枯草芽孢杆菌ccpA基因敲除及对其核黄素产量的影响

CcpA蛋白是介导枯草芽孢杆菌碳分解代谢物阻遏(CCR)的全局调控因子,由ccpA基因编码。CCR效应的存在影响B.subtilis对葡萄糖的利用,降低B.subtilis生产发酵产品的效率。采用基因重组技术敲除了核黄素发酵菌株B.subtilis24/pMX45的ccpA基因,构建了CcpA缺陷株B.subtilis24A1/pMX45。发酵结果显示:B.subtilis24A1/pMX45能够在70h内基本耗尽10%的葡萄糖,生物量达到1.5×109个细胞/mL,溢流代谢产物积累量减少,在8%和10%葡萄糖浓度下,B.subtilis24A1/pMX45核黄素产量分别比B.subtilis24/pMX45提高了62%和95%。CcpA的缺陷,可以缓解葡萄糖引起的CCR效应,显著提高菌株的核黄素产量。     

大肠杆菌碳分解代谢抑制及混合C源共利用的研究进展

总结了大肠杆菌中C源分解代谢(carbon catabolite repression,CCR)现象的原理及特点,综述并分析了如何通过对宿主菌进行基因工程改造以解除碳代谢抑制,以实现大肠杆菌利用多种C源。     

苏云金芽胞杆菌CcpA蛋白对几丁质酶基因chiA和chiB的表达调控作用

【目的】研究苏云金芽胞杆菌Bti75中糖代谢蛋白Ccp A对两种几丁质酶基因chi A和chi B的表达调控。【方法】利用PREDetector软件分析Bti75 chi A和chi B的基因上游区序列,EMSA方法在体外验证Ccp A是否能与chi A和chi B基因的启动子区域片段特异性结合。构建ccp A基因敲除载体以获得敲除突变株Δccp A,运用实时荧光定量PCR技术和Western blot技术比较有无葡萄糖存在的情况下,Ccp A对chi A和chi B基因表达的影响。【结果】计算机分析显示,chi B上游启动子区存在一个潜在的Ccp A结合位点crechi B,而chi A上游启动子区未发现类似序列。体外实验表明,Ccp A蛋白在共阻遏蛋白Hpr-Ser45-P的参与下可与chi B基因启动子区特异性结合,而与chi A基因启动子区没有特异性结合;实时荧光定量PCR和Western blot结果均显示,Bti75中ccp A基因敲除后,同样在葡萄糖存在下chi B的表达量提高而chi A的表达量变化不明显。【结论】在葡萄糖存在的情况下,Ccp A蛋白能抑制苏云金芽胞杆菌中几丁质酶chi B的表达,而chi A的表达不受Ccp A调控。     

大肠杆菌碳分解代谢抑制及混合 C 源共利用的研究进展简

总结了大肠杆菌中C源分解代谢（ carbon catabolite repression,CCR）现象的原理及特点,综述并分析了如何通过对宿主菌进行基因工程改造以解除碳代谢抑制,以实现大肠杆菌利用多种C源。     

植物凋落物影响土壤有机质分解的研究进展

植物凋落物是土壤动物和土壤微生物的主要生命物质和能量来源,其类型、组成以及物理化学等性质直接决定了土壤有机质的品质。对植物凋落物的类型、品质、物理性质、层效应和激发效应以及根际碳淀积与土壤有机质分解的关系进行了总结,可为研究植物凋落物对土壤有机质的影响提供理论参考,指出要在全球变暖背景下进一步加强凋落物分解过程中土壤微生物和酶活性变化的研究。     

杉木凋落物对土壤有机碳分解及微生物生物量碳的影响

利用¹³C稳定同位素示踪技术,研究了杉木凋落物对杉木人工林表层(0～5 cm)和深层(40～45 cm)土壤有机碳分解、微生物生物量碳和可溶性碳动态的影响.结果表明: 杉木人工林中深层土壤有机碳分解速率显著低于表层土壤,但其激发效应却显著高于表层土壤.杉木凋落物添加使土壤总微生物生物量碳和源于原有土壤的微生物生物量碳均显著增加,但对土壤可溶性碳没有显著影响.深层土壤被翻到林地表层,可能加速杉木人工林土壤中碳的损失.     

