线性质粒

在染色体外DNA分子的研究领域中,一支异军正在悄悄突起,这就是线性质粒的研究。 迄今报导的多数质粒都是环状的DNA分子。近年来,人们相继在真核生物中发现了一些线状质粒。例如,玉米线粒体中的SⅠ和SⅡ质粒(1,2)、Sarghum线粒体中质粒(8)、真菌Ascobolus immersus中的质粒。     

细菌线性质粒的复制研究

细菌的线性质粒是近年来基础微生物学研究的一个新热点。线性质粒的复制与环状质粒相比,其最突出的特点就是它的5'末端填补机制——填补因为5'末端引物被切除所留下的缺口。根据线性质粒末端结构的不同将其分为两大类:一类具有共价闭合的发夹末端,另一类具有共价结合的末端蛋白。就细菌线性质粒复制的研究现状作一综述,重点介绍了具有两种不同末端结构的线性质粒5'末端填补机制。     

副干酪乳酪杆菌ZY-1内源性质粒分析及其表达系统的构建与应用

高效稳定的乳杆菌表达载体的构建是实现其菌种改良和个性化菌株开发的关键。本研究从副干酪乳酪杆菌(Lacticaseibacillus paracasei) ZY-1中分离出4个内源性质粒并进行功能分析。通过将pLPZ3与pLPZ4的复制子rep,与pNZ5319质粒的氯霉素乙酰转移酶报告基因cat、pUC19的复制子ori构建大肠杆菌-乳酸菌穿梭载体pLPZ3N与pLPZ4N,进一步加上启动子Pldh3和mCherry红色荧光蛋白,获得表达载体pLPZ3E与pLPZ4E。pLPZ3与pLPZ4质粒大小分别为6 289 bp和5 087 bp,GC含量分别为40.94%和39.51%。2个穿梭载体可成功转化至乳酪杆菌属中,pLPZ4N的转化效率(5.23×10²-8.93×10²CFU/μg)略高于pLPZ3N。乳酸菌表达载体pLPZ3E与pLPZ4E转化至副干酪乳酪杆菌S-NB后,成功获得了mCherry红色荧光蛋白的表达。以P_ldh3为启动子构建的重组表达载体pLPZ4E-lacG转化得到的重组菌,其β-半乳糖苷酶酶活性...     

三种提取乳酸菌中内源性质粒DNA方法的比较

采用3种不同抽提质粒DNA的方法普查了6株不同乳酸菌的内源性质粒DNA,并对3种不同方法进行比较。在相同条件下,琼脂糖凝胶电泳检测的结果显示:效果最佳者为改进的Anderson-Mckay方法,其次为液氮反复冻融方法,最差者为改进的碱裂解方法。为抽提革兰阳性细菌乳酸菌类的内源性质粒DNA提供了行之有效的方法。     

枯草杆菌弹性酶的理化性质研究

对用DEAE纤维素—Sephader G—75层析所得到的4万u/g的纯酶进行分子量、等电点、最适作用条件、稳定性及水解性质等进行试验。     

类杆菌5-硝基咪唑抗性质粒pIP419的研究

5-硝基咪唑类药物广泛应用于厌氧菌感染症的临床治疗,近几年厌氧菌5-硝基咪唑抗性菌株的报道有所增加。本文介绍分离自类杆菌的编码抗5-硝基咪唑抗性质粒pIP419的研究结果。pIP419分子量为10kb,已建立了限制性内切酶物理图谱。该质粒可通过接合和转化在不同的菌种或菌株间转移。质粒上负责质粒复制和转移的基因片段已被定位和克隆。     

大线性质粒pHZ1000,pHZ1001在链霉菌种间的接合转移

从1979年首先在娄彻氏链霉菌(Ｓｔｒｅｐｔｏｍｙｃｅｓｒｏｃｈｅｉ)中发现第一例原核生物线性质粒ｐＳＬＡ2[1]至今,人们已在多种链霉菌中发现了线性质粒,大小12ｋｂ到640ｋｂ不等[2]。从红球菌[3]和诺卡氏菌中[4],在硫杆菌属和螺旋体属中,以及酵母、丝状真菌等真核生物中也都陆续发现了线性质粒的存在[5],大肠杆菌噬菌体Ｎ15的原噬菌体也是一种特殊的线性质粒[6]。线性质粒存在的生物学意义引起了人们的浓厚兴趣。然而20多年以来,链霉菌线性质粒,尤其是大线性质粒的研究始终进展不快。目前有关链霉菌线性质粒复制机理、端粒结构的阐明,都…     

