyltransferase; cps2L, glucose 1-phosphate thymidylyltransferase; cps2K, UDP-glucose 6-dehydrogenase, putative; rfbC, dTDP-4-dehydrorhamnose 3,5-epimerase, putative; rfbB, dTDP-glucose 4,6-dehydratase; rfbD, dTDP-4-dehydrorhamnose reductase; gki, glucokinase; pgi, glucose 6-phosphate isomerase; pfkA, 6-phosphofructokinase; fba, fructose bisphosphate aldolase; tpiA, triosephosphate isomerase; gap, glyceraldehyde-3-phosphate dehydrogenase; pgk, phosphogltcerate kinase; gpmA, phosphoglyceromutase; eno, phosphopyruvate hydratase; pyk, pyruvate kinase; lacA, galactose 6-phosphate isomerase subunit LacA; lacB, galactose 6-phosphate isomerase subunit LacB; lacC, tagatose 6-phosphate kinase; lacD, tagatose 1,6-diphosphate aldolase; ldh, L-lactate dehydrogenase; lctO, lactate oxidase; spxB, pyruvate oxidase; ackA, acetate kinase; pta, phosphotransacetylase; pfl, pyruvate formate-lyase; adh (spd_1834), bifunctional acetaldehyde-CoA/alcohol dehydrogenase; glmS, D-fructose 6-phosphate amidotransferase; glmU, UDP-N-acetylglucosamine pyrophosphorylase; murA-1 and murA-2, UDP-N-acetylglucosamine 1-carboxyvinyltransferase; murB, UDP-N-acetylenolpyruvoylglucosamine reductase; murC, UDP-N-acetylmuramate-L-alanine ligase; murD, UDP-N-acetylmuramoyl-L-alanyl-D-glutamate synthetase; murE, UDP-N-acetylmuramoylalanyl-D-glutamate-2,6-diaminopimelate ligase; murF, UDP-N-acetylmuramoylalanyl-D-glutamyl-2, 6-diaminopimelate–D-alanyl-D-alanyl ligase; tarJ (spd_1126), alcohol dehydrogenase, zinc-containing or TarJ; tarI (spd_1127), 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase or TarI; licB, protein LicB; pck, choline kinase or LicA; licC, CTP:phosphocholine cytidylyltransferase; LicD1, phosphotransferase LicD1; LicD2, phosphotransferase LicD2.
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pone-0026707-g005: Schematic representation of central metabolic pathways in S. pneumoniae D39.Glc is oxidized to pyruvate via the Embden-Meyerhof-Parnas pathway (Glycolysis, orange box). Homolactic fermentation reduces pyruvate into lactate, whereas mixed-acid fermentation leads to other products, such as formate, acetate and ethanol (Pyruvate metabolism, yellow box). Gal is converted to G6P by the Leloir pathway (light pink box) and to dihydroxyacetone phosphate or glyceraldehyde 3-phosphate by the tagatose 6-phosphate pathway (light orange box). Pathways for capsule, peptidoglycan and phosphorylcholine biosynthesis are shown in light blue, light purple and light green boxes, respectively. Putative and functional characterized genes encoding depicted metabolic steps are shown in white boxes. Proposed pathways were reconstructed based on genome information (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi), literature and database surveys (KEGG, MetaCyc). Gene annotation downloaded from NCBI: galM, aldose 1-epimerase; galK, galactokinase; galE-1, UDP-glucose 4-epimerase; galT-2, galactose 1-phosphate uridylyltransferase; pgm, phosphoglucomutase/phosphomannomutase family protein; galU, UTP-glucose 1-phosphate uridylyltransferase; cps2L, glucose 1-phosphate thymidylyltransferase; cps2K, UDP-glucose 6-dehydrogenase, putative; rfbC, dTDP-4-dehydrorhamnose 3,5-epimerase, putative; rfbB, dTDP-glucose 4,6-dehydratase; rfbD, dTDP-4-dehydrorhamnose reductase; gki, glucokinase; pgi, glucose 6-phosphate isomerase; pfkA, 6-phosphofructokinase; fba, fructose bisphosphate aldolase; tpiA, triosephosphate isomerase; gap, glyceraldehyde-3-phosphate dehydrogenase; pgk, phosphogltcerate kinase; gpmA, phosphoglyceromutase; eno, phosphopyruvate hydratase; pyk, pyruvate kinase; lacA, galactose 6-phosphate isomerase subunit LacA; lacB, galactose 6-phosphate isomerase subunit LacB; lacC, tagatose 6-phosphate kinase; lacD, tagatose 1,6-diphosphate aldolase; ldh, L-lactate dehydrogenase; lctO, lactate oxidase; spxB, pyruvate oxidase; ackA, acetate kinase; pta, phosphotransacetylase; pfl, pyruvate formate-lyase; adh (spd_1834), bifunctional acetaldehyde-CoA/alcohol dehydrogenase; glmS, D-fructose 6-phosphate amidotransferase; glmU, UDP-N-acetylglucosamine pyrophosphorylase; murA-1 and murA-2, UDP-N-acetylglucosamine 1-carboxyvinyltransferase; murB, UDP-N-acetylenolpyruvoylglucosamine reductase; murC, UDP-N-acetylmuramate-L-alanine ligase; murD, UDP-N-acetylmuramoyl-L-alanyl-D-glutamate synthetase; murE, UDP-N-acetylmuramoylalanyl-D-glutamate-2,6-diaminopimelate ligase; murF, UDP-N-acetylmuramoylalanyl-D-glutamyl-2, 6-diaminopimelate–D-alanyl-D-alanyl ligase; tarJ (spd_1126), alcohol dehydrogenase, zinc-containing or TarJ; tarI (spd_1127), 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase or TarI; licB, protein LicB; pck, choline kinase or LicA; licC, CTP:phosphocholine cytidylyltransferase; LicD1, phosphotransferase LicD1; LicD2, phosphotransferase LicD2.

