短小芽孢杆菌degQ基因的克隆与鉴定

degQ基因编码一个由46个氨基酸组成的多肽,能增强许多芽孢杆菌胞外酶基因的表达．以pMK4作克隆载体构建短小芽孢杆菌基因文库,并用DNA探针原位杂交法从中钓出degQ基因．对克隆基因的DNA序列进行了分析并证明克隆的短小芽孢杆菌degQ基因具有增强枯草杆菌蛋白酶和果聚糖蔗糖酶基因表达的能力．degQ基因克隆有助于研究芽孢杆菌的正调控机理并可望提高外源基因在芽孢杆菌中表达．     

短小芽孢杆菌degQ基因的克隆与鉴定

ｄｅｇＱ基因编码一个由４６个氨基酸组成的多肽,能增强许多芽孢杆菌胞外酶基因的表达,以ＰＭＫ４作克隆体构建短小芽孢杆菌基因库,并用ＤＮＡ探针原位杂次法从中钓出ｄｅｇＱ基因,对克隆基因的ＤＮＡ序列进行了分析并证明克隆的短小芽孢杆菌ｄｅｇＱ基因具有增强枯草杆菌蛋白酶和果聚糖蔗糖酶基因表达的能力,ｄｅｇＱ基因克隆有助于研究芽孢杆菌的正调控机一并可望提高外源基因在芽孢杆菌中表达。     

耐碱性木聚糖酶基因在巨大芽孢杆菌中的表达及其酶学性质

摘要：【目的】从耐碱性木聚糖酶高产短小芽孢杆菌中克隆得到带有自身启动子的木聚糖酶基因,将其在巨大芽孢杆菌中进行表达,并对表达产物进行性质分析。【方法】将克隆得到的木聚糖酶基因xynA以及带有自身启动子序列的结构基因, 构建在芽孢杆菌表达载体pWH1520和改造后的载体pWG03中,得到重组质粒pWTEJX和pWGXYN,分别转化到巨大芽孢杆菌BM70中,获得重组巨大芽孢杆菌BMJXH9和BMGpp12;经过诱导产酶培养,均得到分泌表达。【结论】重组巨大芽孢杆菌BMGpp12比BMJXH9产酶活力提高了三倍     

球形芽胞杆菌C3-41磷酸果糖激酶的克隆、表达及基本生物活性

[目的]球形芽孢杆菌缺乏EMP、HMP、ED途径的关键酶,如磷酸果糖激酶等被认为是其不能以糖类物质进行生长的主要原因.杀蚊球形芽孢杆菌C3-41全基因组序列分析表明,在染色体DNA上存在的磷酸果糖激酶基因pfk,为了进一步分析球形芽孢杆菌糖酵解途径,进一步确定磷酸果糖激酶在糖酵解途径中的功能.[方法]通过pfk基因在球形芽孢杆菌菌株中的Southern-blot拷贝数鉴定,在C3-41pfk基因克隆的基础上进行pfk基因在大肠杆菌中的融合表达、序列分析和序列比对等方法进行研究.[结果]证明了球形芽孢杆菌pfk基因由960 bp核苷酸组成,表达42 kDa的PFK融合蛋白,有保守的底物结合域和ATP结合域,同时pfk基因重组表达质粒可以回复大肠杆菌pfk缺陷型菌株DFl020代谢糖的能力.[结论]杀蚊球形芽孢杆菌C3-41的pfk表达产物具有磷酸果糖激酶活性,为今后深入研究球形芽孢杆菌产能代谢机理奠定了基础.     

猪圆环病毒衣壳蛋白在枯草芽孢杆菌中的表达研究

研究猪圆病毒衣壳蛋白在枯草芽孢杆菌中的表达情况。以3个不同的质粒为基础,构建不同类型的猪圆环病毒衣壳蛋白基因(PCV2-ORF2)重组表达载体,并分别转化进入枯草芽孢杆菌168和WB800中。利用SDS-PAGE和Western blot检测目的蛋白的表达,并通过实时荧光定量PCR检测目的基因的转录水平上的表达情况。结果显示,ORF2基因原序列在枯草芽孢杆菌中难以表达,经过密码子优化后,构建的截短ORF2基因表达载体在枯草芽孢杆菌内表达系统中成功表达目的蛋白。密码子优化促进了目的基因的表达。     

短小芽孢杆菌实时荧光定量PCR分析中内参基因的筛选

为了利用荧光定量PCR方法分析短小芽孢杆菌的基因表达水平,需要首先确定适合该菌株的荧光定量PCR分析内参基因。以短小芽孢杆菌的16S rRNA、mecA、cadR、rpoB及sphP共5个基因作为候选内参基因,利用实时荧光定量PCR的方法分析这5个候选基因在短小芽孢杆菌发酵培养不同时间点的表达情况,再用geNorm和NormFinder软件评估它们的表达稳定性。结果显示,利用geNorm软件分析得出16S rRNA和mecA是表达最稳定的基因,最适内参基因数为2。NormFindr软件分析得出mecA为最稳定的基因。对短小芽孢杆菌3个功能基因的差异表达分析结果表明,16S rRNA和mecA都是合适的内参基因,而mecA基因由于表达丰度适中,更适合于作为内参基因研究结构基因的表达。为了得到更准确的差异表达结果,也可用两个基因同时进行校正。     