重组枯草杆菌纤溶酶的酶学性质研究

对一种重组枯草杆菌纤溶酶 (rBSFE)的酶学性质进行了初步研究 ,结果表明 :酶作用最适温度为 35℃ ;最适反应pH为 8.0 ;酶活性能被PMSF抑制 ,是典型的丝氨酸蛋白酶。通过与尿激酶进行比较 ,发现该酶对纤维蛋白有直接降解作用 ,而对于纤维蛋白原的敏感性则低于尿激酶 ,提示该酶具有直接溶栓作用 ,又不致引起出血 ,是一种有潜力的新型溶栓剂。     

高效回收苏云金杆菌质粒DNA的方法研究

介绍三种简易、快速和高效回收苏云金杆菌（Bacillus thuringiensis,简称Bt)质粒DNA的方法。这些方法省时、经济、适用范围广,回收的Bt质粒DNA质量高,可直接用于各种分子克隆操作。     

抗氧化型枯草杆菌碱性蛋白酶高产工程菌的构建——Ⅰ.抗氧化型碱性蛋白酶的提纯及其主要性质的研究

枯草芽孢杆菌(Bacillus Subtilis)B135工程菌能产生抗氧化型碱性蛋白酶,粗酶经硫酸铵分级沉淀,CM-52层析,Sephadex G-100层析,得到凝胶电泳均一样品,比活达到1700U/mg,是粗酶比活的7.69倍.该酶在60℃时酶活力最高,最适pH为10.2,在50℃时,温浴10min后,酶活降低到原来的50%.该酶受1M H_2O_2作用20min后,仍保持96%的酶活     

Isolation and Preliminary Characterization of Cryptic Plasmids from Erwinia carotovora

Of the fifty-two Erwinia carotovorastrains studied, sixteen were found to contain extrachromosomal DNA (plasmids) from 2.5 to 129 kbp in size. Some E. carotovorastrains bore two to five different plasmids. Experiments showed that the cryptic plasmids of erwinia are not responsible for their resistance to antibiotics and are not involved in the synthesis of macromolecular colicin-like carotovoricins. At the same time, one of the E. carotovorastrains, 13A, augmented the production of carotovoricin after curing from one of its plasmids, the 47.7-kbp pCA 6-2. Three E. carotovorasubsp.carotovorastrains and one E. carotovorasubsp.atrosepticastrain contained large 129-kbp plasmids, which may play a role in the ecology of phytopathogenic pectinolytic erwinia.     

The Replication System of Plasmids from Bacillus subtilis Environmental Isolates

Restriction enzyme analysis, cloning, and sequencing showed that large (more than 90 kb) plasmids isolated from different Bacillus subtilisstrains are identical in structure of the region ensuring stable inheritance of plasmid replicons and are widespread in Belarussian environmental strains of B. subtilis.     

Intimate Relationship between mtDNA, Plasmids, and the Fusion of Mitochondria

Physarum polycephalum. The conformation of Physarum mtDNA is currently thought to be circular. The inheritance of its mtDNA depends on the multiallelic mating type loci, matA. In a cross with ordinary matA combinations, the strain that has the higher matA status transmits its mtDNA to the progeny (uniparental inheritance). The mF plasmid promotes the fusion of mitochondria in the zygote and during sporulation. When it exists in a strain with a lower status matA, the mF plasmid overcomes the force of uniparental inheritance and is preferentially transmitted to the progeny via mitochondrial fusion. Moreover, the conformation of mtDNA is changed from circular to linear by recombination with the mF plasmid. Since biparental inheritance usually occurs in a cross involving a combination of matA1 and matA15, two types of inheritance of Physarum mtDNA exist. Considering the existence of the mF plasmid, there are four patterns of cytoplasmic inheritance in P. polycephalum: 1) uniparental inheritance of mtDNA, 2) uniparental inheritance of mtDNA and preferential transmission of the mF plasmid, 3) biparental inheritance of mtDNA, and 4) biparental inheritance of mtDNA and the mF plasmid. This article describes the events involved in each pattern. Finally, we discuss a hypothetical mechanism for mitochondrial fusion. The essential protein may be the ORF640 protein encoded in the mF plasmid. Received 8 March 2000/ Accepted in revised form 23 March 2000     

