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Novel roles of cAMP receptor protein (CRP) in regulation of transport and metabolism of carbon sources.

Shimada T, Fujita N, Yamamoto K, Ishihama A - PLoS ONE (2011)

Bottom Line: Using the newly developed Genomic SELEX screening system of transcription factor-binding sequences, however, we have identified a total of at least 254 CRP-binding sites.Based on the functions of novel target genes, we conclude that CRP plays a key regulatory role in the whole processes from the selective transport of carbon sources, the glycolysis-gluconeogenesis switching to the metabolisms downstream of glycolysis, including tricarboxylic acid (TCA) cycle, pyruvate dehydrogenase (PDH) pathway and aerobic respiration.One unique regulation mode is that a single and the same CRP molecule bound within intergenic regions often regulates both of divergently transcribed operons.

View Article: PubMed Central - PubMed

Affiliation: Department of Frontier Bioscience, Hosei University, Koganei, Tokyo, Japan.

ABSTRACT
CRP (cAMP receptor protein), the global regulator of genes for carbon source utilization in the absence of glucose, is the best-studied prokaryotic transcription factor. A total of 195 target promoters on the Escherichia coli genome have been proposed to be under the control of cAMP-bound CRP. Using the newly developed Genomic SELEX screening system of transcription factor-binding sequences, however, we have identified a total of at least 254 CRP-binding sites. Based on their location on the E. coli genome, we predict a total of at least 183 novel regulation target operons, altogether with the 195 hitherto known targets, reaching to the minimum of 378 promoters as the regulation targets of cAMP-CRP. All the promoters selected from the newly identified targets and examined by using the lacZ reporter assay were found to be under the control of CRP, indicating that the Genomic SELEX screening allowed to identify the CRP targets with high accuracy. Based on the functions of novel target genes, we conclude that CRP plays a key regulatory role in the whole processes from the selective transport of carbon sources, the glycolysis-gluconeogenesis switching to the metabolisms downstream of glycolysis, including tricarboxylic acid (TCA) cycle, pyruvate dehydrogenase (PDH) pathway and aerobic respiration. One unique regulation mode is that a single and the same CRP molecule bound within intergenic regions often regulates both of divergently transcribed operons.

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Analysis of promoter regulation in vivo by CRP using LacZ fusion.The predicted CRP-target promoters were inserted into the lacZ expression plasmid to construct a collection of promoter-lacZ fusions. The promoter-dependent expression of LacZ was determined in both wild-type E. coli and crp-defective mutants (Y-axis, β-galactosidase activity in Miller unit). Except for the talA and fbaB promoters (shown under gray background), the activity of all other promoters decreased in the absence of activator CRP, indicating the involvement of CRP as an activator. The newly identified promoters are shown under black background. As references, some known CRP target promoters such as ptsG, dgsA, ptsH and glpF (shown under white background) were examined in parallel under the same conditions.
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pone-0020081-g003: Analysis of promoter regulation in vivo by CRP using LacZ fusion.The predicted CRP-target promoters were inserted into the lacZ expression plasmid to construct a collection of promoter-lacZ fusions. The promoter-dependent expression of LacZ was determined in both wild-type E. coli and crp-defective mutants (Y-axis, β-galactosidase activity in Miller unit). Except for the talA and fbaB promoters (shown under gray background), the activity of all other promoters decreased in the absence of activator CRP, indicating the involvement of CRP as an activator. The newly identified promoters are shown under black background. As references, some known CRP target promoters such as ptsG, dgsA, ptsH and glpF (shown under white background) were examined in parallel under the same conditions.

Mentions: CRP has been identified to regulate some of the enzymes for central carbon metabolism, including fbaA, gapA, and pgk. In addition, we identified in this study the binding of cAMP-CRP to the genes encoding glk (glucokinase), fruK (1-phosphofructokinase), yggF (type II fructose-1,6-bisphosphatase), ppsA (phosphoenolpyruvate synthase), talA (transaldolase) and tktB (transketolase II). In order to further confirm the accuracy of target prediction by the Genomic SELEX screening, we performed the lacZ promoter assay for some of the newly identified target promoters, together with some known targets, in both wild-type and crp deletion mutant. Fig. 3 shows the activity of 12 representative promoters that were predicted to be under the control cAMP-CRP after SELEX-chip analysis, together with 4 known promoters, ptsG (glucose-specific PTS), dgsA (global regulator of carbon metabolism), ptsH (PTS Hpr component) and glpF (glycerol facilitator), that are activated by cAMP-CRP. All the putative target genes examined were indeed found to be under the control of CRP, classified in group-A in Table 2, supporting the prediction that the Genomic SELEX screening allows the identification of regulation targets of cAMP-CRP with high accuracy.


