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

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.

Keywords: Bacillus subtilis; CcpA; CcpA mutants; DNA microarray; transcriptomics.

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.

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.

Figure 3

All differently regulated genes were categorized in Clusters of Orthologous Groups (COGs). [C] Energy production and conversion; [D] Cell cycle control, cell division, chromosome partitioning; [E] Amino acid transport and metabolism; [F] Nucleotide transport and metabolism; [G] Carbohydrate transport and metabolism; [H] Coenzyme transport and metabolism; [I] Lipid transport and metabolism; [J] Translation, ribosomal structure and biogenesis; [K] Transcription; [L] Replication, recombination and repair; [M] Cell wall/membrane/envelope biogenesis; [O] Posttranslational modification, protein turnover, chaperones; [P] Inorganic ion transport and metabolism; [Q] Secondary metabolites biosynthesis, transport and catabolism; [R] General function prediction only; [S] Function unknown; [T] Signal transduction mechanisms; [U] Intracellular trafficking, secretion, and vesicular transport; [V] Defense mechanisms; [X] No prediction. Categorization of genes was done on the MolGen server (http://server.molgenrug.nl/index.php/functional-analysis) (see also Supplementary File, Sheets 9, 10).

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.