An extracellullar nuclease from Bacillus firmus VKPACU-1: specificity and mode of action

An extracellular nuclease from Bacillus firmus VKPACU-1 was multifunctional enzyme, this nuclease hydrolyzed poly U rapidly and more preferentially than the other homopolyribonucleotides. Hydrolysis of RNA this enzyme released mononucleotides in the order 5'UMP > 5'AMP > 5'GMP where as in hydrolysis of DNA the mononucleotides in the order of 5'dAMP > 5'dGMP > 5'dTMP and oligonucleotides. Uridylic linkages in RNA and adenylic linkages in DNA were preferentially cleaved by the nuclease. Nuclease produced oligonucleotides having only 3' hydroxyl and 5' phosphate termini. Present nuclease hydrolyzed RNA and DNA released oligonucleotides as major end products and mononucleotides, suggesting an endo mode of action.     

Mode of Action of Nuclease P1 on Nucleic Acids and Its Specificity for Synthetic Phosphodiesters

Nuclease P₁ was found to attack RNA and heat-denatured DNA in endo- and exonucleolytic manners. The evidence was as follows: (1) In the early stage of digestion both mononucleotides and oligonucleotides with various sizes were formed simultaneously with rapid fragmentation of polynucleotides. (2) The relative amount of the monomer was larger than that of any class of oligomers throughout the process of digestion. Nuclease P₁ showed a preference for the linkages between 3′-hydroxyl group of adenosine or deoxyadenosine and the 5′-phosphoryl group of the adjacent nucleotides. p-Nitrophenyl ester of 3′-dTMP was hydrolyzed to thymidine and p-nitrophenyl phosphate, while p-nitrophenyl ester of 5′-dTMP was not attacked. It is concluded from these findings that the basic structure required for the substrate of nuclease P₁ is a nucleoside 3′-phosphate-containing structure and the enzyme cleaves the diester bond between the phosphate and the 3′-hydroxyl group of the sugar.     

S1核酸酶作用与微环DNA分子的克隆策略

米曲霉来源的S1 核酸酶具有降解单链DNA或RNA的作用。在适当的条件下 ,该酶能将不同的环形DNA分子从超螺旋转变成开环和线形结构 ,对质粒pUC19的实验证明 ,S1 核酸酶的这种转变作用与加入的酶量呈正相关。在 2 5 μL总反应体积中 ,按 10 0ngDNA加入 5u至 17u的S1 核酸酶 ,能获得较高比例的线形DNA。由于微环DNA分子太小 ,单酶切位点的出现率较低 ,很难用常规方式进行克隆 ,以S1 核酸酶进行线形化是微环DNA克隆的途径。pC3是已知最小的真核生物线粒体DNA类质粒 (5 37bp) ,经S1 核酸酶线形化后 ,成功地克隆到pMD18 T载体上。     

Mode of Action and Specificity of a Nuclease Preferential for Thymidine Residue from Neurospora crassa Mycelia

A nuclease from N. crassa mycelia was found to attack both heat-denatured and native DNA in endonucleolytic manner. The products of exhaustive degradation of heat-denatured DNA were mainly di- to pentanucleotides bearing 5′-phosphoryl groups. 5′-Mononucleotides amounted to 4.4% of the total products and the base distribution was in the following order: dTMP > dCMP > dGMP > dAMP. Analysis of the residues at 5′- and 3′-termini of the oligonucleotides showed that thymidine was predominant at both termini, especially at 3′- termini. Also the analysis of terminal residues produced by limited digestion (27% and 55.5 % of the substrate were rendered acid soluble, respectively) gave the same results as above. Therefore, it was suggested that N. crassa nuclease has some preference for thymidine residue to hydrolyze the sequence of ?T ↓ pT? or ?T ↓ pX-predominantly. The activity toward synthetic polymers was in the following order; poly d(A-T) ? poly dA poly dT > poly d(G-C) > poly dGpoly dC. The correlation between GC-contents and the activity was also investigated.     

