Stoichiometric growth model for riboflavin-producing Bacillus subtilis

Rate equations for measured extracellular rates and macromolecular composition data were combined with a stoichiometric model to describe riboflavin production with an industrial Bacillus subtilis strain using errors in variables regression analysis. On the basis of this combined stoichiometric growth model, we explored the topological features of the B. subtilis metabolic reaction network that was assembled from a large amount of literature. More specifically, we simulated maximum theoretical yields of biomass and riboflavin, including the associated flux regimes. Based on the developed model, the importance of experimental data on building block requirements for maximum yield and flux calculations were investigated. These analyses clearly show that verification of macromolecular composition data is important for optimum flux calculations.     

枯草芽孢杆菌代谢工程改造的策略与工具

作为一种食品安全级的典型工业模式微生物,枯草芽孢杆菌Bacillus subtilis由于具有非致病性、胞外分泌蛋白能力强以及无明显的密码子偏爱性等特点,现已被广泛应用于代谢工程领域。近年来,随着分子生物学和基因工程技术等的迅速发展,多种研究策略和工具被用于构建枯草芽孢杆菌底盘细胞进行生物制品的高效合成。文中从启动子工程、基因编辑、基因回路、辅因子工程以及途径酶组装等方面介绍枯草芽孢杆菌在代谢工程领域的研究历程,并总结其在生物制品生产中的相关应用,最后对其未来的研究方向进行展望。     

一种快速分离检测产脂肽类生物表面活性剂枯草芽孢杆菌的方法

脂肽(Lipopeptide)是由枯草芽孢杆菌(Bacillus subtilis)等微生物产生的一类具有较强表面活性的生物表面活性剂.枯革杆菌磷酸泛酰巯基转移酶基因(afp)是枯草芽孢杆菌中参与脂肽代谢的功能性基因.采用sfp基因PCR对从环境中得到的一组产生表面活性剂的微生物进行筛选,结合Tricine-SDS-PAGE电泳对PCR结果呈阳性的菌蛛的代谢粗初提物进行检测,初步鉴定得到两株枯草芽孢杆菌.进一步利用16S rDNA序列的系统发育学分析确定这两种菌株为枯草芽孢杆菌,并利用TLC、HPLC鉴定其产物为脂肽类表面活性剂,从而建立了一套快速分离检测产生脂肽类生物表面活性剂的枯草芽孢杆菌方法.     

Self-assembly product formation of the Bacillus stearothermophilus PV72/p6 S-layer protein SbsA in the course of autolysis of Bacillus subtilis

In order to achieve high level expression and to study the release of a protein capable of self-assembly, the gene encoding the crystalline cell surface (S-layer) protein SbsA of Bacillus stearothermophilus PV72/p6, including its signal sequence, was cloned and expressed in Bacillus subtilis. To obtain high level expression, a tightly regulated, xylose-inducible, stably replicating multicopy-plasmid vector was constructed. After induction of expression, the S-layer protein made up about 15% of the total cellular protein content, which was comparable to the SbsA content of B. stearothermophilus PV72/p6 cells. During all growth stages, SbsA was poorly secreted to the ambient cellular environment by B. subtilis. Extraction of whole cells with guanidine hydrochloride showed that in late stationary growth phase cells 65% of the synthesised SbsA was retained in the peptidoglycan-containing layer, indicating that the rigid cell wall layer was a barrier for efficient SbsA secretion. Electron microscopic investigation revealed that SbsA release from the peptidoglycan-containing layer started in the late stationary growth phase at distinct sites at the cell surface leading to the formation of extracellular self-assembly products which did not adhere to the cell wall surface. In addition, intracellular sheet-like SbsA self-assembly products which followed the curvature of the cell became visible in partly lysed cells. Intracellularly formed self-assembly products remained intact even after complete lysis of the rigid cell envelope layer.     