Mentions: Ratio's of genes in glycolysis, the Leloir and tagatose 6-phosphate pathways, and the pyruvate node, in all four conditions analysed in this study (Glc_M, Gal_M, Glc_TS, Gal_TS) are depicted. On top of the figure, a colour scale is given for the ratio of the expression in the ccpA mutant over that in the wild-type strain. Thus, red means repression by CcpA (upregulation in the microarray analysis) and green means activation by CcpA (downregulation in the microarray analysis). For each gene the D39 locus tag and the gene name is given on the right. No cut-off value was applied for the expression ratios of the genes given in the figure. Genes were considered significantly changed when having a Bayesian p-value and FDR meeting the criteria as outlined in the Materials & Methods. Genes that did not meet these criteria were given a ratio of 1.0 (black colour), meaning no significant change in expression. See Fig. 5 for an overview of these pathways in S. pneumoniae D39.


CcpA ensures optimal metabolic fitness of Streptococcus pneumoniae.

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

Schematic representation of central metabolic pathways in S. pneumoniae D39.Glc is oxidized to pyruvate via the Embden-Meyerhof-Parnas pathway (Glycolysis, orange box). Homolactic fermentation reduces pyruvate into lactate, whereas mixed-acid fermentation leads to other products, such as formate, acetate and ethanol (Pyruvate metabolism, yellow box). Gal is converted to G6P by the Leloir pathway (light pink box) and to dihydroxyacetone phosphate or glyceraldehyde 3-phosphate by the tagatose 6-phosphate pathway (light orange box). Pathways for capsule, peptidoglycan and phosphorylcholine biosynthesis are shown in light blue, light purple and light green boxes, respectively. Putative and functional characterized genes encoding depicted metabolic steps are shown in white boxes. Proposed pathways were reconstructed based on genome information (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi), literature and database surveys (KEGG, MetaCyc). Gene annotation downloaded from NCBI: galM, aldose 1-epimerase; galK, galactokinase; galE-1, UDP-glucose 4-epimerase; galT-2, galactose 1-phosphate uridylyltransferase; pgm, phosphoglucomutase/phosphomannomutase family protein; galU, UTP-glucose 1-phosphate uridylyltransferase; cps2L, glucose 1-phosphate thymidylyltransferase; cps2K, UDP-glucose 6-dehydrogenase, putative; rfbC, dTDP-4-dehydrorhamnose 3,5-epimerase, putative; rfbB, dTDP-glucose 4,6-dehydratase; rfbD, dTDP-4-dehydrorhamnose reductase; gki, glucokinase; pgi, glucose 6-phosphate isomerase; pfkA, 6-phosphofructokinase; fba, fructose bisphosphate aldolase; tpiA, triosephosphate isomerase; gap, glyceraldehyde-3-phosphate dehydrogenase; pgk, phosphogltcerate kinase; gpmA, phosphoglyceromutase; eno, phosphopyruvate hydratase; pyk, pyruvate kinase; lacA, galactose 6-phosphate isomerase subunit LacA; lacB, galactose 6-phosphate isomerase subunit LacB; lacC, tagatose 6-phosphate kinase; lacD, tagatose 1,6-diphosphate aldolase; ldh, L-lactate dehydrogenase; lctO, lactate oxidase; spxB, pyruvate oxidase; ackA, acetate kinase; pta, phosphotransacetylase; pfl, pyruvate formate-lyase; adh (spd_1834), bifunctional acetaldehyde-CoA/alcohol dehydrogenase; glmS, D-fructose 6-phosphate amidotransferase; glmU, UDP-N-acetylglucosamine pyrophosphorylase; murA-1 and murA-2, UDP-N-acetylglucosamine 1-carboxyvinyltransferase; murB, UDP-N-acetylenolpyruvoylglucosamine reductase; murC, UDP-N-acetylmuramate-L-alanine ligase; murD, UDP-N-acetylmuramoyl-L-alanyl-D-glutamate synthetase; murE, UDP-N-acetylmuramoylalanyl-D-glutamate-2,6-diaminopimelate ligase; murF, UDP-N-acetylmuramoylalanyl-D-glutamyl-2, 6-diaminopimelate–D-alanyl-D-alanyl ligase; tarJ (spd_1126), alcohol dehydrogenase, zinc-containing or TarJ; tarI (spd_1127), 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase or TarI; licB, protein LicB; pck, choline kinase or LicA; licC, CTP:phosphocholine cytidylyltransferase; LicD1, phosphotransferase LicD1; LicD2, phosphotransferase LicD2.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198803&req=5