Production of cyclodextrin from starch by cyclodextrin glycosyltransferase from Bacillus firmus and characterization of purified enzyme

During screening for cyclodextrin-forming microorganisms, an alkalophilic Bacillus sp, which produced high activity of cyclodextrin glycosyltransferase, was isolated and identified as Bacillus firmus. The crude enzyme transformed starch to mainly β-and γ-cyclodextrin. The purified enzyme had an optimum pH of 7.5–8.5 and its optimum temperature was 65°C, which is the highest optimum temperature as compared to other cyclodextrin glycosyltransferases except that produced by Bacillus amyloliquefaciens. Received 06 January 1997/ Accepted in revised form 20 March 1997     

Effects and mechanisms of Bacillus thuringiensis crystal toxins for mosquito larvae

Bacillus thuringiensis is a Gram‐positive aerobic bacterium that produces insecticidal crystalline inclusions during sporulation phases of the mother cell. The virulence factor, known as parasporal crystals, is composed of Cry and Cyt toxins. Most Cry toxins display a common 3‐domain topology. Cry toxins exert intoxication through toxin activation, receptor binding and pore formation in a suitable larval gut environment. The mosquitocidal toxins of Bt subsp. israelensis (Bti) were found to be highly active against mosquito larvae and are widely used for vector control. Bt subsp. jegathesan is another strain which possesses high potency against broad range of mosquito larvae. The present review summarizes characterized receptors for Cry toxins in mosquito larvae, and will also discuss the diversity and effects of 3‐D mosquitocidal Cry toxin and the ongoing research for Cry toxin mechanisms generated from investigations of lepidopteran and dipteran larvae.     

Isolation, purification and properties of new restriction endonucleases from Bacillus badius and Bacillus lentus

We tentatively named two enzymes as BbaI and BleI, which were isolated and purified from Gram-positive mesophilic bacteria Bacillus badius 1458 and Bacillus lentus 1689 respectively, by ammonium sulphate precipitation, phosphocellulose and heparin-sepharose column chromatography. SDS-PAGE protein profiles for BbaI and BleI showed denatured molecular weights of 52 and 48 kDa, respectively. BbaI hydrolyzed pUC18 DNA into 1900 and 700 bp, pBR322 DNA into two fragments of 2800 and 1500 bp and Φ×174 DNA into 3800 and 1600 bp. BleI hydrolyzed pUC18 DNA into 1800 and 800 bp, pBR322 DNA into two fragments of 2700 and 1600 bp and Φ×174 DNA into 3700 and 1700 bp. The effects of temperature, ionic strength, pH and Mg²⁺ ion concentrations were studied to demonstrate some biochemical properties of BbaI and BleI. Maximum activities of these enzymes were observed at 37 °C (pH 8.0) with 100 mM NaCl and 10 mM Mg²⁺ concentrations.     

坚强芽胞杆菌主要分泌蛋白的鉴定及其分泌性序列

[目的]坚强芽胞杆菌是一种在自然界普遍存在的益生菌,在对虾养殖中应用较为广泛.为了研究其分泌性蛋白从而为分泌性载体的构建提供理论依据,本文对坚强芽胞杆菌的主要分泌蛋白进行质谱鉴定及分泌性序列的分析.[方法]从本实验室分离保存的1株来自对虾肠道的坚强芽胞杆菌(Bacillus firmus)培养液中获取了分泌性蛋白,进行SDS-PAGE,并对表达量较高的3条蛋白区带进行MALDI-TOF/TOF质谱鉴定及克隆测序,进行生物信息学分析.[结果]鉴定出的蛋白分别是坚强芽胞杆菌几丁质酶(chitinase)、坚强芽胞杆菌肠毒素A(enterotoxin A)和坚强芽胞杆菌BCG9842蛋白(hypothetical protein BCG9842).经在线软件SignaIP 3.0分析,确定chitinase、enterotoxin和BCG9842均存在不同的分泌性信号肽序列bf-43、bf-37和bf-16,通过在线软件PSORT分析表明,bf-43定位于细胞的外膜上,bf-37和bf-16定位于细胞的胞外.[结论]本研究鉴定出了坚强芽胞杆菌的3个主要分泌性蛋白,分析筛选出了3条分泌性序列,为分泌性载体的构建提供理论依据.     

The extracellular and cytoplasmic proteomes of the non-virulent Bacillus anthracis strain UM23C1-2

