乳酸菌载体pMG36e的应用现状

乳酸乳球菌通用表达质粒pMG36e是一个经典的人工构建的组成型表达载体,是以乳酸乳球菌乳脂亚种蛋白酶基因的转录和翻译信号为基础构建而成的。它包含一个强启动子,能够在多种细菌中表达外源蛋白。已用于研究细菌素作用机制,乳酸菌基因工程菌株的改造以及口服疫苗的开发等,应用领域十分广泛,已成为乳酸菌基因工程研究的重要工具质粒之一。本文主要从载体构成、基因表达与食品级载体改造等三方面的应用对其进行综述,旨在为该质粒今后研究提供资料。     

Cloning of lysozyme and lysostaphin genes of Staphylococcus aureus and their expression in Bacillus subtilis cells

The gene of microbial lysozyme (lyz) of S. aureus 118 and the gene of lysostaphin (lzf) of S. aureus RN 3239 were cloned and their expression in B. subtilis cells was shown. Lysozyme production in B. subtilis recombinant clone pLF14-Lyz, obtained as the result of cloning, was 2.5-fold greater than lysozyme production in S. aureus wild strain 118. Lysostaphin production in B. subtilis recombinant strain pLF14-Lzf which had inherited the cloned genes was approximately equal to lysostaphin production observed in S. aureus initial strain RN 3239. The production of lysozyme and lysostaphin in the cells of B. subtilis recombinant strains was observed at 30 degrees C and pH 5.5, while in S. aureus initial strains 118 and RN 3239 bacteria produced lysozyme and lysostaphin at 37 degrees C and pH 7.5 respectively.     

Factors for bile tolerance in <Emphasis Type="Italic">Lactococcus lactis</Emphasis>: Analysis by using plasmid variants

The factors of bile tolerance (as one among the fundamental characteristics of probiotic bacteria) were determined in lactococci by using plasmid variants. Bile tolerance of Lactococcus lactis wild-type (WT) strains 527 and N7 (determined by viability counts on bile-containing agar) was equivalent to the corresponding plasmid-free derivatives. In contrast, L. lactis WT strain DRC1 had lower bile tolerance than its plasmid-free derivative DRC1021. Plasmid pDR1-1B, extracted from strain DRC1, was introduced into strain DRC1021 by co-transformation with the vector plasmid pGKV21 as an indicator. Strain DRC121 (DRC1021 harboring pGKV21) had good bile tolerance as did strain DRC1021, while strain DRC13 (DRC1021 harboring both pDR1-1B and pGKV21) did not. Fatty acid (FA) composition was different between strains DRC121 and DRC13. The plasmid pDR1-1B or plasmid profile and FA composition are key factors for bile tolerance of strain DRC1, and therefore changing the plasmid profile might be a way of modulating bile tolerance in lactococci.     

A host-vector system for gene cloning in the cyanobacterium Synechocystis PCC 6803

Summary Synechocystis 6803 contains at least four cryptic plasmids of 2.27 kb (pUS1, pUS2 and pUS3) and 5.20 kb (pUS4). The 1.70 kb HpaI fragments of the related plasmids pUS2 and pUS3 were cloned into the Ap^r gene of the E. coli plasmid pACYC177, yielding the Km^r hybrid plasmids pUF12 and pUF3 respectively. pUF3 recombines in Synechocystis 6803 with a 2.27 kb plasmid giving the Km^r shuttle vector pUF311. The 1.35 kb HaeII fragment containing the Cm² gene of the E. coli plasmid pACYC184 was cloned in pUF311 generating the Cm^r Km^r shuttle vector pFCLV7. Wild-type cells of Synechocystis 6803 are transformed, albeit poorly, by the plasmids pUF3, pUF12 and pFCLV7. pFCLV7 very efficiently transforms the SUF311 strain of Synechocystis 6803 containing pUF311 as a resident plasmid. This is due to recombination between the homologous parts of pFCLV7 and pUF311. For the same reason the strain SUF311 is also efficiently transformable by E. coli plasmids, as shown for pLF8, provided that they have some homology with the E. coli part of pUF311.The combined use of Synechocystis 6803 strain SUF311 and of plasmids pFCLV7 and pLF8 generates an efficient host-vector system for gene cloning in this facultatively heterotrophic cyanobacterium.     