枯草芽孢杆菌蛋白质分泌机制研究进展

综述了枯草芽孢杆菌不同蛋白质分泌机制,重点讨论了大多数细菌蛋白分泌的Sec途径,包括Sec途径的信号肽,信号肽酶,SecYEG通道,与分泌有关的各种细胞因子以及Sec途径的限制因素,此外还简要讨论了Tat途径,该途径能够转运折叠迅速或归密的蛋白质。     

巨大芽孢杆菌青霉素G酰化酶基因在枯草杆菌中的高表达

用ＰＣＲ方法从巨大芽孢杆菌的基因组ＤＮＡ中扩增到青霉素Ｇ酰化酶基因,并装载到枯草杆菌质粒ｐＰＺＷ１０３中,将其转化到枯草杆菌ＤＢ１０４中进行了分泌表达,重组菌株产酶无需苯乙酸诱导。在３７℃培养２４ｈ,菌液酶活力可达６ｕ／ｍｌ。１０天的连续传代实验表明重组菌株的稳定性很高。     

同源重组法构建枯草芽孢杆菌转酮酶缺失突变菌株

采用同源重组法高效构建枯草芽孢杆菌转酮酶(tkt)缺失突变株。以大肠杆菌(E.coliDH5α)质粒pBlUSKM为框架,构建出基于枯草杆菌(B.S104)tkt基因位点的整合载体pb-Trs-n,将此载体重组到Bacillus subtilis104中,从新霉素抗性平板上挑取转化子,整合载体pb-Trs-n的测序结果与Kunst.F报道的tkt基因高度同源(98.9%)。同源重组后,B.S104染色体上的tkt基因(2 004bp)部分与载体pb-Trs-n的neo基因(1 197bp)发生了同源交换,确定了该转化子为枯草芽孢杆菌转酮酶缺失突变株(tkt-,neo),该方法构建枯草芽孢杆菌转酮酶(tkt)缺失突变株是可行的,为D-核糖工程菌的研究奠定了基础。     

P45, an extracellular 45 kDa protein of Listeria monocytogenes with similarity to protein p60 and exhibiting peptidoglycan lytic activity

A monoclonal antibody obtained by immunization of mice with heat-killed cells of Listeria monocytogenes serotype 4d showed reactivity towards a protein (P45) from L. monocytogenes with an apparent molecular mass of 45 kDa. This protein was detected in the culture supernatant and at the cell surface of L. monocytogenes. Proteins cross-reacting with the monoclonal antibody were present in all Listeria strains investigated, except L. grayi. The structural gene was cloned in Escherichia coli and sequenced. Translation of the gene starts at a TTG initiation codon. The gene was found to code for a protein of 402 amino acid residues with a predicted molecular mass of 42.7 kDa. It has a signal peptide of 27 amino acid residues, resulting in a molecular mass for the mature polypeptide of 39.9 kDa. Protein database searches showed that this protein has 55% similarity and 38% identity to protein p60 of L. monocytogenes and exhibits significant sequence similarities to p54 from Enterococcus faecium and Usp45 from Lactococcus lactis. P45 was shown to have peptidoglycan lytic activity and the encoding gene was named spl (secreted protein with lytic property). Revision received: 11 August 1999 / Accepted: 8 September 1999     

Mutations affecting hemolysin production in Listeria monocytogenes located outside the listeriolysin gene

We have investigated the molecular basis of spontaneous mutations leading to non-hemolytic and avirulent variants of the Listeria monocytogenes serotype 1/2a strain NCTC 7973 using Southern hybridization to DNA fragments that harbor the listeriolysin gene (hlyA) and adjacent regions cloned from a L. monocytogenes serotype 1/2a strain. The analysis of such non-hemolytic variants revealed the presence of a deletion of 300 base pairs, located 1.6 kb upstream of an otherwise intact listeriolysin gene. The importance of regions upstream of the hlyA gene in controlling the expression of the listeriolysin gene was further emphasized by the detection of a transposon-derived nonhemolytic mutant in which the transposon had inserted approximately 200 bp upstream of the listeriolysin gene. We conclude that at least two elements, contained within a region encompassing 1.6 kb of sequences upstream of the hlyA gene, may be required for expression of the listeriolysin gene.     