The recently published genome sequence of Bacillus anthracis Ames has facilitated the prediction of proteins associated with the virulence of this bacterium. The aim of this study was to define reference maps for the extracellular and cytoplasmic proteomes of the avirulent B. anthracis strain UM23C1-2 that are useful for physiological studies and the development of improved vaccines. Using 2-DE and subsequent MALDI-TOF-TOF MS, 64 proteins were identified in the extracellular proteome, only 29 of which were predicted to be exported into the culture medium. The latter included chitinases, proteases, nucleotidases, sulfatases, phosphatases and proteins of unknown function. Of the remaining proteins in the culture medium, 18 were predicted to be associated with the cell wall or anchored on the trans side of the cytoplasmic membrane while 17 other proteins lacked identifiable export signals and were predicted to be cytoplasmic proteins. Among the S-layer proteins, Sap and Eag account for 10% of the total extracellular proteome. Many of the proteins are predicted to contribute to the virulence and antigenic signature of B. anthracis. We have also studied the composition of the cytoplasmic proteome, identifying 300 distinct proteins. The most abundant cytoplasmic proteins are primarily those involved in glycolysis, amino acid metabolism, protein translation, protein folding and stress adaptation. The presence of a variety of proteases, peptidases, peptide binding proteins, as well as enzymes required for the metabolism of amino acids, suggests that B. anthracis is adapted to life in a protein-rich environment rather than the soil. We therefore speculate that proteases and peptidases could be useful targets for the development of improved vaccines. In addition, both of these B. anthracis compartment-specific proteomes can be used as reference maps to monitor changes in the production of secreted and cytosolic proteins that occur, for example, during growth in macrophages.     

Competitive exclusion as a mode of action of a novel Bacillus cereus aquaculture biological agent

Aims: To determine the contribution of potential modes of action of a Bacillus cereus aquaculture biological control agent in inhibition of the fish pathogen, Aeromonas hydrophila. Methods and Results: When B. cereus was tested in plate well inhibition studies, no production of antimicrobial compounds was detected. Bacillus cereus had a high growth rate (0·96 h^?1), whereas Aer. hydrophila concentration decreased by c. 70% in co‐culture experiments. In nutrient limitation studies, B. cereus had a significantly higher growth rate when cultured under glucose (P < 0·05) and iron (P < 0·01) limitation in comparison with Aer. hydrophila. Bacillus cereus glucose (0·30 g l^?1 h^?1) and iron (0·60 mg l^?1 h^?1) uptake rates were also significantly higher (P < 0·01) than the Aer. hydrophila glucose (0·14 g l^?1 h^?1) and iron (0·43 mg l^?1 h^?1) uptake rates. Iron uptake was facilitated by siderophore production shown in time profile studies where relative siderophore production was c. 60% through the late exponential and sporulation phases. Conclusions: Competitive exclusion by higher growth rate, competition for organic carbon and iron, facilitated by siderophore production, could be identified as mechanisms of pathogen growth inhibition by B. cereus. Significance and Impact of the Study: This study is the first elucidation of the mechanism of action of our novel B. cereus biological agent in growth attenuation of pathogenic Aer. hydrophila. This study enhances the application knowledge and attractiveness for adoption of B. cereus NRRL 100132 for exploitation in aquaculture.     

Purification and Properties of a Galacturonic Acid-releasing Exopolygalacturonase from a Strain of Bacillus

An exopolygalacturonase [exo-PGase; poly (1,4-α-D-galacturonide) galacturonohydrolase, EC 3.2.1.67] was found to be extracellularly produced by Bacillus sp. strain KSM-P443. The exo-PGase was purified to homogeneity, as judged by polyacrylamide gel electrophoresis, through sequential column chromatographies. The enzyme had a molecular weight of approximately 45,000 and an isoelectric point of pH 5.8. The N-terminal sequence was Ser-Met-Gln-Lys-Ile-Lys-Asp-Glu-Ile-Leu-Lys-Thr-Leu-Lys-Val-Pro-Val-Phe and had no sequence similarity to those of other petinolytic enzymes reported to date. Maximum activity toward polygalacturonic acid (PGA) was observed at 60°C and at pH 7.0 in 100 mM Tris-HCl buffer without requiring any metal ions. When the chain length of oligogalacturonic acids increased, the apparent Km for them decreased, but the kcat values increased. This is the first bacterial exo-PGase that releases exclusively mono-galacturonic acid from PGA, di-, tri-, tetra-, and penta-galacturonic acids.     