Tn10介导的Bit cryIVA基因在荧光假单胞菌染色体中的整合及表达

苏云金杆菌以色列亚种的杀虫晶体蛋白基因cryIVA被亚克隆到自杀型转座子质粒载体pLOF／Km的TN10中,构建了转座子质粒PLF97A。通过电转化／转座作用,cryIVA随Tn10转座并整合到荧光假单胞菌FP．DE2染色体中,构建了工程菌株FP．DE202。Southernblotting验证了cryIVA在FP．DE202中整合在不同的位点。Westernblotting证明了cryIVA在F．P．DE202中得到了表达,其产物对双翅目害虫韭菜迟眼蕈蚊的3龄幼虫有较强的毒杀效果。     

Obtaining of fluorescent-labeled nodule bacteria strains of wild legumes for their detection in vive and in vitro

A series of expression vectors containing genes of fluorescent proteins TurboGFP and TurboRFP under the phage T5 constitutive promoter regulation, intended for lifetime marking of nodule bacteria is created: a series of vectors based on a broad-host-range replicon BBRI, for marking strains with an expression of reporter gene from a transformed plasmid and a series of vectors based on a plasmid pRL765gfp for marking strains by introduction genes of fluorescent proteins in a bacterial chromosome. It was shown that transformation is the most preferable method of constructions transfer in nodule bacteria cells, as in the presence of mob locus in the vectors necessary for conjugation, exists the possibility of occasional plasmid mobilization and its transition from marked strain cells in other soil bacteria. With application of the created vector constructions we obtained fluorescent tagged strains of Rhizobium sp., Mesorhizobium sp., Ensifer (Sinorhizobium) sp., Bradyrhizobium sp., Phyllobacterium sp., Agrobacterium sp. Also their suitability for experiments in vivo and in vitro is shown.     

Characterization, cloning, curing, and distribution in lactic acid bacteria of pLP1, a plasmid from Lactobacillus plantarum CCM 1904 and its use in shuttle vector construction

A small 2.1-kb plasmid called pLP1 was extracted from Lactobacillus plantarum CCM 1904 (ATCC 8014) and cloned into the Escherichia coli pUC19 plasmid. As determined by DNA-DNA Southern hybridization with a pLP1-radioactively labeled probe, other lactic acid bacteria such as L. curvatus, L. sake, Carnobacterium, and Leuconostoc mesenteroides harbor pLP1-related plasmids. Shuttle vectors based on the pLP1 replicon were constructed by inserting the erythromycin-resistance gene from pVA891 into the various pUC19-pLP1 constructions. pLP1-based shuttle vector transformation efficiencies (TE) by electroporation were compared to TE of a broad-host-range plasmid pGK12 in different lactobacilli strains. Expression of the pUC19-pLP1 plasmids in Escherichia coli maxicells showed that pLP1 encodes for a 37,000 MW protein which can act in trans allowing the replication of plasmids in which this protein is truncated. The pLP1-based shuttle vectors producing this protein replicate in lactobacilli and also in Bacillus subtilis. A pLP1-free strain was obtained by incompatibility with a pLP1-based shuttle vector introduced in L. plantarum CCM 1904 by electroporation. The absence of pLP1 has no incidence on the strain phenotype suggesting that pLP1 is not essential for the strain in our laboratory conditions.     