食物源蛋白肽铁配合物的研究进展

综述食物源蛋白多肽铁配合物的制备、分离纯化、结构分析、生物活性以及安全性研究进展。     

凋落物对土壤有机碳与微生物功能多样性的影响

森林凋落物是影响土壤微生物群落和有机碳含量的重要因素,但其作用的程度和机制尚不清楚,研究该问题对于分析森林生态系统碳循环和资源管理具有重要意义。研究凋落物去除与添加处理下土壤有机碳含量与土壤微生物对碳源利用的差异,明确凋落物去除与添加对土壤微生物群落代谢功能及其多样性的影响,探究不同处理下SOC含量变化的土壤微生物群落代谢机理。选取承德市雾灵山1405-1435 m海拔范围内核桃楸-蒙古栎混交林的表层土壤,采用室内培养结合Biolog-ECO方法,测定了培养第21天的土壤有机碳（soil organic carbon,SOC）含量及微生物群落的AWCD值、Shannon-Wiener多样性指数、Simpson优势度指数、McIntosh均匀度指数、Pielou丰富度指数,分析培养期内凋落物的不同处理下SOC含量与微生物功能多样性的变化特征。结果表明：1）不同凋落物处理对SOC含量与土壤微生物群落多样性具有显著影响（P<0.05）,DL > HL > NL > CK;2）不同凋落物处理下土壤微生物群落代谢活性和土壤微生物对碳源的利用程度具有显著差异（P<0.05）,碳水化合物类和氨基酸类是土壤微生物的主要碳源;3）不同处理的SOC含量与土壤微生物多样性具有正相关关系。双倍凋落物添加在短期内对土壤微生物多样性影响难以达到显著水平且在一定程度上对土壤微生物的代谢活性具有抑制作用,土壤微生物群落功能多样性对SOC含量具有重要影响。     

稀土及其配合物在生物医药上的研究进展

稀土属于化学周期表中镧系元素,具有独特生物活性,能与具有特定生理活性的配体形成稀土配合物。简要归纳了稀土配合物的种类及特点,并阐述了稀土及其配合物在细菌,真菌,癌细胞,正常细胞和病毒方面的生物效应,指出稀土及其配合物在生物医药领域方面有很大的应用前景。     

微生物NAD合成酶及其抑制剂的研究进展

NAD(P)生物代谢在能量代谢,维持氧化还原稳态以及调节细胞寿命等许多细胞进程中有重要作用。因此,NAD生物合成途径的关键酶的抑制剂就成为备受关注的候选新药,如NAD合成酶抑制剂。本文对微生物中的NAD合成酶的催化活性特征,晶体结构,调控因子以及基于晶体结构的抑制剂设计方面进行了综述,以期为基于NAD的治疗领域打开新的思路。     

Expression of recombinant protein A from the lac promoter in Escherichia coli JM83 is not subject to catabolite repression when grown under specific conditions of continuous culture

Although widely used in experimental and industrial situations, genetically engineered plasmids containing the lac promoter from Escherichia coli are subject to catabolite repression when grown in glucose-containing media. Several methods of overcoming this problem have been investigated by studying the expression of the protein A gene from Staphylococcus aureus under the control of the Escherichia coli lac promoter. When glycerol is used as a sole carbon source, the plasmid is unstable and is rapidly lost from the culture. When the bacteria are grown in chemostats under glucose limitation, the plasmid is maintained, even at high dilution rates, and the expression of protein A is similar to that observed when glycerol was used. The balance between metabolic load and protein A expression seems to be maintained by reducing the gene dose to a tolerable level. Depending on the metabolic conditions prevailing in the culture, this is achieved, either by reducing the copy number of the plasmid or in extreme cases by removing the plasmid altogether.     

Regulation of DNA-binding activity of the Staphylococcus aureus catabolite control protein A by copper (II)-mediated oxidation

Catabolite control protein A (CcpA) of the human pathogen Staphylococcus aureus is an essential DNA regulator for carbon catabolite repression and virulence, which facilitates bacterial survival and adaptation to a changing environment. Here, we report that copper (II) signaling mediates the DNA-binding capability of CcpA in vitro and in vivo. Copper (II) catalyzes the oxidation of two cysteine residues (Cys216 and Cys242) in CcpA to form intermolecular disulfide bonds between two CcpA dimers, which results in the formation and dissociation of a CcpA tetramer of CcpA from its cognate DNA promoter. We further demonstrate that the two cysteine residues on CcpA are important for S. aureus to resist host innate immunity, indicating that S. aureus CcpA senses the redox-active copper (II) ions as a natural signal to cope with environmental stress. Together, these findings reveal a novel regulatory mechanism for CcpA activity through copper (II)-mediated oxidation.     