Novel roles of cAMP receptor protein (CRP) in regulation of transport and metabolism of carbon sources.

Shimada T, Fujita N, Yamamoto K, Ishihama A - PLoS ONE (2011)

Analysis of promoter regulation in vivo by CRP using LacZ fusion.The predicted CRP-target promoters were inserted into the lacZ expression plasmid to construct a collection of promoter-lacZ fusions. The promoter-dependent expression of LacZ was determined in both wild-type E. coli and crp-defective mutants (Y-axis, β-galactosidase activity in Miller unit). Except for the talA and fbaB promoters (shown under gray background), the activity of all other promoters decreased in the absence of activator CRP, indicating the involvement of CRP as an activator. The newly identified promoters are shown under black background. As references, some known CRP target promoters such as ptsG, dgsA, ptsH and glpF (shown under white background) were examined in parallel under the same conditions.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105977&req=5

pone-0020081-g003: Analysis of promoter regulation in vivo by CRP using LacZ fusion.The predicted CRP-target promoters were inserted into the lacZ expression plasmid to construct a collection of promoter-lacZ fusions. The promoter-dependent expression of LacZ was determined in both wild-type E. coli and crp-defective mutants (Y-axis, β-galactosidase activity in Miller unit). Except for the talA and fbaB promoters (shown under gray background), the activity of all other promoters decreased in the absence of activator CRP, indicating the involvement of CRP as an activator. The newly identified promoters are shown under black background. As references, some known CRP target promoters such as ptsG, dgsA, ptsH and glpF (shown under white background) were examined in parallel under the same conditions.
Mentions: CRP has been identified to regulate some of the enzymes for central carbon metabolism, including fbaA, gapA, and pgk. In addition, we identified in this study the binding of cAMP-CRP to the genes encoding glk (glucokinase), fruK (1-phosphofructokinase), yggF (type II fructose-1,6-bisphosphatase), ppsA (phosphoenolpyruvate synthase), talA (transaldolase) and tktB (transketolase II). In order to further confirm the accuracy of target prediction by the Genomic SELEX screening, we performed the lacZ promoter assay for some of the newly identified target promoters, together with some known targets, in both wild-type and crp deletion mutant. Fig. 3 shows the activity of 12 representative promoters that were predicted to be under the control cAMP-CRP after SELEX-chip analysis, together with 4 known promoters, ptsG (glucose-specific PTS), dgsA (global regulator of carbon metabolism), ptsH (PTS Hpr component) and glpF (glycerol facilitator), that are activated by cAMP-CRP. All the putative target genes examined were indeed found to be under the control of CRP, classified in group-A in Table 2, supporting the prediction that the Genomic SELEX screening allows the identification of regulation targets of cAMP-CRP with high accuracy.

Bottom Line: Using the newly developed Genomic SELEX screening system of transcription factor-binding sequences, however, we have identified a total of at least 254 CRP-binding sites.Based on the functions of novel target genes, we conclude that CRP plays a key regulatory role in the whole processes from the selective transport of carbon sources, the glycolysis-gluconeogenesis switching to the metabolisms downstream of glycolysis, including tricarboxylic acid (TCA) cycle, pyruvate dehydrogenase (PDH) pathway and aerobic respiration.One unique regulation mode is that a single and the same CRP molecule bound within intergenic regions often regulates both of divergently transcribed operons.

View Article: PubMed Central - PubMed

Affiliation: Department of Frontier Bioscience, Hosei University, Koganei, Tokyo, Japan.

ABSTRACT
CRP (cAMP receptor protein), the global regulator of genes for carbon source utilization in the absence of glucose, is the best-studied prokaryotic transcription factor. A total of 195 target promoters on the Escherichia coli genome have been proposed to be under the control of cAMP-bound CRP. Using the newly developed Genomic SELEX screening system of transcription factor-binding sequences, however, we have identified a total of at least 254 CRP-binding sites. Based on their location on the E. coli genome, we predict a total of at least 183 novel regulation target operons, altogether with the 195 hitherto known targets, reaching to the minimum of 378 promoters as the regulation targets of cAMP-CRP. All the promoters selected from the newly identified targets and examined by using the lacZ reporter assay were found to be under the control of CRP, indicating that the Genomic SELEX screening allowed to identify the CRP targets with high accuracy. Based on the functions of novel target genes, we conclude that CRP plays a key regulatory role in the whole processes from the selective transport of carbon sources, the glycolysis-gluconeogenesis switching to the metabolisms downstream of glycolysis, including tricarboxylic acid (TCA) cycle, pyruvate dehydrogenase (PDH) pathway and aerobic respiration. One unique regulation mode is that a single and the same CRP molecule bound within intergenic regions often regulates both of divergently transcribed operons.

Show MeSH
Related in: MedlinePlus