Cloning in Bacillus subtilis of temperature-dependent promoter fragments from Bacillus stearothermophilus and Bacillus subtilis

Abstract Using promoter-probe plasmids, more than 200 promoter-containing fragments from Bacillus stearothermophilus and Bacillus subtilis were cloned in B. subtilis . Among these, 15 promoter fragments were highly temperature-dependent in activity compared to the promoter sequence (TTGAAA for the −35 region, TATAAT for the −10 region) of the amylase gene, amyT , from B. stearothermophilus . Some fragments exhibited higher promoter activities at elevated temperature (48°C), others showed higher activities at lower temperature (30°C). Active promoter fragments at higher and lower temperatures were obtained mainly from the thermophile ( B. stearothermophilus ) and the mesophile ( B. subtilis ), respectively. A promoter fragment active at high temperature was sequenced, and the feature of the putative promoter region was discussed.     

Co-linear scaffold of the Bacillus licheniformis and Bacillus subtilis genomes and its use to compare their competence genes

We have established the co-linear regions of Bacillus licheniformis, an industrially important bacterium, and Bacillus subtilis, a model bacterium. In the co-linear regions, revealed by PCR, gene content and order are presumed to be conserved. These regions constitute approximately 60% of the compared chromosomes. Sequencing of the competence genes of B. licheniformis allowed us to validate the approach, and to demonstrate how it can be used for the comparative analysis of complex genetic systems. A new insertion sequence, designated IS3Bli1, was discovered in the competence region of the analyzed B. licheniformis strain.     

1株枯草芽胞杆菌体外拮抗6种肠道致病菌的研究

目的研究枯草杆菌BS-3株对大肠埃希菌等6种肠道致病菌的拮抗作用。方法通过在体外BS-3菌株分别与大肠埃希菌等6种致病菌混合培养后,观察不同时间内各菌的菌量变化。结果BS-3菌株与6种肠道致病菌混合培养24、48、72和96h,其菌量逐渐增加;6种致病菌的菌量随着培养时间延续逐渐减少,其中产毒性大肠埃希菌、致病性大肠埃希菌和宋内志贺菌与对照组比较差异更明显。结论BS3菌株在培养生长过程中,可抑制大肠埃希菌等6种肠道致病菌的生长。     

枯草芽孢杆菌抗脯氨酸结构类似物突变株的筛选及突变株proBA基因的克隆和序列分析

利用亚硝基胍对枯草芽孢杆菌93151进行诱变处理,获得了耐NaCl浓度达14％的突变株,同时发现该突变株也是一个抗脯氨酸反馈抑制突变菌株,其胞内自由脯氨酸的含量随着盐浓度的提高显著增加,说明其对渗透压的耐受能力与胞内自由脯氨酸的含量紧密相关。利用PCR方法克隆突变株的proBA基因,得到一个约2.3kb的DNA片段,序列分析表明该片段含有一完整的proB基因和部分proA基因,与野生菌株的proB基因相比,突变株proB基因中有3个碱基发生了改变,其中一个碱基的变化（从起始密码子开始第781位由T→A）导致了一个氨基酸发生改变（Ser→Thr),另外两个碱基变化为沉默位点突变。将该proB基因转入大肠杆菌脯氨酸营养缺陷型菌株,能够与其功能互补。同时对部分proA基因序列分析发现,其与proB基因头尾重叠。在proA基因起始密码子上游第14个碱基处有一个类似于SD的序列,其所编码的氨基酸序列与枯草芽孢杆菌168的同源性为77％。     

枯草杆菌ylyA基因的绿色荧光蛋白标记

[目的]本研究对枯草杆菌ylyA基因进行荧光标记以便对其产物YlyA在菌体中的位置进行初步观察.[方法]以不同菌株基因组DNA为模板,对ylyA基因进行PCR扩增和序列分析;重新设计引物扩增全长的ylyA并将其克隆到载体pSG1729中,形成gfpmut1-ylyA融合而构建重组载体pNG426;将pNG426转化枯草杆菌168菌株,双交换使gfpmut1-ylyA插入染色体的amyE位点,用碘染色法和菌落PCR对阳性转化子BS363进行鉴定.NA固体培养基上生长的BS363经0.5%木糖诱导表达后,利用表面荧光显微镜技术进行观察.[结果]通过对多个PCR产物的序列分析确定了ylyA基因的正确序列以及正确的翻译起始位点;成功将重组载体pNG426转化枯草杆菌得到了BS363菌株;荧光检测结果表明GFP标记的YlyA分布于菌体的外周,在位置上靠近细胞膜并与之平行排列.[结论]生长缓慢的BS363菌体,在0.5%木糖诱导下产生的荧光标记YlyA蛋白分布在细胞外周,可能在膜生物学中发挥作用.     