苏云金芽孢杆菌芽孢萌发相关基因gerM的克隆及功能研究

依照蜡状芽孢杆菌gerM基因的保守序列设计引物,从苏云金芽孢杆菌中扩增出640bp的DNA片段。以此为探针,从苏云金芽孢杆菌部分基因组酶切文库中成功地克隆到了一个4·5kb的DNA片段。序列分析表明,该片段包含一个完整的开放阅读框,其预测的编码产物与枯草芽孢杆菌GerM蛋白具有很高的同源性,将该基因命名为gerM。RT-PCR分析表明,gerM基因仅在芽孢形成的过程中表达。通过同源重组的策略构建了gerM基因的阻断突变株。研究表明,gerM基因的破坏影响苏云金芽孢杆菌芽孢萌发的速率和比例。     

利用枯草芽孢杆菌ytkA和ywoF基因启动子与信号肽重组分泌表达mpd基因

用大肠杆菌-枯草芽孢杆菌穿梭载体pNW33N和去除了信号肽编码序列的成熟mpd基因构建了穿梭启动子探针pNW33N-mpd。用该探针从质粒pMPDP3和pMPDP29上克隆来自于枯草芽孢杆菌ytkA和ywoF基因上游的启动子功能片段,构建了穿梭表达载体pNYTM和pNYWM。将表达载体pNYTM和pNYWM转入枯草芽孢杆菌1A751获得表达菌株1A751(pNYTM)和1A751(pNYTM),mpd基因在ytkA和ywoF基因的启动子和信号肽的带动下实现了分泌表达且具有天然活性,结果表明ytkA基因的启动子强度强于ywoF基因的启动子。利用ytkA基因的强启动子和nprB基因的分泌型信号肽编码序列构建了新的穿梭分泌表达载体pYNMK,并使mpd基因在枯草芽孢杆菌WB800中得到了更高水平的分泌表达,表达菌株WB800(pYNMK)在培养到第84h时甲基对硫磷水解酶酶活达到最高值为10.40u/mL,是出发菌株邻单胞菌M6表达量的10.8倍,重组表达产物有91.4%分泌在培养基中。     

枯草芽孢杆菌中一种新型双向启动子的功能鉴定

本研究旨在通过转录组分析预测的方法,由地衣芽孢杆菌中筛选获得一种新型双向启动子,鉴定其启动强度。以已知强组成型启动子pShuttle-09为对照,检测其对克劳氏芽孢杆菌碱性蛋白酶基因的表达活性。成功构建了3种重组碱性蛋白酶表达载体及对应的工程菌株。在新型启动子pLA和其反向启动子pLB调控转录下,克劳氏芽孢杆菌碱性蛋白酶表达活性达到164 U/mL和111 U/mL。结果表明,pLA的启动强度明显高于pShuttle-09和pLB,pLA启动子与pLB启动子均可表达碱性蛋白酶。从而为枯草芽孢杆菌表达系统中异源基因的表达提供一个新的方向,也为原核生物中共同表达两种基因提供了新的思路。     

β-半乳糖苷酶在枯草芽孢杆菌中的分泌表达

为检测本课题组构建的枯草芽孢杆菌分泌表达载体pGPST中启动子和信号肽的活性,将β-半乳糖苷酶基因插入表达载体的多克隆位点,构建含有β-半乳糖苷酶基因的重组质粒pGPST-lacZ,重组质粒转化枯草芽孢杆菌,分别测定液体培养基中上清液和菌体沉淀中β-半乳糖苷酶的活性。培养上清液中的酶活在22h达到峰值,约为26mU/mL,之后开始下降;菌体沉淀中的酶活在14h最高,达6mU/mL,而阴性对照pGPST没有检测到酶活性。结果表明分泌型表达载体中的启动子和信号肽具有较好的活性,能实现异源基因在枯草芽孢杆菌中的分泌表达。本试验为该表达载体的进一步研究和应用提供重要的数据资料,也为外源基因在枯草芽孢杆菌中的表达提供参考资料。该载体将在研究外源基因在枯草芽孢杆菌中表达发挥重要作用。     

Cloning, sequencing, and characterization of the Bacillus subtilis biotin biosynthetic operon.

A 10-kb region of the Bacillus subtilis genome that contains genes involved in biotin-biosynthesis was cloned and sequenced. DNA sequence analysis indicated that B. subtilis contains homologs of the Escherichia coli and Bacillus sphaericus bioA, bioB, bioD, and bioF genes. These four genes and a homolog of the B. sphaericus bioW gene are arranged in a single operon in the order bioWAFDR and are followed by two additional genes, bioI and orf2. bioI and orf2 show no similarity to any other known biotin biosynthetic genes. The bioI gene encodes a protein with similarity to cytochrome P-450s and was able to complement mutations in either bioC or bioH of E. coli. Mutations in bioI caused B. subtilis to grow poorly in the absence of biotin. The bradytroph phenotype of bioI mutants was overcome by pimelic acid, suggesting that the product of bioI functions at a step prior to pimelic acid synthesis. The B. subtilis bio operon is preceded by a putative vegetative promoter sequence and contains just downstream a region of dyad symmetry with homology to the bio regulatory region of B. sphaericus. Analysis of a bioW-lacZ translational fusion indicated that expression of the biotin operon is regulated by biotin and the B. subtilis birA gene.     