一株拮抗多重耐药菌的芽孢杆菌的初步研究

目的 对一株具有拮抗多重耐药菌的芽孢杆菌的特性进行研究.方法 采用菌落法测试多株芽孢杆菌对18种不同来源致病菌的抑制效应,并对其中一株可拮抗多重耐药菌的芽孢杆菌(KC株)进行鉴定和电泳分析.结果 该芽孢杆菌(KC株)经生理生化以及16S rDNA鉴定为枯草芽孢杆菌;KC株对人源、鱼源、畜禽源和鸡粪源的多重耐药菌有不同程度的拮抗效应;SDS-PAGE分析提示,35 kD左右的分泌蛋白可能参与了KC株的抑菌活性.结论 具有拮抗多重耐药菌的芽孢杆菌为枯草芽孢杆菌,且该菌分泌的35 kD蛋白可能参与抑菌活性.     

Characterization of a Theta Plasmid Replicon with Homology to All Four Large Plasmids ofBacillus megateriumQM B1551

A replicon from one of an array of seven indigenous compatible plasmids ofBacillus megateriumQM B1551 has been cloned and sequenced. The replicon hybridized with all four of the large plasmids (165, 108, 71, and 47 kb) of strain QM B1551. The cloned 2374-bpHindIII fragment was sequenced and contained two upstream palindromes and a large (>419-amino-acid) open reading frame (ORF) truncated at the 3′ end. Unlike most plasmid origins, a region of four tandem 12-bp direct repeats was located within the ORF. The direct repeats alone were incompatible with the replicon, suggesting that they are iterons and that the plasmid probably replicates by theta replication. The ORF product was shown to act intrans.A small region with similarity to theB. subtilischromosomal origin membrane binding region was detected as were possible binding sites for DnaA and IHF proteins. Deletion analysis showed the minimal replicon to be a 1675-bp fragment containing the incomplete ORF plus 536 bp upstream. The predicted ORF protein of >48 kDa was basic and rich in glutamate + glutamine (16%). There was no significant amino acid similarity to any gene, nor were there any obvious motifs present in the ORF. The data suggest that this is a theta replicon with an expressedrepgene required for replication. The replicon contains its iterons within the gene and has no homology to reported replicons. It is the first characterization of aB. megateriumreplicon.     

Identification and distribution of sequences having similarity to mitochondrial plasmids in mitochondrial genomes of filamentous fungi

Mitochondrial plasmids are autonomously replicating genetic elements commonly associated with fungal and plant species. Analysis of several plant and fungal mitochondrial genomes has revealed regions that show significant homology to mitochondrial plasmids, suggesting that plasmids have had a long-term association with their mitochondrial hosts. To assess the degree to which plasmids have invaded fungal mitochondrial genomes, BLAST search parameters were modified to identify plasmid sequences within highly AT-rich mtDNAs, and output data were parsed by E value, score, and sequence complexity. High scoring hits were evaluated for the presence of shared repetitive elements and location within plasmids and mtDNAs. Our searches revealed multiple sites of sequence similarity to four distinct plasmids in the wild-type mtDNA of Neurospora crassa, which collectively comprise more than 2% of the mitochondrial genome. Regions of plasmid similarity were not restricted to plasmids known to be associated with senescence, indicating that all mt plasmids can potentially integrate into mitochondrial DNA. Unexpectedly, plasmid-related sequences were found to be clustered in regions that have disproportionately low numbers of PstI palindromic sequences, suggesting that these repetitive elements may play a role in eliminating foreign DNA. A separate class of GC-rich palindromes was identified that appear to be mobile, as indicated by their occurrence within regions of plasmid homology. Sites of sequence similarity to mitochondrial plasmids were also detected in other filamentous fungi, but to a lesser degree. The tools developed here will be useful in assessing the contribution plasmids have made to mitochondrial function and in understanding the co-evolution of mitochondrial plasmids and their hosts.Electronic Supplementary Material Supplementary material is available for this article at     

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.