Catabolite repression in Bacillus subtilis: a global regulatory mechanism for the Gram-positive bacteria?

Three components involved in catabolite repression (CR) of gene expression in Bacillus have been identified. The cis-acting catabolite responsive element (CRE), which is present in many genes encoding carbon catabolic enzymes in various species of the Gram-positive bacteria, mediates CR of several genes in Bacillus subtilis, Bacillus megaterium, and Staphylococcus xylosus. CR of most genes regulated via CRE is also affected by the trans-acting factors CcpA and HPr. Similarities between CcpA and Lac and Gal repressors suggest binding of CcpA to CRE. HPr, a component of the phosphoenol pyruvate:sugar phosphotransferase system, undergoes regulatory phosphorylation at a serine residue by a fcuctose-1,6-diphosphate-activated kinase. A mutant of HPr, which is not phosphorylatable at this position because of an exchange of serine to alanine, lacks CR of several catabolic activities. This mutant phenotype is similar to the one exhibited by a ccpA mutant. Direct protein-protein interaction between CcpA and HPr(Ser-P) was recently demonstrated and constitutes a link between metabolic activity and CR.     

Protein kinase-dependent HPr/CcpA interaction links glycolytic activity to carbon catabolite repression in Gram-positive bacteria

CcpA, the repressor/activator mediating carbon catabolite repression and glucose activation in many Gram-positive bacteria, has been purified from Bacillus megaterium after fusing it to a His tag. CcpA-his immobilized on a Ni-NTA resin specifically interacted with HPr phosphorylated at seryl residue 46. HPr, a phosphocarrier protein of the phosphoenolpyruvate: glycose phosphotransferase system (PTS), can be phosphorylated at two different sites: (i) at His-15 in a PEP-dependent reaction catalysed by enzyme I of the PTS; and (ii) at Ser-46 in an ATP-dependent reaction catalysed by a metabolite-activated protein kinase. Neither unphosphorylated HPr nor HPr phosphorylated at His-15 nor the doubly phosphorylated HPr bound to CcpA. The interaction with seryl-phosphorylated HPr required the presence of fructose 1,6-bisphosphate. These findings suggest that carbon catabolite repression in Gram-positive bacteria is a protein kinase-triggered mechanism. Glycolytic intermediates, stimulating the corresponding protein kinase and the P-ser-HPr/CcpA complex formation, provide a link between glycolytic activity and carbon catabolite repression. The sensitivity of this complex formation to phosphorylation of HPr at His-15 also suggests a link between carbon catabolite repression and PTS transport activity.     

Carbon catabolite repression of type IV pilus-dependent gliding motility in the anaerobic pathogen Clostridium perfringens

Clostridium perfringens is an anaerobic, gram-positive, spore-forming bacterium responsible for the production of severe histotoxic and gastrointestinal diseases in humans and animals. In silico analysis of the three available genome-sequenced C. perfringens strains (13, SM101, and ATCC13124) revealed that genes that encode flagellar proteins and genes involved in chemotaxis are absent. However, those strains exhibit type IV pilus (TFP)-dependent gliding motility. Since carbon catabolite regulation has been implicated in the control of different bacterial behaviors, we investigated the effects of glucose and other readily metabolized carbohydrates on C. perfringens gliding motility. Our results demonstrate that carbon catabolite regulation constitutes an important physiological regulatory mechanism that reduces the proficiencies of the gliding motilities of a large number of unrelated human- and animal-derived pathogenic C. perfringens strains. Glucose produces a strong dose-dependent inhibition of gliding development without affecting vegetative growth. Maximum gliding inhibition was observed at a glucose concentration (1%) previously reported to also inhibit other important behaviors in C. perfringens, such as spore development. The inhibition of gliding development in the presence of glucose was due, at least in part, to the repression of the genes pilT and pilD, whose products are essential for TFP-dependent gliding proficiency. The inhibitory effects of glucose on pilT and pilD expression were under the control of the key regulatory protein CcpA (catabolite control protein A). The deficiency in CcpA activity of a ccpA knockout C. perfringens mutant strain restored the expressions of pilT and pilD and gliding proficiency in the presence of 1% glucose. The carbon catabolite repression of the gliding motility of the ccpA mutant strain was restored after the introduction of a complementing plasmid harboring a wild-type copy of ccpA. These results point to a central role for CcpA in orchestrating the negative effect of carbon catabolite regulation on C. perfringens gliding motility. Furthermore, we discovered a novel positive role for CcpA in pilT and pilD expression and gliding proficiency in the absence of catabolite regulation. Carbon catabolite repression of gliding motility and the dual role of CcpA, either as repressor or as activator of gliding, are analyzed in the context of the different social behaviors and diseases produced by C. perfringens.     

