黄色粘球菌发育阶段外膜蛋白表达的变化

【目的】黄色粘球菌是研究原核发育的一种模式生物,对其膜蛋白的研究仍然十分缺乏。【方法】利用6种预测软件,在黄色粘球菌的基因组中筛选编码外膜蛋白(OMP)的基因。根据报告基因lacZ,检测这些基因在营养性生长和发育阶段的表达。【结果】基于生物信息学分析,筛选出11个编码外膜蛋白的基因。其中2个基因(MXAN3106和MXAN3883)在发育阶段表达量上升,它们分别编码Secretin家族和Fimbrial usher protein (FUP)家族转运蛋白。其余9个基因在发育起始阶段表达量降低或保持较低水平,它们均编码TonB依赖型受体或外排蛋白。【结论】这些数据提示,黄色粘球菌由生长到发育的转换过程,伴随着膜蛋白表达的显著变化。     

铜绿假单胞菌水解弹性蛋白能力相关基因的筛选与鉴定

【目的】研究铜绿假单胞菌弹性蛋白水解能力相关基因。【方法】应用人工Mu转座技术构建铜绿假单胞菌野生型菌株PA68的转座突变文库,从2000多个突变子中筛选得到4株弹性蛋白水解能力改变的突变子,并通过克隆及测序获得转座子插入位点侧翼的序列。将铜绿假单胞菌弹性蛋白酶结构基因lasB的转录启始区序列整合入载体pDN19lacΩ并将该重组质粒电转化入野生型菌株PA68及4个突变株中,对报告基因在不同菌株中的表达水平进行测定。【结果】发现4个突变株中Mu转座子分别插入lasA、galU、xcpZ和ptsP 4个基因。ptsP基因失活的突变株中,lasB基因的转录水平是野生型菌株的7%,xcpZ和lasA基因的失活使lasB基因的转录水平分别降低为野生株的54%和75%,galU基因的插入失活使lasB基因的转录上升了1倍。【结论】推测ptsP和galU基因很可能直接或间接地调控着弹性蛋白酶的生物合成。     

融合基因gfp-lacZ在克隆载体pGEM3Z-f(+)中高效表达

目的:将gfp基因克隆到pGEM3Z-f( )载体。方法:将PCR扩增得到的gfp基因插入到pGEM3Z-f( )质粒的SmaⅠ位点,构建重组质粒转化大肠杆菌DH5α。结果:含重组质粒的大肠杆菌在自然光下呈现黄绿色,而在涂X-gal的培养基上它不仅发出黄绿色荧光,而且还出现蓝色。结论:gfp基因的插入没有引起载体上lacZ基因的失活,而且形成的gfp-lacZ融合基因在大肠杆菌得到了表达。表达产物不仅具有GFP活性,而且保持了β-半乳糖苷酶的生物学活性。     

极端嗜盐古菌质粒pSCM201衍生穿梭表达载体的构建及应用

摘要：【目的】利用有自主知识产权的嗜盐古菌θ型复制质粒和启动子,构建在极端嗜盐古菌模式菌株西班牙盐盒菌（Haloarcula hispanica）中方便使用、功能完善的基因表达载体。【方法】以pSCM201的最小复制子为基础,通过引入莫维诺林抗性基因,大肠杆菌质粒复制子以及氨苄抗性基因,构建了一个新的嗜盐古菌-大肠杆菌穿梭载体。利用定点突变和末端补平法依次将其中多余的酶切位点去除后,再添加hsp5启动子核心序列、人工合成的多克隆位点以及蛋白纯化标签His?Tag等重要元件成功构建了嗜盐古菌表达载体pSCM307。将报告基因bgaH插入到该载体的多克隆位点中并转化H. hispanica AS2049,通过X-gal平板筛选和β-半乳糖苷酶酶活实验检测pSCM307的表达能力。【结果】pSCM307具有独立的自主复制能力,其多克隆位点方便实用,报告基因bgaH在hsp5启动子控制下实现了高效表达。【结论】成功构建了嗜盐古菌领域中第一个方便使用的基因表达载体。     

