Dataset Information

Structure of the apo form of the catabolite control protein A (CcpA) from Bacillus megaterium with a DNA-binding domain.

ABSTRACT: Crystal structure determination of catabolite control protein A (CcpA) at 2.6 A resolution reveals for the first time the structure of a full-length apo-form LacI-GalR family repressor protein. In the crystal structures of these transcription regulators, the three-helix bundle of the DNA-binding domain has only been observed in cognate DNA complexes; it has not been observed in other crystal structures owing to its mobility. In the crystal packing of apo-CcpA, the protein-protein contacts between the N-terminal three-helix bundle and the core domain consisted of interactions between the homodimers that were similar to those between the corepressor protein HPr and the CcpA N-subdomain in the ternary DNA complex. In contrast to the DNA complex, the apo-CcpA structure reveals large subdomain movements in the core, resulting in a complete loss of contacts between the N-subdomains of the homodimer.

SUBMITTER: Singh RK

PROVIDER: S-EPMC2330204 | biostudies-literature | 2007 Apr

REPOSITORIES: biostudies-literature

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Structure of the apo form of the catabolite control protein A (CcpA) from Bacillus megaterium with a DNA-binding domain.

Singh Rajesh Kumar RK Palm Gottfried J GJ Panjikar Santosh S Hinrichs Winfried W

Acta crystallographica. Section F, Structural biology and crystallization communications 20070312 Pt 4

Crystal structure determination of catabolite control protein A (CcpA) at 2.6 A resolution reveals for the first time the structure of a full-length apo-form LacI-GalR family repressor protein. In the crystal structures of these transcription regulators, the three-helix bundle of the DNA-binding domain has only been observed in cognate DNA complexes; it has not been observed in other crystal structures owing to its mobility. In the crystal packing of apo-CcpA, the protein-protein contacts betwee ...[more]

PMID: 17401189

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Project description:BACKGROUND: The catabolite control protein CcpA is a transcriptional regulator conserved in many Gram-positives, controlling the efficiency of glucose metabolism. Here we studied the role of Bacillus cereus ATCC 14579 CcpA in regulation of metabolic pathways and expression of enterotoxin genes by comparative transcriptome analysis of the wild-type and a ccpA-deletion strain. RESULTS: Comparative analysis revealed the growth performance and glucose consumption rates to be lower in the B. cereus ATCC 14579 ccpA deletion strain than in the wild-type. In exponentially grown cells, the expression of glycolytic genes, including a non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase that mediates conversion of D-glyceraldehyde 3-phosphate to 3-phospho-D-glycerate in one single step, was down-regulated and expression of gluconeogenic genes and genes encoding the citric acid cycle was up-regulated in the B. cereus ccpA deletion strain. Furthermore, putative CRE-sites, that act as binding sites for CcpA, were identified to be present for these genes. These results indicate CcpA to be involved in the regulation of glucose metabolism, thereby optimizing the efficiency of glucose catabolism. Other genes of which the expression was affected by ccpA deletion and for which putative CRE-sites could be identified, included genes with an annotated function in the catabolism of ribose, histidine and possibly fucose/arabinose and aspartate. Notably, expression of the operons encoding non-hemolytic enterotoxin (Nhe) and hemolytic enterotoxin (Hbl) was affected by ccpA deletion, and putative CRE-sites were identified, which suggests catabolite repression of the enterotoxin operons to be CcpA-dependent. CONCLUSION: The catabolite control protein CcpA in B. cereus ATCC 14579 is involved in optimizing the catabolism of glucose with concomitant repression of gluconeogenesis and alternative metabolic pathways. Furthermore, the results point to metabolic control of enterotoxin gene expression and suggest that CcpA-mediated glucose sensing provides an additional mode of control in moderating the expression of the nhe and hbl operons in B. cereus ATCC 14579.