产SEB金黄色葡萄球菌α-溶血毒素基因缺失菌株的构建

构建产肠毒素B(Staphylococcal enterotoxin B ,SEB)的金黄色葡萄球菌α-溶血毒素（α-hemolysin, α-HL)缺失菌株。首先构建用于α-HL基因敲除的同源重组质粒pMHL-α,经金黄色葡萄球菌RN4220修饰后再通过原生质体转入金黄色葡萄球菌SM-01。含重组质粒pMHL-α的金黄色葡萄球菌SM-01在42℃诱导条件下培养多代,最终筛选出α-溶血毒素基因缺失菌株。经序列分析和血平板溶血实验结果证明最终获得产SEB金黄色葡萄球菌α-HL缺失菌株。为野生型金黄色葡萄球菌的体内遗传操作及构建产超抗原药物金黄色葡萄球菌基因工程菌株提供了一定的理论基础和方法。     

Expression of bacillar glutamyl endopeptidase genes in Bacillus subtilis by a new mobilizable single-replicon vector pLF

The pLF1311 natural plasmid from Lactobacillus fermentum 1311 was used to construct a single-replicon vector suitable for rapid cloning in a wide range of gram-positive hosts and Escherichia coli. The new vector is capable of conjugative mobilization from E. coli to various hosts by conjugal transfer. The final vector (3.4 kb) showed a high segregational and structural stability and a high copy number. Glutamyl endopeptidase genes from Bacillus licheniformis (gseBL) and B. intermedius (gseBI) were cloned in both pLF9 and pLF14 vectors and introduced to B. subtilis. The yield of enzymes in the pLF-derived producers was 6- to 30-fold more than in the natural producers and reached 100-150 mg/L of mature protease.     

Construction and use of a new vector/transposon, pLBT::mim-Tn10:lac:kan, to identify environmentally responsive genes in a marine bacterium

Abstract The previously described pLOFKm transposon delivery plasmid (J. Bacteriol. (1990) 172, 6557–6567) was engineered such that a promoterless lacZ gene was cloned within the transposon cassette, generating the vector pLBT. Using pLBT, stable insertion mutations were generated at high frequencies in Vibrio sp. S141 and Pseudomonas sp. S91, and the interrupted genes could be monitored for their pattern of regulation. Genetic screens isolated mutants defective in a variety of activities. We describe the construction and use of pLBT as a tool for reporter gene mutant analysis in bacteria other than well-characterized laboratory strains.     

A method for introduction of unmarked mutations in the genome of Paracoccus denitrificans: construction of strains with multiple mutations in the genes encoding periplasmic cytochromes c550, c551i, and c553i.

A new suicide vector, pRVS1, was constructed to facilitate the site-directed introduction of unmarked mutations in the chromosome of Paracoccus denitrificans. The vector was derived from suicide vector pGRPd1, which was equipped with the lacZ gene encoding beta-galactosidase. The reporter gene was found to be a successful screening marker for the discrimination between plasmid integrant strains and mutant strains which had lost the plasmid after homologous recombination. Suicide vectors pGRPd1 and pRVS1 were used in gene replacement techniques for the construction of mutant strains with multiple mutations in the cycA, moxG, and cycB genes encoding the periplasmic cytochromes c550, c551i, and c553i, respectively. Southern analyses of the DNA and protein analyses of the resultant single, double, and triple mutant strains confirmed the correctness of the mutations. The wild type and mutant strains were all able to grow on succinate and choline chloride. In addition, all strains grew on methylamine and displayed wild-type levels of methylamine dehydrogenase activities. cycA mutant strains, however, showed a decreased maximum specific growth rate on the methylamine substrate. The wild-type strain, cycA and cycB mutant strains, and the cycA cycB double mutant strain were able to grow on methanol and showed wild-type levels of methanol dehydrogenase activities. moxG mutant strains failed to grow on methanol and had low levels of methanol dehydrogenase activities. The maximum specific growth rate of the cycA mutant strain on methanol was comparable with that of the wild-type strain. The data indicate the involvement of the soluble cytochromes c in clearly defined electron transport routes.     