苏云金芽胞杆菌BkdR和CcpA对bkd基因簇的转录调控

【目的】通过分析苏云金芽胞杆菌(Bacillus thuringiensis,Bt)转录调控因子BkdR和多效调控因子CcpA对亮氨酸、异亮氨酸、缬氨酸代谢基因簇bkd的转录调控,明确bkd基因簇的转录调控机制。【方法】通过β-半乳糖苷酶活性测定分析bkd基因簇启动子的诱导转录活性,采用同源重组技术敲除Bt HD73菌株的ccpA基因,通过融合His标签的方法在大肠杆菌中表达纯化BkdR和CcpA蛋白,通过凝胶阻滞实验明确BkdR和CcpA蛋白与bkd基因簇启动子的结合作用。【结果】亮氨酸、异亮氨酸、缬氨酸可诱导bkd基因簇启动子Pptb的转录活性。Pptb的诱导活性在bkdR突变体中明显降低,而在ccpA突变体中明显上升。BkdR和CcpA蛋白与Pptb均有结合作用。【结论】bkd基因簇的转录活性受BkdR正调控,而受CcpA负调控。     

Catabolite repression in Lactobacillus casei ATCC 393 is mediated by CcpA.

The chromosomal ccpA gene from Lactobacillus casei ATCC 393 has been cloned and sequenced. It encodes the CcpA protein, a central catabolite regulator belonging to the LacI-GalR family of bacterial repressors, and shows 54% identity with CcpA proteins from Bacillus subtilis and Bacillus megaterium. The L. casei ccpA gene was able to complement a B. subtilis ccpA mutant. An L. casei ccpA mutant showed increased doubling times and a relief of the catabolite repression of some enzymatic activities, such as N-acetylglucosaminidase and phospho-beta-galactosidase. Detailed analysis of CcpA activity was performed by using the promoter region of the L. casei chromosomal lacTEGF operon which is subject to catabolite repression and contains a catabolite responsive element (cre) consensus sequence. Deletion of this cre site or the presence of the ccpA mutation abolished the catabolite repression of a lacp::gusA fusion. These data support the role of CcpA as a common regulatory element mediating catabolite repression in low-GC-content gram-positive bacteria.     

Catabolite repression of the Bacillus subtilis gnt operon exerted by two catabolite-responsive elements

Catabolite repression of Bacillus subtilis catabolic operons is supposed to occur via a negative regulatory mechanism involving the recognition of a cis-acting catabolite-responsive element (cre) by a complex of CcpA, which is a member of the GalR-LacI family of bacterial regulatory proteins, and the seryl-phos-phorylated form of HPr (P-ser-HPr), as verified by recent studies on catabolite repression of the gnt operon. Analysis of the gnt promoter region by deletions and point mutations revealed that in addition to the ere in the first gene (gntR) of the gnt operon (cre_down), this operon contains another ere located in the promoter region (cre_up). A translational gntR-lacZ fusion expressed under the control of various combinations of wild-type and mutant cre_down and cre_up was integrated into the chromosomal amyE locus, and then catabolite repression of p-galac-tosidase synthesis in the resultant integrants was examined. The in vivo results implied that catabolite repression exerted by cre_up was probably independent of catabolite repression exerted by cre_down; both creup and cre_down catabolite repression involved CcpA. Catabolite repression exerted by cre_up was independent of P-ser-HPr, and catabolite repression exerted by cre_down was partially independent of P-ser-HPr. DNase I footprinting experiments indicated that a complex of CcpA and P-ser-HPr did not recognize cre_up, in contrast to its specific recognition of cre_down. However, CcpA complexed with glucose-6-phosphate specifically recognized cre_up as well as cre_down, but the physiological significance of this complexing is unknown.     