Project description:Cellobiose, a β-1,4-linked glucose dimer, is a major cellodextrin resulting from the enzymatic hydrolysis of cellulose. It is a major source of carbon for soil bacteria. In bacteria, the phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS), encoded by the cel operon, is responsible for the transport and utilization of cellobiose. In this study, we analyzed the transcription and regulation of the cel operon in Bacillus thuringiensis (Bt). The cel operon is composed of five genes forming one transcription unit. β-Galactosidase assays revealed that cel operon transcription is induced by cellobiose, controlled by Sigma54, and positively regulated by CelR. The HTH-AAA+ domain of CelR recognized and specifically bound to three possible binding sites in the celA promoter region. CelR contains two PTS regulation domains (PRD1 and PRD2), which are separated by two PTS-like domains-the mannose transporter enzyme IIA component domain (EIIAMan) and the galactitol transporter enzyme IIB component domain (EIIBGat). Mutations of His-546 on the EIIAMan domain and Cys-682 on the EIIBGat domain resulted in decreased transcription of the cel operon, and mutations of His-839 on PRD2 increased transcription of the cel operon. Glucose repressed the transcription of the cel operon and catabolite control protein A (CcpA) positively regulated this process by binding the cel promoter. In the celABCDE and celR mutants, PTS activities were decreased, and cellobiose utilization was abolished, suggesting that the cel operon is essential for cellobiose utilization. Bt has been widely used as a biological pesticide. The metabolic properties of Bt are critical for fermentation. Nutrient utilization is also essential for the environmental adaptation of Bt. Glucose is the preferred energy source for many bacteria, and the presence of the phosphotransferase system allows bacteria to utilize other sugars in addition to glucose. Cellobiose utilization pathways have been of particular interest owing to their potential for developing alternative energy sources for bacteria. The data presented in this study improve our understanding of the transcription patterns of cel gene clusters. This will further help us to better understand how cellobiose is utilized for bacterial growth.

Project description:The two-component regulatory system, involving the histidine sensor kinase DegS and response regulator DegU, plays an important role to control various cell processes in the transition phase of Bacillus subtilis. The degU32 allele in strain 1A95 is characterized by the accumulation of phosphorylated form of DegU (DegU-P).Growing 1A95 cells elevated the pH of soytone-based medium more than the parental strain 168 after the onset of the transition phase. The rocG gene encodes a catabolic glutamate dehydrogenase that catalyzes one of the main ammonia-releasing reactions. Inactivation of rocG abolished 1A95-mediated increases in the pH of growth media. Thus, transcription of the rocG locus was examined, and a novel 3.7-kb transcript covering sivA, rocG, and rocA was found in 1A95 but not 168 cells. Increased intracellular fructose 1,6-bisphosphate (FBP) levels are known to activate the HPr kinase HPrK, and to induce formation of the P-Ser-HPr/CcpA complex, which binds to catabolite responsive elements (cre) and exerts CcpA-dependent catabolite repression. A putative cre found within the intergenic region between sivA and rocG, and inactivation of ccpA led to creation of the 3.7-kb transcript in 168 cells. Analyses of intermediates in central carbon metabolism revealed that intracellular FBP levels were lowered earlier in 1A95 than in 168 cells. A genome wide transcriptome analysis comparing 1A95 and 168 cells suggested similar events occurring in other catabolite repressive loci involving induction of lctE encoding lactate dehydrogenase.Under physiological conditions the 3.7-kb rocG transcript may be tightly controlled by a roadblock mechanism involving P-Ser-HPr/CcpA in 168 cells, while in 1A95 cells abolished repression of the 3.7-kb transcript. Accumulation of DegU-P in 1A95 affects central carbon metabolism involving lctE enhanced by unknown mechanisms, downregulates FBP levels earlier, and inactivates HPrK to allow the 3.7-kb transcription, and thus similar events may occur in other catabolite repressive loci.

Dataset Information

Structure of the apo form of the catabolite control protein A (CcpA) from Bacillus megaterium with a DNA-binding domain.

Publications

Structure of the apo form of the catabolite control protein A (CcpA) from Bacillus megaterium with a DNA-binding domain.

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