rpoS基因插入突变对铜绿假单胞菌两个吩嗪合成基因簇的调控

【目的】为了研究铜绿假单胞菌rpoS基因对吩嗪(Phenazine)合成基因簇phz1和phz2的调控方式与机制。【方法】采用抗庆大霉素基因(gentamycin resistance cassette,aacC1)插入失活的策略构建了rpoS基因突变株PA-SG;同时利用lacZ的翻译融合表达载体pME6015,构建了phz1′-′lacZ和phz2′-′lacZ翻译融合表达载体pMEZ1和pMEZ2。采用电转化法分别将pMEZ1、pMEZ2和pME6015导入铜绿假单胞菌突变株PA-SG和野生株PAO1,用Miller法检测融合β-半乳糖苷酶活性。【结果】在KMB或PPM培养基中,pMEZ1在突变株PA-SG中的表达均增强,为野生株的4-5倍;而pMEZ2在突变株PA-SG中的表达均降低,野生株是突变株的2-3倍。【结论】由此推测,铜绿假单胞菌rpoS基因对两个不同吩嗪合成基因簇的调控作用具有特异性,在一定程度上,rpoS负调控phz1,正调控phz2。     

黄色黏细菌转录因子FruA对自身基因的表达不具自调节作用

 粘细菌是研究多细胞结构形态发生机制的良好模型.FruA是粘细菌发育所必需的一种 关键性转录因子, 调节一系列发育相关基因的表达,本文研究FruA对自身基因是否存在反馈调节从而导致发育后期fruA表达水平的下调.以野生型粘细菌模式菌株DK1622为基础构建fruA基因敲除突变株DK1622ΔfruA,再将fruA-lacZ转录融合载体pMF1A整合入fruA突变株染色体attB, 获得重组菌株DK1622ΔfruA/pMF1A,通过检测β-半乳糖苷酶活性来确认FruA对自身基因的表达水平是否有影响. 结果表明fruA调控序列完整的fruA-lacZ转录融合体β-半乳糖苷酶活性在DK1622/pMF1A和DK1622ΔfruA/pMF1A之间无明显差异, 即fruA表达产物作为一种转录因子对自身基因的转录没有调节作用,黏细菌发育后期fruA表达水平的下降存在其它调节机制.     

大肠杆菌启动基因的重组和表达 Ⅰ.不同启动基因对β-半乳糖苷酶结构基因表达的影响

质粒pKL6带有β-半乳糖苷酶结构基因(lacZ),启动基因突变失活,lacZ不能表达。在lacZ前插入外源DNA片段,观察lacZ的表达,可以研究启动基因的功能作用。我们用大肠杆菌染色体DNA的HindⅢ片段与pKL6重组,获得一批重组体,对这些重组体的β-半乳糖苷酶的活力水平和限制性图谱作了分析,对它们用作表达载体的可能性进行了讨论。     

苏云金芽胞杆菌sigK 基因插入失活突变体的特点及其对cry3A基因启动子活性的影响

摘要: 【目的】构建苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt) sigK 基因插入失活突变体,分析突变体特点并明确其对cry3A 基因启动子的影响。【方法】采用同源重组技术在苏云金芽胞杆菌HD-73 菌株sigK 基因中插入卡那霉素抗性基因,构建了sigK 基因插入失活突变体。通过生长曲线测定、扫描电子显微镜观察晶体、芽胞形成情况和芽胞计数及SDS-PAGE 等方法分析了突变体的特点; 构建了遗传恢复菌株对上述性状进行了功能验证; 利用启动子融合lacZ 技术检测了cry3A 基因启动子的转录活性。【结果】获得了苏云金芽胞杆菌HD-73 菌株sigK 基因突变体,生长曲线测定表明,突变体较出发菌株在稳定期后期生长较慢; 扫描电子显微镜观察和芽胞计数分析显示,突变体丧失了形成芽胞和晶体的能力; SDS-PAGE 分析表明突变体中伴胞晶体蛋白的表达量明显低于出发菌株和恢复菌株。利用载体pHT315 携带sigK 基因及其启动子在突变株中表达,所获得的遗传恢复菌株恢复了突变株产生芽胞和晶体的能力; sigK 基因的突变可以提高cry3A 基因启动子在产胞后期的转录活性,对cry3A 启动子指导的Cry 蛋白表达量没有显著影响。【结论】本研究证明sigK 基因为苏云金芽胞杆菌芽胞形成所必需,并影响伴胞晶体蛋白的产量; sigK 基因功能的丧失有利于cry3A 基因启动子在产胞后期的转录。     

