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Probing the regulatory effects of specific mutations in three major binding domains of the pleiotropic regulator CcpA of Bacillus subtilis.

Detert Oude Weme R, Seidel G, Kuipers OP - Front Microbiol (2015)

Bottom Line: The following three amino acids were mutated to characterize their function: M17(R) which is involved in DNA binding, T62(H) which is important for the allosteric switch in CcpA upon HPr-Ser46-P binding, and R304(W) which is important for binding of the coeffectors HPr-Ser46-P and fructose-1,6-bisphosphate.CcpA-M17R showed a small relief of Carbon Catabolite Control; the CcpA-M17R mutant regulates fewer genes than the CcpA-wt and the palindromicity of the cre site is less important for CcpA-M17R.The results presented here provide detailed information on alterations in gene regulation for each CcpA-mutant.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics Group, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen Groningen, Netherlands.

ABSTRACT
Carbon catabolite control is required for efficient use of available carbon sources to ensure rapid growth of bacteria. CcpA is a global regulator of carbon metabolism in Gram-positive bacteria like Bacillus subtilis. In this study the genome-wide gene regulation of a CcpA knockout and three specific CcpA mutants were studied by transcriptome analysis, to further elucidate the function of specific binding sites in CcpA. The following three amino acids were mutated to characterize their function: M17(R) which is involved in DNA binding, T62(H) which is important for the allosteric switch in CcpA upon HPr-Ser46-P binding, and R304(W) which is important for binding of the coeffectors HPr-Ser46-P and fructose-1,6-bisphosphate. The results confirm that CcpA was also involved in gene regulation in the absence of glucose. CcpA-M17R showed a small relief of Carbon Catabolite Control; the CcpA-M17R mutant regulates fewer genes than the CcpA-wt and the palindromicity of the cre site is less important for CcpA-M17R. CcpA-T62H was a stronger repressor than CcpA-wt and also acted as a strong repressor in the absence of glucose. CcpA-R304W was shown here to be less dependent on HPr-Ser46-P for its carbon catabolite control activities. The results presented here provide detailed information on alterations in gene regulation for each CcpA-mutant.

No MeSH data available.


Related in: MedlinePlus

Venn diagram showing the genes that were differentially regulated in each one of the CcpA mutant strains. Numbers represent the genes that were differentially regulated in each CcpA mutant strain. Only genes with a fold change larger than 1.7 or smaller than -1.7 were used in the analysis. (A) Overview of the differentially regulated genes when the strains with the CcpA mutant were grown on LB + 1% glucose (see also Supplementary File, Sheet 2). (B) Overview of the differentially regulated genes when the strains with the CcpA mutant were grown on LB (see also Supplementary File, Sheet 5). Venn diagrams were made via http://bioinfogp.cnb.csic.es/tools/venny/. More detailed information on up- or down-regulated genes is shown in Table 4.
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Figure 2: Venn diagram showing the genes that were differentially regulated in each one of the CcpA mutant strains. Numbers represent the genes that were differentially regulated in each CcpA mutant strain. Only genes with a fold change larger than 1.7 or smaller than -1.7 were used in the analysis. (A) Overview of the differentially regulated genes when the strains with the CcpA mutant were grown on LB + 1% glucose (see also Supplementary File, Sheet 2). (B) Overview of the differentially regulated genes when the strains with the CcpA mutant were grown on LB (see also Supplementary File, Sheet 5). Venn diagrams were made via http://bioinfogp.cnb.csic.es/tools/venny/. More detailed information on up- or down-regulated genes is shown in Table 4.

Mentions: Most likely CcpA-M17R lost part of its capacity to repress genes, as can be concluded from the large number of derepressed genes. The gene regulation of the CcpA-M17R mutant differed significantly from the ΔCcpA mutant. Of the genes that were affected by CcpA-M17R 53% differed from the genes that were affected in the ΔCcpA strain (Figure 2A and Supplementary File, Sheet 2). The number of affected genes was smaller (Table 5) indicating that the function of the CcpA-M17R mutant was similar to the CcpA-wt, but with lowered affinity for most binding sites. When the CcpA-M17R mutant strain was grown in LB + 1% glucose the gene regulation for 129 genes (115 up and 14 down) differed from CcpA-wt (Table 5, Supplementary File, Sheets 1–3). Only 27 of these genes belonged to the CcpA regulon from SubtiWiki. Especially genes from COG categories: [C] Energy production and conversion, [R] General function prediction, and [T] Signal transduction mechanisms showed a weaker regulation by CcpA-M17R (Figure 3).


