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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

(A) The crystal structure of CcpA-HPr-Ser46-P in complex with the AckA2-cre site, viewed along the DNA (left) and perpendicular to the DNA (right). The two CcpA monomers were shown in red and purple, the two HPr-Ser46-P monomers were shown in yellow and green, and the DNA was shown in black. The amino acids that were mutated in this study were highlighted in ribbon style in blue (adapted from PDB 3OQM (Schumacher et al., 2011). (B) The expression levels of the different CcpA mutants were shown on a Western Blot. Crude extracts of the B. subtilis strains with the different ccpA mutants were loaded on gel, transferred to a membrane and the CcpA proteins were visualized via chemiluminescence with a CcpA specific antibody. The size of the CcpA protein was 37 kDa. CcpA in lane 4–7 was expressed from plasmid. The B. subtilis ccpA::spec strain was utilized in lane 2–7, and complemented with ccpA on the indicated pHT304 derived plasmid. The empty pHT304 vector served as negative control.
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Figure 1: (A) The crystal structure of CcpA-HPr-Ser46-P in complex with the AckA2-cre site, viewed along the DNA (left) and perpendicular to the DNA (right). The two CcpA monomers were shown in red and purple, the two HPr-Ser46-P monomers were shown in yellow and green, and the DNA was shown in black. The amino acids that were mutated in this study were highlighted in ribbon style in blue (adapted from PDB 3OQM (Schumacher et al., 2011). (B) The expression levels of the different CcpA mutants were shown on a Western Blot. Crude extracts of the B. subtilis strains with the different ccpA mutants were loaded on gel, transferred to a membrane and the CcpA proteins were visualized via chemiluminescence with a CcpA specific antibody. The size of the CcpA protein was 37 kDa. CcpA in lane 4–7 was expressed from plasmid. The B. subtilis ccpA::spec strain was utilized in lane 2–7, and complemented with ccpA on the indicated pHT304 derived plasmid. The empty pHT304 vector served as negative control.

Mentions: In this study, we examined the regulons of specific CcpA mutants. Therefore, three specific amino acids in CcpA were mutated (Figure 1A) and examined by transcriptome analyses to study the effects on CCC. Two of these mutants, CcpA-M17R and CcpA-R304W, have been shown in a previous study to differentially regulate gntR, xynP, alsS, and ackA (Sprehe et al., 2007). Interestingly, these mutants are located in different regions: M17 is in the DNA binding domain and contacts the cre site specifically, while R304 makes an important contact to the Ser46-P of HPr. The third mutant, CcpA-T62H was found to repress xynP very strongly in the absence of glucose (unpublished data). Threonine 62 is the last residue of the allosteric switch domain, mediating the signal of HPr-Ser46-P binding to the DNA binding domain (Schumacher et al., 2011). The aim of the transcriptome analysis presented here was to study the effect of the pointmutations on a genome-wide level and elucidate the mutant specific regulons. Furthermore we examined the presence of specific correlations between the deregulated or regulated genes and altered cre site binding. Will all genes and operons be affected equally by a specific mutation in CcpA or are some genes of the regulon more affected than others? This will provide novel insights on the importance of the residues M17, T62, and R304.


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)

(A) The crystal structure of CcpA-HPr-Ser46-P in complex with the AckA2-cre site, viewed along the DNA (left) and perpendicular to the DNA (right). The two CcpA monomers were shown in red and purple, the two HPr-Ser46-P monomers were shown in yellow and green, and the DNA was shown in black. The amino acids that were mutated in this study were highlighted in ribbon style in blue (adapted from PDB 3OQM (Schumacher et al., 2011). (B) The expression levels of the different CcpA mutants were shown on a Western Blot. Crude extracts of the B. subtilis strains with the different ccpA mutants were loaded on gel, transferred to a membrane and the CcpA proteins were visualized via chemiluminescence with a CcpA specific antibody. The size of the CcpA protein was 37 kDa. CcpA in lane 4–7 was expressed from plasmid. The B. subtilis ccpA::spec strain was utilized in lane 2–7, and complemented with ccpA on the indicated pHT304 derived plasmid. The empty pHT304 vector served as negative control.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: (A) The crystal structure of CcpA-HPr-Ser46-P in complex with the AckA2-cre site, viewed along the DNA (left) and perpendicular to the DNA (right). The two CcpA monomers were shown in red and purple, the two HPr-Ser46-P monomers were shown in yellow and green, and the DNA was shown in black. The amino acids that were mutated in this study were highlighted in ribbon style in blue (adapted from PDB 3OQM (Schumacher et al., 2011). (B) The expression levels of the different CcpA mutants were shown on a Western Blot. Crude extracts of the B. subtilis strains with the different ccpA mutants were loaded on gel, transferred to a membrane and the CcpA proteins were visualized via chemiluminescence with a CcpA specific antibody. The size of the CcpA protein was 37 kDa. CcpA in lane 4–7 was expressed from plasmid. The B. subtilis ccpA::spec strain was utilized in lane 2–7, and complemented with ccpA on the indicated pHT304 derived plasmid. The empty pHT304 vector served as negative control.
Mentions: In this study, we examined the regulons of specific CcpA mutants. Therefore, three specific amino acids in CcpA were mutated (Figure 1A) and examined by transcriptome analyses to study the effects on CCC. Two of these mutants, CcpA-M17R and CcpA-R304W, have been shown in a previous study to differentially regulate gntR, xynP, alsS, and ackA (Sprehe et al., 2007). Interestingly, these mutants are located in different regions: M17 is in the DNA binding domain and contacts the cre site specifically, while R304 makes an important contact to the Ser46-P of HPr. The third mutant, CcpA-T62H was found to repress xynP very strongly in the absence of glucose (unpublished data). Threonine 62 is the last residue of the allosteric switch domain, mediating the signal of HPr-Ser46-P binding to the DNA binding domain (Schumacher et al., 2011). The aim of the transcriptome analysis presented here was to study the effect of the pointmutations on a genome-wide level and elucidate the mutant specific regulons. Furthermore we examined the presence of specific correlations between the deregulated or regulated genes and altered cre site binding. Will all genes and operons be affected equally by a specific mutation in CcpA or are some genes of the regulon more affected than others? This will provide novel insights on the importance of the residues M17, T62, and R304.

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