苏云金芽胞杆菌LM1212菌株cry基因启动子的活性分析

【背景】前期发现一株具有独特分化表型的苏云金芽胞杆菌(Bacillusthuringiensis,Bt)LM1212,该菌株共有14种杀虫基因,组成了10个转录单元。利用源自菌株LM1212的晶体产生细胞调控因子(crystal producing cell regulator, CpcR)以及其可以激活的cry35-like基因启动子,已在典型Bt菌株HD73中成功构建了非芽胞杀虫蛋白表达体系。【目的】比较菌株LM1212的不同杀虫基因启动子的转录活性,明确可被转录因子CpcR激活的且转录活性较高的启动子,以此为基础优化非芽胞杀虫蛋白表达体系。【方法】将10个启动子区域分别与lacZ报告基因融合构建在pHT304-18Z载体上,得到了10个重组质粒;然后将cpcR基因及其启动子(PcpcR-cpcR)分别反向构建在选取的启动子区域与lacZ报告基因的上游,得到可以表达CpcR且与上述构建相对应的10个重组质粒,将这些重组质粒分别转入不含CpcR的菌株HD73,即获得20个可用于测定β-半乳糖苷酶活性的重组菌株。通过光学显微镜观察和SDS-PAGE检测明确杀虫蛋白表达的情况。【结果】在...     

木酮糖激酶基因整合表达载体构建及在酿酒酵母中的过表达

【目的】以载体p406ADH1为构建骨架,构建一个酿酒酵母(Saccharomyces cerevisiae)工业菌株的整合表达载体。【方法】通过酶切连接的方式,将4个元件片段:作为筛选标记的G418抗性基因KanR,用于基因表达的ADH1终止子片段,酿酒酵母W5自身木酮糖激酶基因,18S rDNA介导的同源整合区,插入到骨架质粒p406ADH1中,得到多拷贝整合表达载体pCXS-RKTr。将该载体线性转化酿酒酵母后,对转化子中木酮糖激酶酶活进行测定,检测其表达情况。【结果】重组质粒在酿酒酵母体内实现了木酮糖激酶的高水平稳定表达,其酶活力是初始菌株的2.87倍。【结论】本实验构建了一个酿酒酵母工业菌株整合表达载体,并用此载体过表达了其自身的木酮糖激酶基因。该重组质粒载体的构建可以有效解决酿酒酵母中自身木酮糖激酶酶活较低的情况,这为利用木糖高产乙醇酿酒酵母基因工程菌株的构建和其它酵母重组质粒载体的构建奠定基础。     

Comprehensive set of integrative plasmid vectors for copper-inducible gene expression in Myxococcus xanthus

Myxococcus xanthus is widely used as a model system for studying gliding motility, multicellular development, and cellular differentiation. Moreover, M. xanthus is a rich source of novel secondary metabolites. The analysis of these processes has been hampered by the limited set of tools for inducible gene expression. Here we report the construction of a set of plasmid vectors to allow copper-inducible gene expression in M. xanthus. Analysis of the effect of copper on strain DK1622 revealed that copper concentrations of up to 500 μM during growth and 60 μM during development do not affect physiological processes such as cell viability, motility, or aggregation into fruiting bodies. Of the copper-responsive promoters in M. xanthus reported so far, the multicopper oxidase cuoA promoter was used to construct expression vectors, because no basal expression is observed in the absence of copper and induction linearly depends on the copper concentration in the culture medium. Four different plasmid vectors have been constructed, with different marker selection genes and sites of integration in the M. xanthus chromosome. The vectors have been tested and gene expression quantified using the lacZ gene. Moreover, we demonstrate the functional complementation of the motility defect caused by lack of PilB by the copper-induced expression of the pilB gene. These versatile vectors are likely to deepen our understanding of the biology of M. xanthus and may also have biotechnological applications.     