A cloning vector for creation of Escherichia coli lacZ translational fusions and generation of linear template for chromosomal integration

A novel cloning vector to aid in the construction of single copy β-galactosidase reporter systems for gene expression studies in lactose metabolizing Escherichia coli strains, including STEC, is described. The plasmid allows construction of translational fusions of cloned gene promoters to a short segment of E. coli lacZ. A selectable spectinomycin resistance marker flanked by a short lacI segment is positioned 5' to the cloning site. PCR amplification using opposing primers complementary to the upstream lacI fragment and the downstream lacZ fragment generates a linear template suitable for integration using pRedET recombination. Integration of linear template derived from the recombinant plasmid into host strains replaces the entire native lacZ promoter and fuses the promoter of interest in-frame with the lacZ gene, thus simultaneously producing a single-copy, chromosomal reporter system and eliminating background lacZ expression. Studies comparing ahpC expression from a chromosomal fusion in the lac open with that on a plasmid in E. coli strain EDL933 are shown.     

Tagging of Bacillus subtilis soil isolates through illegitimate recombination for PCR detection

Although there are numerous bacteria of the genus Bacillus of great importance for biological control, little is known about their ecology in the soil. We wanted to test illegitimate recombination as a tagging system that would allow us to study selected or genetically engineered Bacillus soil isolates. Strains carrying the plasmid integrated into the chromosome were obtained by growing at a non-permissive temperature after transformation with a plasmid carrying a thermo-sensitive replication origin with selection for erythromycin. A laboratory strain, a commercial strain (Kodiak), and four other soil isolates were generated through this procedure and analysed. In all of these strains the integrated plasmid was maintained in multicopy. The erythromycin resistance gene (ermB) placed on the plasmid was used as a target for polymerase chain reaction (PCR). The tagged strains could be then detected when inoculated into microcosms prepared with non-sterile soil.     

Biological effects of interferon,produced by recombinant bacteria of the probiotic preparation subalin

The present review deals with the analysis of biological and functional activities of recombinant bacteria Bacillus subtilis IF-alpha 2335 are producing a human interferon. The interferon-producing bacteria are constructed on a basis commercial probiotic strain B.subtilis 2335, carrying a recombinant plasmid pMBM 105 with the gene of human alpha-2 interferon. The implementation of the recombinant strain in the preparation probiotic, received a designation "Subalin", necessitates to verify a number of immunologic activities and to perform successive protective effects. Interferon, synthesized by recombinant bacteria shows the activity on macroorganism at oral and rectal application of preparation. Subalin was shown antivirus and antitumor activity and preservation by recombinat bacteria of antagonistic properties. The mechanisms of the positive effect of subalin were considered: this effect was shown to be due to the action of interferon excreted by recombinant bacteria into the mucous of different biotopes of host.     

鸟分枝杆菌PhoP功能分析及其基因突变株的构建

【目的】对鸟分枝杆菌PhoP的功能进行分析及构建PhoP基因突变株,为深入研究PhoP的调控机制打下基础。【方法】利用PCR扩增出鸟分枝杆菌PhoP DNA结合区(PhoPC)编码序列,与表达载体p GEX-4T-3连接后,转化入大肠杆菌BL21(DE3)中表达GST-PhoPC融合蛋白。用凝血酶去除GST标签,制备PhoPC蛋白;利用PCR扩增出鸟分枝杆菌PhoP基因及其下游基因MAV0127、PhoU和Amt的启动子片段,采用凝胶迁移率移动试验(EMSA)分别检测PhoPC与PhoP、MAV0127、PhoU和Amt的启动子结合的情况。通过PCR扩增PhoP基因上、下游片段,构建PhoP基因缺失性同源核苷酸片段,与自杀质粒p GMB151连接后,通过电转化导入鸟分枝杆菌进行同源交换,利用PCR筛选出PhoP基因缺失突变株。【结果】EMSA结果显示,鸟分枝杆菌PhoP能与PhoP、MAV0127及Amt基因启动子结合,不能与PhoU结合。通过PCR和序列分析证实基因突变株的PhoP基因缺失了309个碱基。【结论】PhoP不仅可调控其下游基因MAV0127和Amt的转录水平,还可调控其自身基因的转录,但不参与调节PhoU二元调控系统。构建了PhoP基因缺失突变株,为进一步研究其在鸟分枝杆菌的调控功能奠定了基础。