拟诺卡氏菌线型质粒pNPL1的测序和分析

【目的】检测和分析稀有放线菌中新的线型质粒。【方法】从植物内生菌中分离链霉菌之外的放线菌菌株,检测、测序和分析线型质粒。【结果】从中草药植物紫花前胡的叶片中分离到一株内生放线菌25L-1-1c,经过16S rRNA基因序列比对属于拟诺卡氏菌。从该菌株中检测到一个约25 kb的线型质粒pNPL1。克隆和测序了pNPL1新的端粒,含有多个小的回文序列。测序获得全长为24 621 bp的线型质粒pNPL1,预测编码22个基因,其中2个基因与链霉菌质粒的端粒复制基因同源,1个基因与链霉菌质粒主要的接合转移基因相似,其余19个基因为未知功能。携带pNPL1端粒复制基因的质粒不能转化变铅青链霉菌,暗示需要发展拟诺卡氏菌的遗传操作系统。【结论】这是首次在拟诺卡氏菌中发现和描述线型质粒。     

小葱植物内生链霉菌线型质粒pYY8L的端粒与复制区的克隆和鉴定

从小葱植物中分离到一株编号为36R-2-1B的链霉菌菌株,该菌株含有一个约为280kb的线型质粒pYY8L。【目的】克隆、测序和分析pYY8L新的端粒和复制区。【方法】采用改良的"在凝胶中进行DNA碱处理与酶切"的方法来克隆大的线型质粒pYY8L的端粒,通过构建基因组柯斯文库和次级克隆的方法来缩小和鉴定pYY8L的复制区。【结果】在小葱植物内生链霉菌36R-2-1B中检测到约为280kb的线型质粒pYY8L,克隆了pYY8L的端粒。其末端的152bp包含6个小的回文序列,可以形成复杂的二级结构。利用柯斯文库构建、次级克隆和测序获得了4891bp的pYY8L的复制区。该复制区含有6个基因,其中2个与天蓝色链霉菌线型质粒SCP1的复制基因非常相似,但是邻近的重复序列不同。【结论】采用新的改良的方法克隆和鉴定了pYY8L新的端粒和复制区。本文首次报道了植物内生链霉菌线型质粒的端粒和复制基因。     

游动双孢菌线型质粒pPR2的全序列测定及分析

[目的]获得游动双孢菌线型质粒pPR2的全序列,并揭示新型的端粒复制蛋白和可能的中间复制位点.[方法]用分段克隆的方法和序列拼接获得pPR2的全序列,利用软件分析端粒DNA的二级结构和可能的端粒复制蛋白,利用链霉菌原生质体转化的方法检测可能的中间复制的位点.[结果]pPR2全长为15520 bp,(G C)含量为68.1%.其端粒末端反向重复序列的长度为329 bp,不能像多数链霉菌的线型质粒那样能形成保守的"折返"的二级结构.pPR2虽然没有参与链霉菌端粒复制的保守的tap/tpg基因,但是pPR2.3c基因编码了一个双结构域蛋白,分别同链霉菌的端粒复制相关蛋白Tap和嗜血杆菌的解旋酶具有相似性.pPR2缺少典型的链霉菌重复序列-复制基因(iteron-rep)区段,将几乎覆盖全长pPR2的两段DNA进行克隆后,不能转化变铅青链霉菌.此外,pPR2基因还编码可能参与线型DNA复制的调控的单链结合蛋白(SSB)和与放线菌质粒接合转移相关的主要蛋白(Tva).[结论]pPR2是链霉菌之外的放线菌中最小的线型质粒,其序列在游动双孢菌属的线型质粒中是首次报道.pPR2可能具有新型的端粒复制的机制,其中pPR2.2c和pPR2.3c编码可能的端粒复制蛋白.     

西藏土壤中分离的链霉菌含有丰富的线型和环型质粒

【目的】研究极端自然环境对链霉菌线型和环型质粒分布的影响。【方法】从西藏高原采集了20份土壤样品,分离和初步鉴定链霉菌,提取和检测质粒DNA。【结果】从中分离到46株链霉菌,其中有23株菌含有1 4个线型质粒,大小在19 650 kb之间,8个菌株含有1 4个环型质粒,大小在4 80 kb之间。【结论】西藏土壤来源的链霉菌含有大量的、多样的线型质粒和环型质粒,暗示极端环境中诸如强紫外辐射等可能会引发DNA损伤和修复,进而造成质粒的多样性。     