Site-specific integration and expression of a developmental promoter in Myxococcus xanthus.

A series of intercellular signals are involved in the regulation of gene expression during fruiting body formation of Myxococcus xanthus. Mutations which block cell interactions, such as csgA (formerly known as spoC), also prevent expression of certain developmentally regulated promoters. csgA+ cells containing Tn5 lac omega DK4435, a developmentally regulated promoter fused to lacZ, began synthesizing lacZ mRNA 12 to 18 h into the developmental cycle. beta-Galactosidase specific activity increased about 12 h later. Neither lacZ mRNA nor beta-galactosidase activity was detected in a developing csgA mutant containing omega DK4435. The developmental promoter and its fused lacZ reporter gene were cloned into a pBR322-derived plasmid vector containing a portion of bacteriophage Mx8. These plasmids preferentially integrated into the M. xanthus chromosome by site-specific recombination at the bacteriophage Mx8 attachment site and maintained a copy number of 1 per chromosome. The integrated plasmids were relatively stable, segregating at a frequency of 0.0007% per generation in the absence of selection. The cloned and integrated promoter behaved like the native promoter, expressing beta-galactosidase at the proper time during wild-type development and failing to express the enzyme during development of a csgA mutant. The overall level of beta-galactosidase expression in merodiploid cells containing one native promoter and one promoter fused to lacZ was about half that of cells containing a single promoter fused to lacZ. These results suggest that the timing of developmentally regulated gene expression is largely independent of the location of this gene within the chromosome. Furthermore, they show that site-specific recombination can be a useful tool for establishing assays for promoter or gene function in M. xanthus.     

Discovery of the autonomously replicating plasmid pMF1 from Myxococcus fulvus and development of a gene cloning system in Myxococcus xanthus

Myxobacteria are very important due to their unique characteristics, such as multicellular social behavior and the production of diverse and novel bioactive secondary metabolites. However, the lack of autonomously replicating plasmids has hindered genetic manipulation of myxobacteria for decades. To determine whether indigenous plasmids are present, we screened about 150 myxobacterial strains, and a circular plasmid designated pMF1 was isolated from Myxococcus fulvus 124B02. Sequence analysis showed that this plasmid was 18,634 bp long and had a G+C content of 68.7%. Twenty-three open reading frames were found in the plasmid, and 14 of them were not homologous to any known sequence. Plasmids containing the gene designated pMF1.14, which encodes a large unknown protein, were shown to transform Myxococcus xanthus DZ1 and DK1622 at high frequencies ( approximately 10(5) CFU/microg DNA), suggesting that the locus is responsible for the autonomous replication of pMF1. Shuttle vectors were constructed for both M. xanthus and Escherichia coli. The pilA gene, which is essential for pilus formation and social motility in M. xanthus, was cloned into the shuttle vectors and introduced into the pilA-deficient mutant DK10410. The transformants subsequently exhibited the ability to form pili and social motility. Autonomously replicating plasmid pMF1 provides a new tool for genetic manipulation in Myxococcus.     

Transfer of plasmid RP4 to Myxococcus xanthus and evidence for its integration into the chromosome.

The broad-host-range plasmid RP4 and its derivative R68.45 were transferred to Myxococcus xanthus DK101 and DZ1; RP4 was maintained integrated in the chromosome. Loss of plasmid markers occurred during the growth of the transconjugants, which could be prevented by selective pressure with oxytetracycline. The integrated plasmid was transferred back to Escherichia coli often as RP4-prime plasmids carrying various segments of the M. xanthus chromosome. It also mediated chromosomal transfer between M. xanthus strains.     

Further characterization and in situ localization of chain-like aggregates of the gliding bacteria Myxococcus fulvus and Myxococcus xanthus.

