Regulation of pho regulon gene expression by the carbon control protein A, CcpA, in Bacillus subtilis

Bacterial regulons involved in carbon, nitrogen and phosphorus metabolism must interact for purposes of coordination, but the mechanisms involved are not understood. We here report that the carbon control pro-tein-A (CcpA) of Bacillus subtilis, primarily concerned with carbon metabolism, influences expression of various phosphorus (pho) regulon genes including the two alkaline phosphatase structural genes, phoA and phoB. The directions and magnitudes of the effects of glucose and the loss of CcpA on these two genes depend on growth conditions, but they always correlate inversely. Absolute expression levels of phoA and phoB depend on a rich nitrogen source, and gene activation by a fermentable substrate such as glucose depends on the presence of a respiratory substrate such as succinate. We show that these CcpA-dependent glucose effects can be explained by the effects of glucose and CcpA acting on the phoPR operon. Although a good CcpA-binding site (CRE) is found in the control region of the phoPR operon, direct regulation of phoPR gene expression by CcpA via this CRE could not account for the effects of glucose and CcpA on phoA and phoB gene expression. We conclude that CcpA exerts indirect control over the pho regulon by a mechanism that involves CcpA and PhoRP but does not involve the phoPR operon CRE.     

Mechanism of CcpA-mediated glucose repression of the resABCDE operon of Bacillus subtilis

The resABCDE operon of Bacillus subtilis encodes a three-protein complex involved in cytochrome c biogenesis as well as the ResE sensor kinase and the ResD response regulator that control electron transfer and other functions in response to oxygen availability. We have investigated the mechanism of CcpA-mediated control of res operon expression which occurs maximally in the stationary phase of growth. Two CcpA-binding (CRE) sites were found in the res operon, one (CRE1) in the control region in front of the resA promoter, the other (CRE2) in the resB structural gene. Both CRE sites proved to be essential for full CcpA-mediated glucose repression of res operon expression. We propose that both looping and road block mechanisms are involved in res operon control by CcpA.     

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.     

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.     

Functions of the internal pre-S domain of the large surface protein in hepatitis B virus particle morphogenesis.

The large hepatitis B virus (HBV) surface protein (L) forms two isomers which display their N-terminal pre-S domain at the internal and external side of the viral envelope, respectively. The external pre-S domain has been implicated in binding to a virus receptor. To investigate functions of the internal pre-S domain, a secretion signal sequence was fused to the N terminus of L (sigL), causing exclusive expression of external pre-S domains. A fusion construct with a nonfunctional signal (s25L), which corresponds in its primary sequence to sigL cleaved by signal peptidase, was used as a control. SigL was N glycosylated in transfected COS cells at both potential sites in pre-S in contrast to s25L or wild-type L, confirming the expected transmembrane topologies of sigL and s25L. Phenotypic characterization revealed the following points. (i) SigL lost the inhibitory effect of L or s25L on secretion of subviral hepatitis B surface antigen particles, suggesting that the retention signal mapped to the N terminus of L is recognized in the cytosol and not in the lumen of the endoplasmic reticulum. (ii) SigL was secreted into the culture medium even in the absence of the major HBV surface protein (S), while release of an L mutant lacking the retention signal was still dependent on S coexpression. (iii) s25L but not sigL could complement an L-negative HBV genome defective for virion secretion in cotransfections. This suggests that the cytosolic pre-S domain, like a matrix protein, is involved in the interaction of the viral envelope with preformed cytosolic nucleocapsids during virion assembly.     