五个链霉菌线型质粒端粒的克隆和分析

本室从西藏采集的土壤样品中分离到了一批链霉菌,利用脉冲电泳确定了其中5株链霉菌含有较小的线型质粒。【目的】克隆、测序和分析5个线型质粒的端粒。【方法】采用改良的"在凝胶中进行DNA碱处理和限制性内切酶酶切"的方法来克隆线型质粒的端粒DNA。【结果】克隆和测序了5个线型质粒的端粒DNA。通过与链霉菌典型端粒进行比较,发现这5个新的线型质粒的端粒序列同样含有多个回文序列。但是有的端粒保守的回文序列I不一定能够"折返"与内部序列配对形成"超级发卡"结构,回文序列的"突出环"不一定都为3nt。【结论】采用改良的方法克隆和鉴定了5个线型质粒新的端粒序列,这些新端粒的特征暗示:回文序列I的"折返"和3nt的回文序列的"突出环"不是端粒复制必需的。     

链霉菌质粒的复制与进化

近年来, 随着大质粒提取和检测技术的发展, 尤其是高通量DNA测序技术的应用, 使得链霉菌大的环型质粒和线型质粒的研究取得了较快的进展。相比于研究透彻的细菌Theta型复制的质粒, 链霉菌Theta型质粒在复制区的结构、复制蛋白和调控蛋白作用的分子机理等方面具有多样性和新颖性。新鉴定的许多线型质粒的中心复制区表明中心复制的起始可以靠近端粒, 一个质粒也可以有2个以上的复制区。新分离的端粒序列显示端粒“折返”不是必需的, 而形成二级结构对于端粒复制是重要的。链霉菌环型和线型质粒的测序分析显示它们之间存在亲缘关系。环型质粒可以与噬菌体共整合, 实验证明它们在一定条件下可以相互转换。这些研究结果表明, 链霉菌环型、线型质粒和噬菌体从结构到功能到进化具有多样性、新颖性和亲缘关系。     

吸水链霉菌应城变种的四个内源质粒及其逐个消除的研究

在改良质粒DNA提取方法的基础上,从三种农用抗生素的同一产生菌——吸水链霉菌应城变种中同时发现四个内源质粒,用双向电泳技术确定了它们均为CCC构型,根据分子量从大到小的顺序将它们分别命名为pHZ1、pHZ2、pHZ3和pHZ4,与已知分子量的CCC质粒分子同步电泳估计它们的分子量依次分别为61kb、4.7kb、4.1kb和3.3kb,基于这四个内源质粒中至少部分个体可能为接合性质粒,可在没有某个质粒的衍生菌株的菌坪上形成“麻点”的假设,我们分离和鉴定了三个质粒逐个消除的10-22衍生菌株,并在光学显微镜下确证了二种类型的麻点。     

红球菌线型质粒pNSL1的克隆、测序和复制区的鉴定

从红球菌NS1中检测到两个线型质粒pNSL1和pNSL2。【目的】克隆、测序和分析pNSL1,并鉴定质粒的复制区。【方法】利用脉冲电泳方法从凝胶中回收大量的质粒DNA,进行鸟枪法克隆、测序和拼接,通过生物信息学分析和实验证明质粒的自主复制区。【结果】克隆、测序和拼接获得pNSL1全长为117252bp的序列,包括在红球菌中保守的1282bp端粒的序列。序列预测含有103个蛋白编码区,包括质粒的复制、分配、转移等功能基因。将pNSL1中一个与分枝杆菌质粒的复制基因同源的pNSL1.038及其上游的767bp非编码序列克隆到大肠杆菌质粒,电击转化珊瑚诺卡氏菌4.1037,获得了抗性转化子。【结论】克隆、测序了全长的线型质粒pNSL1,鉴定了质粒的复制区。     

嗜热链霉菌质粒的研究

从分属于12个种的28株嗜热链霉菌中检测到热灰紫链霉菌(S.thermogriseoviolaceus)T272带有3个质粒(pST1,pST2,pST3),热藤黄链霉菌(S.thermoluteus)T422带有1个质粒(pST4),经电镜观察得到证实,并按经验公式求得其分子量分别为27、8.3、6.9、25.7kb。实验数据表明,T272与利福平抗性有关的基因可能位于质粒pST1;而pST4可能与rRNA甲基化酶合成有关,推测该酶使T422具有红霉素、螺旋霉素、林可霉素抗性。未发现这些质粒与宿主的高温生长特性有关。     