For the first time, chain-like aggregates, called "strands," have been enriched from crude cell wall preparations of liquid-grown vegetative cells of two strains of Myxococcus xanthus. These strands are highly isomorphic to macromolecular structures, previously described for Myxococcus fulvus (Lünsdorf and Reichenbach, J. Gen. Microbiol. 135:1633-1641, 1989). The strands are morphologically composed of ring elements, consisting of six or more peripheral protein masses and possibly three small central masses. The ring elements are linked by two parallel strings of filamentous proteins, called elongated elements, which keep the ring elements at a constant distance. The overall dimensions of the ring elements are 16.6 +/- 1.0 nm (n = 55) for M. xanthus Mx x48 and 16.4 +/- 1.5 nm (n = 37) for M. xanthus DK 1622. The distance between the ring elements, as a measure of the length of the elongated elements, is 16.6 +/- 1.1 nm (n = 59) for strain Mx x48 and 15.5 +/- 0.6 nm (n = 41) for strain DK 1622. Characteristically, the strands and oligomeric forms thereof show a strict association with the outer membrane. In situ studies of freeze-fractured cells of M. fulvus showed ring elements, isomorphic to those described for M. xanthus, within the periplasm; they appeared in parallel rows just below the outer membrane but not in direct contact with the cytoplasmic membrane. A three-dimensional model summarizes the morphological data. It is hypothesized that the chain-like strands, as building blocks of a more complex belt-like continuum, represent the peripheral part of the gliding machinery, which transforms membrane potential energy into mechanical work.     

Novel lipids in Myxococcus xanthus and their role in chemotaxis

Organisms that colonize solid surfaces, like Myxococcus xanthus, use novel signalling systems to organize multicellular behaviour. Phosphatidylethanolamine (PE) containing the fatty acid 16:1omega5 (Delta11) elicits a chemotactic response. The phenomenon was examined by observing the effects of PE species with varying fatty acid pairings. Wild-type M. xanthus contains 17 different PE species under vegetative conditions and 19 at the midpoint of development; 13 of the 17 have an unsaturated fatty acid at the sn-1 position, a novelty among Proteobacteria. Myxococcus xanthus has two glycerol-3-phosphate acyltransferase (PlsB) homologues which add the sn-1 fatty acid. Each produces PE with 16:1 at the sn-1 position and supports growth and fruiting body development. Deletion of plsB1 (MXAN3288) results in more dramatic changes in PE species distribution than deletion of plsB2 (MXAN1675). PlsB2 has a putative N-terminal eukaryotic fatty acid reductase domain and may support both ether lipid synthesis and PE synthesis. Disruption of a single sn-2 acyltransferase homologue (PlsC, of which M. xanthus contains five) results in minor changes in membrane PE. Derivatization of purified PE extracts with dimethyldisulfide was used to determine the position of the double bonds in unsaturated fatty acids. The results suggest that Delta5 and Delta11 desaturases may create the double bonds after synthesis of the fatty acid. Phosphatidylethanolamine enriched for 16:1 at the sn-1 position stimulates chemotaxis more strongly than PE with 16:1 enriched at the sn-2 position. It appears that the deployment of a rare fatty acid (16:1omega5) at an unusual position (sn-1) has facilitated the evolution of a novel cell signal.     

An improved beta-galactosidase alpha-complementation system for molecular cloning in Bacillus subtilis

The recently described beta-galactosidase alpha-complementation system for molecular cloning in Bacillus subtilis [Haima et al., Gene 86 (1990) 63-69]was optimized in several ways. First, the efficiency of translation of the lac Z delta M15 gene was improved. Second, the plasmid-borne lacZ delta M15 gene was segregationally stabilized by integration into the B. subtilis chromosome. Third, a new lacZ alpha complementing cloning vector was constructed, containing more unique target sites. It was shown that large heterologous DNA fragments (up to at least 29 kb) could be cloned with lacZ alpha-complementing vectors carrying the replication functions of the cryptic B. subtilis plasmid pTA1060, and that these inserts were structurally stably maintained for at least 100 generations of growth.