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.     

In vitro binding of the CcpA protein of Bacillus megaterium to cis-acting catabolite responsive elements (CREs) of Gram-positive bacteria

Abstract Using DNA band migration retardation assays, specific binding of the CcpA protein of Bacillus megaterium to the ds-acting catabolite responsive element (CRE) of the xyl operon of B. subtilis has been demonstrated. Binding of CcpA was specifically inhibited by addition of unlabeled DNA fragments containing CREs of other operons but not by DNA fragments lacking a CRE. Binding was stimulated by high concentrations of phosphate, pyrophosphate, and organic phosphate esters and specifically inhibited by serine phosphorylated HPr and its conformational analogue, S46D HPr. This report therefore documents the specific binding of CcpA to a target CRE and defines its regulation by HPr(ser-P) and phosphorylated metabolites.     

Sugar uptake and carbon catabolite repression in Bacillus megaterium strains with inactivated ptsHI

We have determined the role played by the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in carbon catabolite repression (CCR) of xylose utilization in Bacillus megaterium. For that purpose we have cloned, sequenced and inactivated the genes ptsH and ptsl of B. megaterium, encoding HPr and EI of the PTS, respectively. While glucose uptake of a ptsHI mutant is not affected at 12.5 mM of glucose, CCR of the xyl operon is reduced in this mutant from 16-fold to 3-fold. This may be attributed to the loss of the corepressor of CcpA, HPr(Ser-P), or could result from the slower growth rate of the mutant. In contrast, CCR exerted by fructose or mannitol is completely abolished. We conclude that glucose triggers additional mechanisms of CCR than fructose or mannitol. The remaining 3-fold glucose repression is relieved in a strain in which ptsHI and glk, encoding glucokinase, are inactivated. This result indicates that glucose metabolism is necessary for CCR. The ability of the ptsHI mutant to take up glucose suggests the existence of a second, non-PTS glucose uptake system. The Km and vmax values of this transporter ranged between 2 and 5 mM and 154 to 219 nmol/[(mg protein)*min], respectively.     

hprK基因敲除及对其枯草芽孢杆菌核黄素发酵的影响

枯草芽孢杆菌在葡萄糖丰富的环境中,胞内糖分解代谢物浓度的提高将引起碳分解代谢物阻遏效应(CCR)及糖吸收的抑制,对核黄素等发酵过程产生不利影响。通过缺陷细胞的分解代谢物控制蛋白A(CcpA)可以解除CCR效应,但不能解除糖吸收的抑制。磷酸烯醇式丙酮酸-糖磷酸转移酶系统(PTS)是枯草芽孢杆菌主要的糖吸收方式,HPr蛋白和双功能的HPr激酶/HPr-Ser46-P磷酸酶(HprK/P)参与PTS系统的调控。在葡萄糖丰富的条件下,HprK/P的激酶活性受1,6-二磷酸果糖激活,催化HPr蛋白46位丝氨酸残基磷酸化,形成HPr-Ser46-P。HPr-Ser46-P抑制某些碳源透过酶基因的表达;同时HPr-Ser46-P难以被酶Ⅰ在His15磷酸化,不能在PTS系统中发挥转移磷酸基团的作用,使细胞的糖吸收受到抑制。在CcpA缺陷的背景下,敲除核黄素生产菌株B.subtilis24A1/pMX45的HprK/P编码基因hprK,构建了CcpA和HprK/P双缺陷的重组菌B.subtilisZHc/pMX45。摇瓶发酵显示,B.subtilisZHc/pMX45核黄素发酵的最适葡萄糖浓度由24A1/pMX45的8%提高到10%;核黄素产量达到4.374mg/mL,比24A1/pMX45提高了19.2%。结果表明,CcpA和HprK/P的双缺陷可有效解除高浓度葡萄糖所引起的CCR效应和糖吸收抑制,有助于提高细胞对葡萄糖的耐受力,并提高核黄素产量。     

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.     

Quantification of the influence of HPrSer46P on CcpA-cre interaction

Carbon catabolite repression (CCR) of the Bacillus megateriumxyl operon is dependent on the catabolite responsive element cre, the catabolite control protein (CcpA) and the histidine-containing phosphocarrier protein phosphorylated at the serine 46 residue (HPrSer46P). The latter is formed in the presence of glucose and mediates CCR via CcpA. We present evidence for the presence of HPrSer46P in a ternary complex with CcpA and cre. We also demonstrate increased stability of this complex compared to the CcpA-cre complex by electrophoretic mobility shift analysis (EMSA). This stabilization by HPrSer46P is the same for the xyl cre and an improved cre. Thus, HPrSer46P is a co-repressor for CcpA. In addition, surface plasmon resonance (SPR) experiments yielded binding constants of CcpA and the CcpA-HPrSer46P complex with cre. HPrSer46P stimulated CcpA binding to cre 50-fold. The binding constant is 4.9(+/- 0.5) x 10(6) M(-1). Non-phosphorylated HPr did not affect the complex formation between CcpA and cre. Previously proposed effects by glucose-6-phosphate, fructose-1,6-diphosphate and NADP on CcpA-cre or CcpA-HPrSer46P-cre formation were not found in EMSA and SPR experiments.     

Significance of HPr in catabolite repression of alpha-amylase.

CcpA and HPr are presently the only two proteins implicated in Bacillus subtilis global carbon source catabolite repression, and the ptsH1 mutation in the gene for the HPr protein was reported to relieve catabolite repression of several genes. However, alpha-amylase synthesis by B. subtilis SA003 containing the ptsH1 mutation was repressed by glucose. Our results suggest HPr(Ser-P) may be involved in but is not required for catabolite repression of alpha-amylase, indicating that HPr(Ser-P) is not the sole signaling molecule for CcpA-mediated catabolite repression in B. subtilis.     

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.     

Catabolite regulation of the pta gene as part of carbon flow pathways in Bacillus subtilis

In Bacillus subtilis, the products of the pta and ackA genes, phosphotransacetylase and acetate kinase, play a crucial role in the production of acetate, one of the most abundant by-products of carbon metabolism in this gram-positive bacterium. Although these two enzymes are part of the same pathway, only mutants with inactivated ackA did not grow in the presence of glucose. Inactivation of pta had only a weak inhibitory effect on growth. In contrast to pta and ackA in Escherichia coli, the corresponding B. subtilis genes are not cotranscribed. Expression of the pta gene was increased in the presence of glucose, as has been reported for ackA. The effects of the predicted cis-acting catabolite response element (CRE) located upstream from the promoter and of the trans-acting proteins CcpA, HPr, Crh, and HPr kinase on the catabolite regulation of pta were investigated. As for ackA, glucose activation was abolished in ccpA and hprK mutants and in the ptsH1 crh double mutant. Footprinting experiments demonstrated an interaction between CcpA and the pta CRE sequence, which is almost identical to the proposed CRE consensus sequence. This interaction occurs only in the presence of Ser-46-phosphorylated HPr (HPrSer-P) or Ser-46-phosphorylated Crh (CrhSer-P) and fructose-1,6-bisphosphate (FBP). In addition to CcpA, carbon catabolite activation of the pta gene therefore requires at least two other cofactors, FBP and either HPr or Crh, phosphorylated at Ser-46 by the ATP-dependent Hpr kinase.