高温链霉菌质粒pTSC2的测序分析和滚环复制方式的鉴定

从猪粪堆肥中分离到一株编号为X3-3的可以在50℃高温生长的链霉菌菌株,该菌株含有一个约7kb的环型质粒pTSC2。【目的】克隆、测序和分析pTSC2,以及鉴定质粒的复制方式。【方法】利用分段克隆和引物延伸获得pTSC2的全序列,利用多序列比对寻找复制元件rep、dso和sso,利用中性转移和Southern杂交检测复制中间体。【结果】克隆和测序获得了全长为7516bp的pTSC2序列,预测编码8种蛋白,其中4种蛋白与链霉菌滚环复制的质粒pIJ101中负责复制和接合转移的蛋白非常相似。pTSC2的复制元件rep、dso和sso也与pIJ101的相似。克隆、转化变铅青链霉菌ZX7以及高温链霉菌2C证明了rep和dso为复制所必需元件。Southern杂交检测到pTSC2复制过程中积累了大量的单链DNA。【结论】高温链霉菌质粒pTSC2以滚环方式进行复制。这是首次在高温链霉菌中克隆和测序质粒,以及鉴定其复制方式。     

Giant linear plasmids of β-lactam antibiotic producing Streptomyces

Abstract A survey of the total cellular DNA from five β-lactam antibiotic-producing Streptomyces spp. by pulsed field gel electrophoresis was conducted to investigate the presence of linear plasmids. Streptomyces clavuligerus NRRL 3585 contained two giant linear plasmids of 120 and 430 kb, in addition to the well-characterized 11.7 kb linear plasmid. Streptomyces griseus NRRL 3851 contained a single giant linear plasmid of 120 kb, and Streptomyces jumonjinensis NRRL 5741 contained two giant linear plasmids (220 and 280 kb), and two smaller linear plasmids. No plasmids were identified in Streptomyces cattleya NRRL 3841 or Streptomyces lipmannii NRRL 3584. Southern hybridization did not reveal any homology shared by these plasmids, and β-lactam antibiotic synthesis gene clusters were located on the chromosome.     

A linear plasmid temperature-sensitive for replication in Streptomyces hygroscopicus 10-22

Streptomyces hygroscopicus 10-22 harbors a conjugative, autonomously replicating linear plasmid pHZ6 of ca. 70 kb, which shows no obvious homology with chromosomal DNA and is temperature-sensitive for replication, being stable in the host at 28 degrees C but easily lost at 37 degrees C. On a lawn of the wild-type S. hygroscopicus 10-22 cured of pHZ6, pHZ6 elicit pocks. Temperature sensitivity seemed to be a unique property for pHZ6 among six linear plasmids tested, including the well-known linear plasmids SLP2 in Streptomyces lividans 1326 and SCP1 in Streptomyces coelicolor A3(2). The distinct identity of pHZ6 from previously identified pHZ1-pHZ5 was demonstrated by the profile of relevant plasmids in six well-defined strains originated from S. hygroscopicus 10-22.     

Diversity of telomere palindromic sequences and replication genes among Streptomyces linear plasmids

Streptomyces sp. linear plasmids and linear chromosomes usually contain conserved terminal palindromic sequences bound by the conserved telomeric proteins Tap and Tp, encoded by the tap and tpg genes, respectively, as well as plasmid loci required for DNA replication in circular mode when the telomeres are deleted. These consist of iterons and an adjacent rep gene. By using PCR, we found that 8 of 17 newly detected linear plasmids in Streptomyces strains lack typical telomeric tap and tpg sequences. Instead, two novel telomeres in plasmids pRL1 and pRL2 from the eight strains and one conserved telomere in pFRL1 from the other strains were identified, while multiple short palindromes were also found in the plasmids. The complete nucleotide sequence of pRL2 revealed a gene encoding a protein containing two domains, resembling Tap of Streptomyces and a helicase of Thiobacillus, and an adjacent gene encoding a protein similar to Tpg of Streptomyces and a portion of the telomere terminal protein pTP of adenoviruses. No typical iterons-rep loci were found in the three plasmids. These results indicate an unexpected diversity of telomere palindromic sequences and replication genes among Streptomyces linear plasmids.     

The isolation and restriction mapping of a miniplasmid from the Actinomycete Nocardia corallina

Abstract A variety of plasmids has been identified as covalently closed circular and linear DNA in certain Actinomycetes, such as Streptomyces . This paper describes the first isolation and characterisation of a plasmid from the genus Nocardia . The plasmid pKU100 isolated from Nocardia corallina is a cccDNA molecule, 2.7 kb in length. This plasmid has been mapped with a wide variety of restriction enzymes and contains a number of unique restriction sites making it suitable for development as a cloning vector.     