pone-0026707-g005: Schematic representation of central metabolic pathways in S. pneumoniae D39.Glc is oxidized to pyruvate via the Embden-Meyerhof-Parnas pathway (Glycolysis, orange box). Homolactic fermentation reduces pyruvate into lactate, whereas mixed-acid fermentation leads to other products, such as formate, acetate and ethanol (Pyruvate metabolism, yellow box). Gal is converted to G6P by the Leloir pathway (light pink box) and to dihydroxyacetone phosphate or glyceraldehyde 3-phosphate by the tagatose 6-phosphate pathway (light orange box). Pathways for capsule, peptidoglycan and phosphorylcholine biosynthesis are shown in light blue, light purple and light green boxes, respectively. Putative and functional characterized genes encoding depicted metabolic steps are shown in white boxes. Proposed pathways were reconstructed based on genome information (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi), literature and database surveys (KEGG, MetaCyc). Gene annotation downloaded from NCBI: galM, aldose 1-epimerase; galK, galactokinase; galE-1, UDP-glucose 4-epimerase; galT-2, galactose 1-phosphate uridylyltransferase; pgm, phosphoglucomutase/phosphomannomutase family protein; galU, UTP-glucose 1-phosphate uridylyltransferase; cps2L, glucose 1-phosphate thymidylyltransferase; cps2K, UDP-glucose 6-dehydrogenase, putative; rfbC, dTDP-4-dehydrorhamnose 3,5-epimerase, putative; rfbB, dTDP-glucose 4,6-dehydratase; rfbD, dTDP-4-dehydrorhamnose reductase; gki, glucokinase; pgi, glucose 6-phosphate isomerase; pfkA, 6-phosphofructokinase; fba, fructose bisphosphate aldolase; tpiA, triosephosphate isomerase; gap, glyceraldehyde-3-phosphate dehydrogenase; pgk, phosphogltcerate kinase; gpmA, phosphoglyceromutase; eno, phosphopyruvate hydratase; pyk, pyruvate kinase; lacA, galactose 6-phosphate isomerase subunit LacA; lacB, galactose 6-phosphate isomerase subunit LacB; lacC, tagatose 6-phosphate kinase; lacD, tagatose 1,6-diphosphate aldolase; ldh, L-lactate dehydrogenase; lctO, lactate oxidase; spxB, pyruvate oxidase; ackA, acetate kinase; pta, phosphotransacetylase; pfl, pyruvate formate-lyase; adh (spd_1834), bifunctional acetaldehyde-CoA/alcohol dehydrogenase; glmS, D-fructose 6-phosphate amidotransferase; glmU, UDP-N-acetylglucosamine pyrophosphorylase; murA-1 and murA-2, UDP-N-acetylglucosamine 1-carboxyvinyltransferase; murB, UDP-N-acetylenolpyruvoylglucosamine reductase; murC, UDP-N-acetylmuramate-L-alanine ligase; murD, UDP-N-acetylmuramoyl-L-alanyl-D-glutamate synthetase; murE, UDP-N-acetylmuramoylalanyl-D-glutamate-2,6-diaminopimelate ligase; murF, UDP-N-acetylmuramoylalanyl-D-glutamyl-2, 6-diaminopimelate–D-alanyl-D-alanyl ligase; tarJ (spd_1126), alcohol dehydrogenase, zinc-containing or TarJ; tarI (spd_1127), 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase or TarI; licB, protein LicB; pck, choline kinase or LicA; licC, CTP:phosphocholine cytidylyltransferase; LicD1, phosphotransferase LicD1; LicD2, phosphotransferase LicD2.
Mentions: Ratio's of genes in glycolysis, the Leloir and tagatose 6-phosphate pathways, and the pyruvate node, in all four conditions analysed in this study (Glc_M, Gal_M, Glc_TS, Gal_TS) are depicted. On top of the figure, a colour scale is given for the ratio of the expression in the ccpA mutant over that in the wild-type strain. Thus, red means repression by CcpA (upregulation in the microarray analysis) and green means activation by CcpA (downregulation in the microarray analysis). For each gene the D39 locus tag and the gene name is given on the right. No cut-off value was applied for the expression ratios of the genes given in the figure. Genes were considered significantly changed when having a Bayesian p-value and FDR meeting the criteria as outlined in the Materials & Methods. Genes that did not meet these criteria were given a ratio of 1.0 (black colour), meaning no significant change in expression. See Fig. 5 for an overview of these pathways in S. pneumoniae D39.

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