透明颤菌血红蛋白基因在提高枯草芽孢杆菌产脂肽功能中的研究

脂肽是一类具有特殊作用的生物表面活性剂。本实验将血红蛋白基因(vhb)置于RDR细菌启动子驱动下的质粒PSET中,构建PSET-RDR-vhb重组质粒,并通过电激作用转入脂肽代谢菌株-枯草芽孢杆菌株(bacillus subtilis)ZW-3中,转化菌株经酶切和PCR电泳检测鉴定,Southern-blot杂交显示部分转化菌株中外源基因插入基因组DNA,采用一氧化碳差异色谱法测定了血红蛋白的表达量。实验进一步对转化菌株的生长曲线、总蛋白量、过氧化氢酶活性、脂肽的产率进行了测定,结果显示,相比于原始菌株,转化菌株数据均有明显提高。     

Diversity of the growth patterns of Bacillus subtilis colonies on agar plates

Abstract A Bacillus subtilis strain showed a variety of colony growth patterns on agar plates. The bacterium grew to a fractal colony through the diffusion-limited aggregation process, a round colony reminiscent of the Eden model, a colony with a straight and densely branched structure similar to the dence branching, morphology, a colony spreading without any openings, and a colony with concentric rings, on plates with various agar and nutrient concentrations. The microstructures of these colonies were also characteristic and dynamic. The patterns of these bacterial colonies were thought to grow in relation to the diffusion of nutrient in the agar plate.     

Isolation of glucosidase and phospholipase positive Bacillus circulans on ALOA medium

Aim: To report the growth of glucosidase and phospholipase positive bacteria on agar Listeria according to Ottaviani and Agosti (ALOA) different from Listeria monocytogenes, Listeria ivanovii and Bacillus cereus. Methods and Results: Raw water‐buffalo milk was analysed according to EN ISO 11290. Streaking of Fraser broth on ALOA resulted in green colonies with an opaque halo after 48 h at 30°C. Colonies were transferred onto Tryptone soya yeast extract agar and showed cultural characteristics atypical for L. monocytogenes. Results of confirmation tests according to EN ISO 11290 method: negative haemolysis test, weak positive camp test in correspondence with Staphylococcus aureus, no fermentation of rhamnose, fermentation of xylose. Gram staining showed tapered, curved, Gram‐positive rods with subterminal to terminal ellipsoidal spores, 0·5–0·7 μm diameter 4–5 μm. API 50CH CHB kit (99·9% percentage of identification) and the sequence of the 833 bp PCR product (portion of 16S rRNA, generic primers 1492‐r; p27‐f) showed 97% identity with Bacillus circulans ATCC 4513 (GenBank AY724690 ). Conclusions: Some B. circulans strains can grow on ALOA, according to ISO 11290, confirmation tests readily differentiate B. circulans from L. monocytogenes. Significance and Impact of the Study: The different morphology of the colonies must be kept in mind to select true L. monocytogenes for confirmation test and to avoid overestimation of L. monocytogenes count.     

蛋白激发子PeaT1在枯草芽胞杆菌中的分泌表达及重组菌株提高小麦抗旱和促生的作用

PeaT1是从极细链格孢菌Alternaria tenuissima中分离的一种蛋白激发子,具有促进植物生长和诱导植物产生系统获得抗性的功能,为了实现peaT1基因在枯草芽胞杆菌Bacillus subtilis中的分泌表达,增加其应用途径,从枯草芽胞杆菌基因组DNA中分别扩增获得P43启动子和nprB基因的信号肽序列,并用SOE (Splicing by over lapping extension) 方法与peaT1基因连接,将连接产物克隆到大肠杆菌-枯草芽胞杆菌穿梭表达载体pHY300-PLK上,构建了重组表达载体pHY43N-peaT1。将重组载体转化枯草芽胞杆菌WB800菌株,SDS-PAGE和Western blotting分析证实,在NprB信号肽的引导下,枯草芽胞杆菌成功分泌表达了PeaT1蛋白。构建的重组菌株能够显著增强幼苗抗旱性,提高小麦株高。