Characterization of replication and conjugation of Streptomyces circular plasmids pFP1 and pFP11 and their ability to propagate in linear mode with artificially attached telomeres

Many Streptomyces species harbor circular plasmids (8 to 31 kb) as well as linear plasmids (12 to 1,700 kb). We report the characterization of two newly detected circular plasmids, pFP11 (35,139 bp) and pFP1 (39,360 bp). As on linear plasmids, their replication loci comprise repA genes and adjacent iterons, to which RepA proteins bind specifically in vitro. Plasmids containing the minimal iterons plus the repA locus of pFP11 were inherited extremely unstably; par and additional loci were required for stable inheritance. Surprisingly, plasmids containing replication loci from pFP11 or Streptomyces circular plasmid SCP2 but not from pFP1, SLP1, or pIJ101 propagated in a stable linear mode when the telomeres of a linear plasmid were attached. These results indicate bidirectional replication for pFP11 and SCP2. Both pFP11 and pFP1 contain, for plasmid transfer, a major functional traB gene (encoding a DNA translocase typical for Streptomyces plasmids) as well as, surprisingly, a putative traA gene (encoding a DNA nickase, characteristic of single-stranded DNA transfer of gram-negative plasmids), but this did not appear to be functional, at least in isolation.     

变铅青链霉菌内源线性质粒接合转移必需功能区的鉴定

通常细菌间环型质粒在接合转移过程中,单链质粒DNA在质粒内部“oriT”接合转移起始位点发生缺刻.随后,打开的单链质粒DNA通过细胞膜的Ⅳ型分泌系统转移到受体菌中.但是,链霉菌中的接合型线型质粒带有游离3′端,5′端与末端蛋白结合,因而不能以细胞-细胞间方式转移单链缺刻DNA.报道了变铅青链霉菌线型质粒SLP2衍生的环型质粒,与SLP2一样可以高频高效接合转移,并鉴定了接合转移功能区.质粒有效的接合转移功能区包含6个共转录的基因,分别编码一个Tra样的DNA转移酶、胞壁水解酶、2个膜蛋白(可以与ATP结合蛋白相互作用)和一个功能未知的蛋白质.从SalⅠR-/M-向SalⅠR/M宿主转移的质粒频率下降表明,线型和环型的质粒都是以双链的形式转移的.上述研究结果表明SLP2衍生的线型质粒和环型质粒以相似的与细胞膜/胞壁功能相关的机理进行接合转移.     

Isolation and characterization of a linear plasmid from Streptomyces rimosus

Abstract A linear plasmid was isolated from a strain of Streptomyces rimosus . This plasmid was separated by agarose gel electrophoresis and its size, about 43 kb, determined both by this method and by electron microscopy. The cleavage pattern of the linear plasmid with 5 restriction endonucleases is given. A protein, which is removed by proteinase K, is probably associated to this plasmid. By ethidium bromides or acridine orange treatment we obtained mutants which had lost their aerial mycelium and their linear plasmid.     

Interspecific transfer of Streptomyces giant linear plasmids in sterile amended soil microcosms

The interspecific transfer of two giant linear plasmids was investigated in sterile soil microcosms. Plasmids pRJ3L (322 kb) and pRJ28 (330 kb), both encoding mercury resistance, were successfully transferred in amended soil microcosms from their streptomycete hosts, the isolates CHR3 and CHR28, respectively, to a plasmidless and mercury-sensitive strain, Streptomyces lividans TK24. Transconjugants of S. lividans TK24 were first observed after 2 to 3 days of incubation at 30 degrees C, which corresponded to the time taken for the formation of mycelia in soil. Transfer frequencies were 4.8 x 10(-4) and 3.6 x 10(-5) CFU/donor genome for pRJ3L and pRJ28, respectively. Transconjugants were analyzed by pulsed-field gel electrophoresis for the presence of plasmids, and plasmid identity was confirmed by restriction digests. Total genomic DNA digests confirmed that transconjugants were S. lividans TK24. The mercury resistance genes were shown to be on the plasmid in the transconjugants by hybridization analysis and were still functional. This is the first demonstration of transfer of giant linear plasmids in sterile soil microcosms. Giant linear plasmids were detected in many Streptomyces spp. isolated from mercury-contaminated sediments from Boston Harbor (United States), Townsville Harbor (Australia), and the Sali River (Tucuman, Argentina). Mercury resistance genes were shown to be present on some of these plasmids. Our findings that giant linear plasmids can be transferred between Streptomyces spp. and are common in environmental Streptomyces isolates suggest that these plasmids are important in gene transfer between streptomycetes in the environment.