Probing the regulatory effects of specific mutations in three major binding domains of the pleiotropic regulator CcpA of Bacillus subtilis.

Detert Oude Weme R, Seidel G, Kuipers OP - Front Microbiol (2015)

Venn diagram showing the genes that were differentially regulated in each one of the CcpA mutant strains. Numbers represent the genes that were differentially regulated in each CcpA mutant strain. Only genes with a fold change larger than 1.7 or smaller than -1.7 were used in the analysis. (A) Overview of the differentially regulated genes when the strains with the CcpA mutant were grown on LB + 1% glucose (see also Supplementary File, Sheet 2). (B) Overview of the differentially regulated genes when the strains with the CcpA mutant were grown on LB (see also Supplementary File, Sheet 5). Venn diagrams were made via http://bioinfogp.cnb.csic.es/tools/venny/. More detailed information on up- or down-regulated genes is shown in Table 4.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591507&req=5

Figure 2: Venn diagram showing the genes that were differentially regulated in each one of the CcpA mutant strains. Numbers represent the genes that were differentially regulated in each CcpA mutant strain. Only genes with a fold change larger than 1.7 or smaller than -1.7 were used in the analysis. (A) Overview of the differentially regulated genes when the strains with the CcpA mutant were grown on LB + 1% glucose (see also Supplementary File, Sheet 2). (B) Overview of the differentially regulated genes when the strains with the CcpA mutant were grown on LB (see also Supplementary File, Sheet 5). Venn diagrams were made via http://bioinfogp.cnb.csic.es/tools/venny/. More detailed information on up- or down-regulated genes is shown in Table 4.
Mentions: Most likely CcpA-M17R lost part of its capacity to repress genes, as can be concluded from the large number of derepressed genes. The gene regulation of the CcpA-M17R mutant differed significantly from the ΔCcpA mutant. Of the genes that were affected by CcpA-M17R 53% differed from the genes that were affected in the ΔCcpA strain (Figure 2A and Supplementary File, Sheet 2). The number of affected genes was smaller (Table 5) indicating that the function of the CcpA-M17R mutant was similar to the CcpA-wt, but with lowered affinity for most binding sites. When the CcpA-M17R mutant strain was grown in LB + 1% glucose the gene regulation for 129 genes (115 up and 14 down) differed from CcpA-wt (Table 5, Supplementary File, Sheets 1–3). Only 27 of these genes belonged to the CcpA regulon from SubtiWiki. Especially genes from COG categories: [C] Energy production and conversion, [R] General function prediction, and [T] Signal transduction mechanisms showed a weaker regulation by CcpA-M17R (Figure 3).

Bottom Line: The following three amino acids were mutated to characterize their function: M17(R) which is involved in DNA binding, T62(H) which is important for the allosteric switch in CcpA upon HPr-Ser46-P binding, and R304(W) which is important for binding of the coeffectors HPr-Ser46-P and fructose-1,6-bisphosphate.CcpA-M17R showed a small relief of Carbon Catabolite Control; the CcpA-M17R mutant regulates fewer genes than the CcpA-wt and the palindromicity of the cre site is less important for CcpA-M17R.The results presented here provide detailed information on alterations in gene regulation for each CcpA-mutant.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics Group, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen Groningen, Netherlands.

ABSTRACT
Carbon catabolite control is required for efficient use of available carbon sources to ensure rapid growth of bacteria. CcpA is a global regulator of carbon metabolism in Gram-positive bacteria like Bacillus subtilis. In this study the genome-wide gene regulation of a CcpA knockout and three specific CcpA mutants were studied by transcriptome analysis, to further elucidate the function of specific binding sites in CcpA. The following three amino acids were mutated to characterize their function: M17(R) which is involved in DNA binding, T62(H) which is important for the allosteric switch in CcpA upon HPr-Ser46-P binding, and R304(W) which is important for binding of the coeffectors HPr-Ser46-P and fructose-1,6-bisphosphate. The results confirm that CcpA was also involved in gene regulation in the absence of glucose. CcpA-M17R showed a small relief of Carbon Catabolite Control; the CcpA-M17R mutant regulates fewer genes than the CcpA-wt and the palindromicity of the cre site is less important for CcpA-M17R. CcpA-T62H was a stronger repressor than CcpA-wt and also acted as a strong repressor in the absence of glucose. CcpA-R304W was shown here to be less dependent on HPr-Ser46-P for its carbon catabolite control activities. The results presented here provide detailed information on alterations in gene regulation for each CcpA-mutant.

No MeSH data available.


Related in: MedlinePlus