Analysis of catabolite control protein A-dependent repression in Staphylococcus xylosus by a genomic reporter gene system

A single-copy reporter system for Staphylococcus xylosus has been developed, that uses a promoterless version of the endogenous β-galactosidase gene lacH as a reporter gene and that allows integration of promoters cloned in front of lacH into the lactose utilization gene cluster by homologous recombination. The system was applied to analyze carbon catabolite repression of S. xylosus promoters by the catabolite control protein CcpA. To test if lacH is a suitable reporter gene, β-galactosidase activities directed by two promoters known to be subject to CcpA regulation were measured. In these experiments, repression of the malRA maltose utilization operon promoter and autoregulation of the ccpA promoters were confirmed, proving the applicability of the system. Subsequently, putative CcpA operators, termed catabolite-responsive elements (cres), from promoter regions of several S. xylosus genes were tested for their ability to confer CcpA regulation upon a constitutive promoter, P_vegII. For that purpose, cre sequences were placed at position +3 or +4 within the transcribed region of P_vegII. Measurements of β-galactosidase activities in the presence or absence of glucose yielded repression ratios between two- and eightfold. Inactivation of ccpA completely abolished glucose-dependent regulation. Therefore, the tested cres functioned as operator sites for CcpA. With promoters exclusively regulated by CcpA, signal transduction leading to CcpA activation in S. xylosus was examined. Glucose-dependent regulation was measured in a set of isogenic mutants showing defects in genes encoding glucose kinase GlkA, glucose uptake protein GlcU, and HPr kinase HPrK. GlkA and GlcU deficiency diminished glucose-dependent CcpA-mediated repression, but loss of HPr kinase activity abolished regulation. These results clearly show that HPr kinase provides the essential signal to activate CcpA in S. xylosus. Glucose uptake protein GlcU and glucose kinase GlkA participate in activation, but they are not able to trigger CcpA-mediated regulation independently from HPr kinase.     

Contributions of XylR,CcpA and cre to diauxic growth of Bacillus megaterium and to xylose isomerase expression in the presence of glucose and xylose

Bacillus megaterium shows diauxic growth in minimal medium containing glucose and xylose. We have examined the influence of three elements that regulate xyl operon expression on diauxic growth and expression of a xylA-lacZ fusion. xylA is 13-fold repressed during growth on glucose. Induction occurs at the onset of the lag phase after glucose is consumed. Inactivation of xylR yields a two-fold increase in expression of xylA on glucose. Deletion of the catabolite responsive element (cre) has a more pronounced effect, reducing glucose repression from 13-fold in the wild type to about 2.5-fold. When xylR and cre are inactivated together a residual two-fold repression of xylA is found. Inactivation of xylR affects diauxic growth by shortening the lag phase from 70 to 40?min. In-frame deletion of ccpA results in the loss of diauxic growth, an increase in doubling time and simultaneous use of both sugars. In contrast, a strain with an inactivated cre site in xylA exhibits diauxic growth without an apparent lag phase on glucose and xylose, whereas fructose and xylose are consumed simultaneously.     

Identification and inactivation of pleiotropic regulator CcpA to eliminate glucose repression of xylose utilization in Clostridium acetobutylicum

d-xylose utilization is a key issue for lignocellulosic biomass fermentation, and a major problem in this process is carbon catabolite repression (CCR). In this investigation, solvent-producing bacterium Clostridium acetobutylicum ATCC 824 was metabolically engineered to eliminate d-glucose repression of d-xylose utilization. The ccpA gene, encoding the pleiotropic regulator CcpA, was experimentally characterized and then disrupted. Under pH-controlled conditions, the ccpA-disrupted mutant (824ccpA) can use a mixture of d-xylose and d-glucose simultaneously without CCR. Moreover, this engineered strain produced acetone, butanol and ethanol (ABE) at a maximal titer of 4.94, 12.05 and 1.04 g/L, respectively, which was close to the solvent level of maize- or molasses-based fermentation by wild type C. acetobutylicum. Molar balance analysis for improved process of mixed sugars utilization also revealed less acid accumulation and more butanol yield by the engineered strain as compared to the wild type. This study offers a genetic modification strategy for improving simultaneous utilization of mixed sugars by Clostridium, which is essential for commercial exploitation of lignocellulose for the production of solvents and biofuels.