Bacillus pumilus WL-11木聚糖酶A的纯化、鉴定及其底物降解方式

对一株BacilluspumilusWL_11木聚糖酶的纯化、酶学性质及其底物降解模式进行了研究。经过硫酸铵盐析、CM_Sephadex及SephadexG_75层析分离纯化,获得一种纯化的WL_11木聚糖酶A ,其分子量为2 6 0kD ,pI值9 5 ,以燕麦木聚糖为底物时的表观Km 值为16 6mg mL ,Vmax值为12 6 3μmol (min·mg)。木聚糖酶A的pH稳定范围为6 0至10 4 ,最适作用pH范围则在7 2至8 0之间,是耐碱性木聚糖酶;最适作用温度为4 5℃～5 5℃,在37℃、4 5℃以下时该酶热稳定性均较好;5 0℃保温时,该酶活力的半衰期大约为2h ,在超过5 0℃的环境下,该酶的热稳定较差,5 5℃和6 0℃时的酶活半衰期分别为35min和15min。WL_11木聚糖酶A对来源于燕麦、桦木和榉木的可溶性木聚糖的酶解结果发现,木聚糖酶A对几种不同来源的木聚糖的降解过程并不一致。采用HPLC法分析上述底物的降解产物生成过程发现木聚糖酶A为内切型木聚糖酶,不同底物的降解产物中都无单糖的积累,且三糖的积累量都较高;与禾本科的燕麦木聚糖底物降解不同的是,木聚糖酶A对硬木木聚糖降解形成的五糖的继续降解能力较强。采用TLC法分析了WL_11粗木聚糖酶降解燕麦木聚糖的过程,结果表明燕麦木聚糖能够被WL_11粗木聚糖酶降解生成系列木寡糖,未检出木糖,这说明WL_11主要合成内切型木聚     

产纤溶酶枯草杆菌液体发酵工艺优化研究

采用单因子实验和正交实验对高产纤溶酶的枯草杆菌最佳发酵工艺进行了优化,结果表明,该菌株分泌的胞外酶具有较强的体外溶栓作用,产纤溶酶最佳的发酵条件为：3％可溶性淀粉,2％豆浆全汁(鲜豆),0．02％CaCl2,培养温度为37℃,初始pH8．3,装液量为250mL三角瓶50mL,发酵时间44h左右,Ca^2 、Mg^2 和Mn^2 对酶活力有促进作用,Cu^2 对酶活性有强烈抑制作用。     

Processing of Cry1Ab delta-endotoxin from Bacillus thuringiensis by Manduca sexta and Spodoptera frugiperda midgut proteases: role in protoxin activation and toxin inactivation

Activation of Cry protoxins is carried out by midgut proteases. This process is important for toxicity and in some cases for specificity. Commercial proteases have been used for in vitro protoxin activation. In the case of Cry1A protoxins, trypsin digestion generates a toxic fragment of 60–65 kDa. Here, we have analyzed the in vitro and in vivo activation of Cry1Ab. We found differences in the processing of Cry1Ab protoxin by Manduca sexta and Spodoptera frugiperda midgut proteases as compared to trypsin. Midgut juice proteases produced two additional nicks at the N-terminal end removing helices 1 and 2a to produce a 58 kDa protein. A further cleavage within domain II splits the toxin into two fragments of 30 kDa. The resulting fragments were not separated, but instead coeluted with the 58 kDa monomer, in size-exclusion chromatography. To examine if this processing was involved in the activation or degradation of Cry1Ab toxin, binding, pore formation, and toxicity assays were performed. Pore formation assays showed that midgut juice treatment produced a more active toxin than trypsin treatment. In addition, it was determined that the 1 helix is dispensable for Cry1Ab activity. In contrast, the appearance of the 30 kDa fragments correlates with a decrease in pore formation and insecticidal activities. Our results suggest that the cleavage in domain II may be involved in toxin inactivation, and that the 30 kDa fragments are stable intermediates in the degradation pathway.     

枯草芽孢杆菌B1-41对小麦纹枯病菌的抑制作用

从小麦根际土壤分离得到枯草芽孢杆菌(Bacillus subtilis)B1-41拮抗菌株,室内测定其带菌培养液对小麦纹枯病菌(Rhizoctonia cerealis)抑制效果为72%,盆栽试验中,其防治效果为60.3%,高于井冈霉素处理的51%的效果。试验表明,该菌株可以造成病菌菌丝发生畸变和菌丝细胞壁瓦解。     

情绪调节时间知觉的作用方式及认知神经机制

人类的时间知觉会受到情绪的调节发生主观扭曲,这种调节可以通过情绪的体验与预期来实现.本文区分了体验性情绪和预期性情绪调节时间知觉的作用方式和认知机制,基于标量计时理论,提出了在不同加工阶段情绪调节时间知觉的认知理论模型,并梳理了情绪调节时间知觉的神经生理学基础和脑机制方面的证据.未来研究需更关注预期性情绪对时间知觉的调节作用,考察注意、唤醒和效价等因素的交互影响,并进一步探究情绪调节时间知觉的神经机制.