Solution structure and dynamics of Crh, the Bacillus subtilis catabolite repression HPr

The solution structure and dynamics of the Bacillus subtilis HPr-like protein, Crh, have been investigated using NMR spectroscopy. Crh exhibits high sequence identity (45 %) to the histidine-containing protein (HPr), a phospho-carrier protein of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system, but contains no catalytic His15, the site of PEP-dependent phosphorylation in HPr. Crh also forms a mixture of monomers and dimers in solution whereas HPr is known to be monomeric. Complete backbone and side-chain assignments were obtained for the monomeric form, and 60 % of the dimer backbone resonances; allowing the identification of the Crh dimer interface from chemical-shift mapping. The conformation of Crh was determined to a precision of 0.46(+/-0.06) A for the backbone atoms, and 1.01(+/-0.08) A for the heavy atoms. The monomer structure is similar to that of known HPr 2.67(+/-0.22) A (C(alpha) rmsd), but has a few notable differences, including a change in the orientation of one of the helices (B), and a two-residue shift in beta-sheet pairing of the N-terminal strand with the beta4 strand. This shift results in a shortening of the surface loop present in HPr and consequently provides a flatter surface in the region of dimerisation contact, which may be related to the different oligomeric nature of these two proteins. A binding site of phospho-serine(P-Ser)-Crh with catabolite control protein A (CcpA) is proposed on the basis of highly conserved surface side-chains between Crh and HPr. This binding site is consistent with the model of a dimer-dimer interaction between P-Ser-Crh and CcpA. (15)N relaxation measured in the monomeric form also identified differential local mobility in the helix B which is located in the vicinity of this site.     

Biological Activities of Sex Hormone Produced by Tremella mesenteriea

d-Aminoacylase was found to be produced not only by S. olivaceus 62–3 isolated from soil but also by three strains of type culture of Streptomyces species. All four of these strains produced d-aminoacylase intracellularly only when an inducer was added to the culture medium. d-Amino acids or N-acetyl-d-amino acids were effective as inducers.

As S. tuirus showed the highest d-aminoacylase activity, the enzyme extract of this strain was subjected to further investigation to determine the optimal conditions for optical resolution of N-acetyl-dl-phenylglycine. Almost all contaminating l-aminoacylase in the enzyme extract could be eliminated by DEAE-Sephadex adsorption. d-Phenylglycine of 99.9% optical purity was obtained after complete hydrolysis of d-isomer with the use of d-aminoacylase solution.     

大肠杆菌分解代谢产物阻遏效应研究进展

细菌在多种碳源共存的环境中优先利用一种(通常是葡萄糖)的现象被称为分解代谢产物阻遏效应。国内现有分子生物学及相关课程教材普遍对该效应的机理解释不清甚至给出错误的解释。大肠杆菌葡萄糖-乳糖分解代谢产物阻遏效应产生的根本原因不是胞内葡萄糖的存在, 而是葡萄糖经PTS(Phosphoenolpyruvate: carbohydrate phosphotransferase system)系统向胞内运输同时藕联磷酸化的过程。磷酸向葡萄糖的传递导致PTS关键组分EⅡA^Glc去磷酸化形式的积累。该形式的EⅡA^Glc可以与质膜上本底表达的乳糖透性酶LacY结合, 阻止诱导物乳糖的吸收。cAMP的影响也是通过激活参与PTS系统的关键基因而加强了诱导物排斥作用。此外, 去磷酸化形式的EⅡBGlc和YeeⅠ对全局性转录阻遏蛋白Mlc活性的抑制也保证了PTS系统关键组分蛋白的基因表达。文章综述了近年来有关大肠杆菌分解代谢产物阻遏效应机理的最新研究进展, 并对相关教材有关这一内容的阐述提出了修改建议。     

A Crh-specific function in carbon catabolite repression in Bacillus subtilis

Carbon catabolite repression in Bacillus subtilis is mediated by phosphorylation of the phosphoenolpyruvate:carbohydrate phosphotransferase system intermediate HPr at a serine residue catalyzed by HPr kinase. The orthologous protein Crh functions in a similar way, but, unlike HPr, it is not functional in carbohydrate uptake. A specific function for Crh is not known. The role of HPr and Crh in repressing the citM gene encoding the Mg(2+)-citrate transporter was investigated during growth of B. subtilis on different carbon sources. In glucose minimal medium, full repression was supported by both HPr and Crh. Strains deficient in Crh or the regulatory function of HPr revealed the same repression as the wild-type strain. In contrast, in a medium containing succinate and glutamate, repression was specifically mediated via Crh. Repression was relieved in the Crh-deficient strain, but still present in the HPr mutant strain. The data are the first demonstration of a Crh-specific function in B. subtilis and suggest a role for Crh in regulation of expression during growth on substrates other than carbohydrates.     

Immunological crossreactivity to the catabolite control protein CcpA from Bacillus megaterium is found in many Gram-positive bacteria

Abstract The catabolite control protein CcpA from Bacillus megaterium was overproduced as a fusion protein to a 6xhis affinity tag and purified to homogeneity. Polyclonal antibodies of high affinity and specificity were raised against the purified protein. The serum did not crossreact with purified Lac repressor despite the fact that CcpA and LacI belong to the same protein family. Using this antiserum we identified proteins that share antigenic determinants with CcpA in many Gram-positive bacteria, including bacilli, staphylococci, streptococci, lactic acid bacteria, and some actinomycetes.