The Influence of Mycobacterium tuberculosis Sigma Factors on the Promotion Efficiency of ptpAt Promoter in Mycobacterium smegmatis

It was found in a previous study that Mycobacterium tuberculosis protein tyrosine phosphatase ptpAt promoter is a highly active promoter in slow-growing species of mycobacteria, such as M. tuberculosis and M. bovis BCG, but inert in fast-growing mycobacterial species, such as M. smegmatis. This difference is presumed to be due to the differences between sigma factors systems of slow-growing pathogenic mycobacteria and the fast-growing saprophyte M. smegmatis. Therefore, we constructed a series of plasmids, named pOLYG-13x, which can express various M. tuberculosis sigma factors and also contain a P(ptpAt)-gfp reporter gene construct. By inducing different sigma factor genes of M. tuberculosis in M. smegmatis, we were able to explore the influences of various sigma factors on the expression efficiency of the ptpAt promoter. The result show that of the 10 sigma factors evaluated, only sigF and sigL were able to weakly drive the ptpAt promoter in M. smegmatis and other sigma factors were unable to drive the promoter.     

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.     

单核细胞增生李斯特菌中CcpA缺失株的构建及对毒力的影响

CcpA是革兰氏阳性菌中由ccpA基因编码的介导碳分解代谢物阻遏的全局调控因子。近年来的研究表明,CcpA不仅参与CCR效应,还直接或间接参与病原细菌毒力基因的表达调控。为探讨CcpA对单核细胞增生李斯特菌（Lm）毒力的影响,应用同源重组方法构建CcpA缺失菌株。以BLAB/c小鼠为实验动物模型,检测野生株EGDe和缺失株EGDeΔccpA侵染小鼠后的半数致死剂量 LD₅₀和肝脾细菌载菌量,观察小鼠肝脏和脾脏的病理形态变化。结果显示：缺失CcpA后,Lm的LD₅₀降低了10倍,虽然肝脾细菌载菌量没有显著变化,但EGDe△ccpA对小鼠肝和脾的损害更为严重,表明CcpA缺失增强了细菌的毒力,CcpA 对Lm 毒力基因的表达可能具有间接或者直接的调控作用。     

Identifying sigma factors in Mycobacterium smegmatis by comparative genomic analysis

Mycobacterium smegmatis is a saprophytic species that has been used for 15 years as a model to perform heterologous regulation and virulence studies of Mycobacterium tuberculosis. Members of the extracytoplasmic sigma factors family, which are required for adaptive responses to various environmental stresses, are responsible for some of the virulence traits of M. tuberculosis. A bioinformatic search on the genome of M. smegmatis has predicted the existence of 26 sigma factors, which is twice the number that are present in M. tuberculosis. A phylogenetic analysis has shown that despite this high number of sigma factors the orthologs of the genes sigC, sigI and sigK of M. tuberculosis are absent in the M. smegmatis genome. Several sigma factors are specific for M. smegmatis, with a special enrichment in the sigH and, to a lesser extent, in the sigJ and sigL subfamily, pinpointing the potential variability of the repertoire of adaptive response in this saprophytic species.     

Environmental influences on competitive hydrogen peroxide production in Streptococcus gordonii

Streptococcus gordonii is an important member of the oral biofilm. One of its phenotypic traits is the production of hydrogen peroxide (H2O2). H2O2 is an antimicrobial component produced by S. gordonii that is able to antagonize the growth of cariogenic Streptococcus mutans. Strategies that modulate H2O2 production in the oral cavity may be useful as a simple therapeutic mechanism to improve oral health, but little is known about the regulation of H2O2 production. The enzyme responsible for H2O2 production is pyruvate oxidase, encoded by spxB. The functional studies of spxB expression and SpxB abundance presented in this report demonstrate a strong dependence on environmental oxygen tension and carbohydrate availability. Carbon catabolite repression (CCR) modulates spxB expression carbohydrate dependently. Catabolite control protein A (CcpA) represses spxB expression by direct binding to the spxB promoter, as shown by electrophoretic mobility shift assays (EMSA). Promoter mutation studies revealed the requirement of two catabolite-responsive elements (CRE) for CcpA-dependent spxB regulation, as evaluated by spxB expression and phenotypic H2O2 production assays. Thus, molecular mechanisms for the control of S. gordonii spxB expression are presented for the first time, demonstrating the possibility of manipulating H2O2 production for increased competitive fitness.