The genes encoding the biotin carboxyl carrier protein and biotin carboxylase subunits of Bacillus subtilis acetyl coenzyme A carboxylase, the first enzyme of fatty acid synthesis.

The genes encoding two subunits of acetyl coenzyme A carboxylase, biotin carboxyl carrier protein, and biotin carboxylase have been cloned from Bacillus subtilis. DNA sequencing and RNA blot hybridization studies indicated that the B. subtilis accB homolog which encodes biotin carboxyl carrier protein, is part of an operon that includes accC, the gene encoding the biotin carboxylase subunit of acetyl coenzyme A carboxylase.     

Expression of the biotin biosynthetic operon of Escherichia coli is regulated by the rate of protein biotination

In Escherichia coli biotin biosynthesis is repressed by high concentrations of exogenous biotin. This paper reports that upon high level production of the apo form of a biotinated protein, biotin operon expression was derepressed by 8-10-fold. The biotinated protein studied was the 1.3 S subunit of Propionibacterium shermanii, and transcarboxylase derepression was assayed by beta-galactosidase production in strains which carry a lacZ gene altered such that it is transcribed from biotin operon promoters. Depression of beta-galactosidase synthesis upon production of the apo 1.3 S protein was observed over a several hundred-fold range of biotin concentrations and also resulted in an increased level of biotin operon expression at maximally repressing biotin concentrations. Biotin operon derepression by apobiotin protein production seems a direct consequence of the properties of the biotin repressor protein which also functions as the ligase catalyzing the covalent attachment of biotin to apoproteins.     

Genetic analysis of an incomplete bio operon in a biotin auxotrophic strain of Bacillus subtilis natto OK2

We describe the genetic analysis of the bio operon of the biotin auxotrophic Bacillus subtilis natto OK2 strain. The OK2 strain would only cross-feed with the Escherichia coli bioB mutant and also grew well in medium containing dethiobiotin. Sequencing analysis revealed two significant genetic alterations in the bioW and bioF genes within the bio operon of the OK2 strain. Complementation analysis with B. subtilis 168 bio mutants demonstrated that only the bioB gene could complement, but other bio operon genes could not. A bio(+) transformant, isolated from an OK2 strain, has biotin autotrophy.     

Use of bio-lac fusion strains to study regulation of biotin biosynthesis in Escherichia coli.

The technique developed by Casadaban (M. J. Casadaban, J. Mol. Biol. 104: 541-555, 1976) has been employed to construct Escherichia coli K-12 derivatives in which the genes determining lactose utilization are fused to the regulatory region of the biotin operon. Fusions of the lac genes to either arm of this divergently transcribed operon have been isolated. When the operon is derepressed, expression of the lac genes is sufficient to permit growth on lactose minimal medium. Repressing conditions prevent growth on lactose. This property of bio-lac fusion strains, as well as the ease of determining the level of operon expression by assaying beta-galactosidase, was used for the isolation and characterization of mutants defective in repression. Preliminary analyses of several newly isolated regulatory mutants are presented. For the several birA mutants examined, there appeared to be no direct correlation between effects on minimum biotin requirement and alterations in repressibility, suggesting a possible dual function for the gene. Parallel attempts to obtain fusions of lac to bioH were unsuccessful, indicating lack of direct biotin control at the bioH locus.     

Molecular characterization of Lactobacillus plantarum genes for beta-ketoacyl-acyl carrier protein synthase III (fabH) and acetyl coenzyme A carboxylase (accBCDA), which are essential for fatty acid biosynthesis

Genes for subunits of acetyl coenzyme A carboxylase (ACC), which is the enzyme that catalyzes the first step in the synthesis of fatty acids in Lactobacillus plantarum L137, were cloned and characterized. We identified six potential open reading frames, namely, manB, fabH, accB, accC, accD, and accA, in that order. Nucleotide sequence analysis suggested that fabH encoded beta-ketoacyl-acyl carrier protein synthase III, that the accB, accC, accD, and accA genes encoded biotin carboxyl carrier protein, biotin carboxylase, and the beta and alpha subunits of carboxyltransferase, respectively, and that these genes were clustered. The organization of acc genes was different from that reported for Escherichia coli, for Bacillus subtilis, and for Pseudomonas aeruginosa. E. coli accB and accD mutations were complemented by the L. plantarum accB and accD genes, respectively. The predicted products of all five genes were confirmed by using the T7 expression system in E. coli. The gene product of accB was biotinylated in E. coli. Northern and primer extension analyses demonstrated that the five genes in L. plantarum were regulated polycistronically in an acc operon.