Limits...
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

Growth of the strains with the CcpA mutants (A) on C-medium, (B) on C-medium supplemented with glutamate, (C) on C-medium supplemented with glutamate and branched chain amino acids, and (D) on C-medium supplemented with glutamate, branched chain amino acids and uridine 5′-monophosphate. All strains are B.subtilis ccpA::spec and strain 2-5 have ccpA on a plasmid. 1, ΔccpA; 2, ccpAwt; 3, ccpA-M17R; 4, ccpA-T62H; 5, ccpA-R304W; 6, empty.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4591507&req=5

Figure 4: Growth of the strains with the CcpA mutants (A) on C-medium, (B) on C-medium supplemented with glutamate, (C) on C-medium supplemented with glutamate and branched chain amino acids, and (D) on C-medium supplemented with glutamate, branched chain amino acids and uridine 5′-monophosphate. All strains are B.subtilis ccpA::spec and strain 2-5 have ccpA on a plasmid. 1, ΔccpA; 2, ccpAwt; 3, ccpA-M17R; 4, ccpA-T62H; 5, ccpA-R304W; 6, empty.

Mentions: A ccpA knockout strain shows a growth defect in glucose minimal medium (Faires et al., 1999; Fujita, 2009). Here we tested how the CcpA mutant strains performed in glucose minimal medium. In the strains with the CcpA mutants, some genes coding for proteins responsible for amino acid catabolism were deregulated. One of them was the gene rocG coding for glutamate dehydrogenase. Normally, the rocG gene is repressed by CcpA, but in both growth conditions used in this study and in strains with all CcpA variants except CcpA-T62H, the repression of rocG was released. Higher levels of RocG decrease the level of glutamate, which impairs the growth of these strains on a glucose minimal medium (Fujita, 2009). The altered gene regulation of rocG caused a growth deficiency of the CcpA mutant strains on minimal medium, which was shown on C-medium agar plates for the strains with the ΔCcpA, CcpA-M17R, and CcpA-R304W mutations (Figure 4A). The CcpA-T62H strain still repressed the rocG expression and did not have a growth defect (Figures 4A,B). The growth deficiency of the mutants was almost fully restored to the same extent as CcpA-wt when the C-medium agar plates were supplemented with glutamate, except for CcpA-M17R (Figure 4B) and fully restored when the C-medium agar plates were supplemented with glutamate and branched chain amino acids (BCAA), except for CcpA-M17R (Figure 4C). The growth defect of the CcpA-M17R mutant strain was partially restored by the addition of glutamate (Figure 4B); however the addition of only glutamate was not enough. The remaining growth defect of the CcpA-M17R strain might be explained by the pyrE gene. PyrE is responsible for pyrimidine biosynthesis (Subtiwiki, 2014) and its gene expression was only down-regulated in the CcpA-M17R strain. This strain did not show a growth defect in LB because LB is rich in nucleotides from the yeast extract. Therefore the C-medium was also supplemented with uridine 5′-monophosphate in addition to glutamate and BCAA, but the growth defect of the CcpA-M17R mutant strain was not restored (Figure 4D). The residual growth deficiency remains elusive and is probably dependent on more additives.


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)

Growth of the strains with the CcpA mutants (A) on C-medium, (B) on C-medium supplemented with glutamate, (C) on C-medium supplemented with glutamate and branched chain amino acids, and (D) on C-medium supplemented with glutamate, branched chain amino acids and uridine 5′-monophosphate. All strains are B.subtilis ccpA::spec and strain 2-5 have ccpA on a plasmid. 1, ΔccpA; 2, ccpAwt; 3, ccpA-M17R; 4, ccpA-T62H; 5, ccpA-R304W; 6, empty.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Growth of the strains with the CcpA mutants (A) on C-medium, (B) on C-medium supplemented with glutamate, (C) on C-medium supplemented with glutamate and branched chain amino acids, and (D) on C-medium supplemented with glutamate, branched chain amino acids and uridine 5′-monophosphate. All strains are B.subtilis ccpA::spec and strain 2-5 have ccpA on a plasmid. 1, ΔccpA; 2, ccpAwt; 3, ccpA-M17R; 4, ccpA-T62H; 5, ccpA-R304W; 6, empty.
Mentions: A ccpA knockout strain shows a growth defect in glucose minimal medium (Faires et al., 1999; Fujita, 2009). Here we tested how the CcpA mutant strains performed in glucose minimal medium. In the strains with the CcpA mutants, some genes coding for proteins responsible for amino acid catabolism were deregulated. One of them was the gene rocG coding for glutamate dehydrogenase. Normally, the rocG gene is repressed by CcpA, but in both growth conditions used in this study and in strains with all CcpA variants except CcpA-T62H, the repression of rocG was released. Higher levels of RocG decrease the level of glutamate, which impairs the growth of these strains on a glucose minimal medium (Fujita, 2009). The altered gene regulation of rocG caused a growth deficiency of the CcpA mutant strains on minimal medium, which was shown on C-medium agar plates for the strains with the ΔCcpA, CcpA-M17R, and CcpA-R304W mutations (Figure 4A). The CcpA-T62H strain still repressed the rocG expression and did not have a growth defect (Figures 4A,B). The growth deficiency of the mutants was almost fully restored to the same extent as CcpA-wt when the C-medium agar plates were supplemented with glutamate, except for CcpA-M17R (Figure 4B) and fully restored when the C-medium agar plates were supplemented with glutamate and branched chain amino acids (BCAA), except for CcpA-M17R (Figure 4C). The growth defect of the CcpA-M17R mutant strain was partially restored by the addition of glutamate (Figure 4B); however the addition of only glutamate was not enough. The remaining growth defect of the CcpA-M17R strain might be explained by the pyrE gene. PyrE is responsible for pyrimidine biosynthesis (Subtiwiki, 2014) and its gene expression was only down-regulated in the CcpA-M17R strain. This strain did not show a growth defect in LB because LB is rich in nucleotides from the yeast extract. Therefore the C-medium was also supplemented with uridine 5′-monophosphate in addition to glutamate and BCAA, but the growth defect of the CcpA-M17R mutant strain was not restored (Figure 4D). The residual growth deficiency remains elusive and is probably dependent on more additives.

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