土壤中质粒pLV1016 在快生型大豆根瘤菌间的水平转移

在滤膜、液体培养基和土壤微宇宙3种系统中,研究了接合型质粒pLV1016 由快生型大豆根瘤菌(Rhizobiumfredii)QB1131 向R.frediilux Lux3的水平转移及pLV1016 由QB1131 向土著细菌的转移.接合培养1d后,分别计算供、受体菌的生长速率和质粒转移速率常数(γ).结果表明,相同接种浓度下,滤膜接合时γ值最高,土壤中γ值最低,γ值不受土壤是否灭菌和是否有大豆植株的影响,γ值与初始接种浓度负相关,与供、受体的生长速率正相关.在未灭菌土中检测到pLV1016 可转移到土著细菌,土著接合子分别属于根瘤菌属和假单胞菌属.     

利用发光酶基因监测根瘤菌重组质粒的转移性

经三亲本杂交,比较测定了重组大豆根瘤菌HN01DNL和TA11DNL中所含重组质粒pHN307在人工滤膜和灭菌土壤杂交条件下、向华癸中生根瘤菌7653R和荧光假单胞菌Pf.X1-5的转移频率;并初步跟踪了pHN307在根盒-土壤缩影、小区试验和环境释放中向土著细菌的转移性,为考察所构建重组根瘤菌在田间应用时的安全性提供了一定的实验依据。     

几类快生型根瘤菌质粒的研究

用一种重现性好而较简便的方法,对国内的紫云英、豌豆、三叶草、大豆等快生型根瘤菌及根癌农杆菌共40多号菌进行了质粒分离,所得质粒图谱具有一定的菌株特异性。每株菌有1—3个质粒,其中至少有一个大质粒或巨大质粒。用质粒消除法获得失去结瘤(或致癌)能力的变异株,均缺少一个大质粒。快生型根瘤菌的结瘤基因定位在大质粒或巨大质粒上。用接合法将Rpl：：Tn501等质粒转入根瘤菌中,能诱动根瘤菌的结瘤基因转移给失去结瘤能力的变异株,可表达功能,质粒图谱也发生变化,与供体或受体都不同。     

xylE基因用于监测根瘤菌在土壤中存活的研究

通过细菌接合将质粒ｐＬＶ１０１６（含ｘｖｌＥ基因）转移至ＲｈｉｚｏｂｉｕｍｆｒｅｄｉｉＱＢ１１３０和Ｒｈｉｚｏｂｉｕｍｌｅｇｕｍｉｎｏｓａｒｕｍｂｖ．ｖｉｃｉａｅＢ４０,ｘｙｌＥ基因在根瘤菌内表达活性较高．质粒ｐＬＶ１０１６携带的ＸｙｌＥ基因用于监测根瘤菌在灭菌和未灭菌土壤中的存活,结果表明,在灭菌土壤中含质粒与不含质粒菌株存活菌数量之间无显著差异（Ｐ＜０．０５）,大接种量有利于细菌的生长与繁殖,Ｂ４０（ｐＬＶ１０１６）质粒丢失比例很低．以低于１０６ＣＦＵ·ｇ－１干土浓度接种时,ＱＢ１１３０（ｐＬＶ１０１６）质粒丢失率随接种浓度的降低而增大．未灭菌土壤中生物因素抑制了释放菌株的生长,大接种量有利于细菌存活．以低浓度（１０６ＣＦＵ·ｇ－１干土）接种时,ＱＢ１１３０（ｐＬＶ１０１６）质粒丢失比例高于Ｂ４０（ｐＬＶ１０１６）．     

三叶草素基因(tfx)在快生型大豆根瘤菌(Sinorhizobium fredii)H12-2中的表达与稳定性测定

将含有三叶草条基因的重组质粒pT2TFXK和pT2TX3K以接合转移的方式导入快生型大豆根瘤菌H12－2。转移接合子H12－2（pXK）能产三叶草素并具有抑菌活性;H12－2（P3K）表现出对三叶草素的抗性。抑菌谱试验结果表明：82％的供试快生型大豆根瘤菌菌株对三叶草素敏感;所有供试慢生型大豆根瘤菌则表现抗性。稳定性检测结果表明：在共生与人工培养条件下,导入的pXK和p3K质粒均可在宿主菌中稳定存在和表达。     

豌豆根瘤菌共生基因pJB5JI与华癸中生根瘤菌7653R共生质粒的相互关系

华癸中生根瘤菌(Mesorhizobium huakuii)7653R是分离自我国南方水稻田的一株根瘤菌,含有2个内源质粒:p7653Ra和p7653Rb,其中7653Rb是共生质粒.通过Tn5-sacB的插入方法来消除质粒,获得7653Rb消除的突变株7653RD.将豌豆根瘤菌T83K3的共生质粒pJB5JI导入7653R和7653RD中,盆栽结果表明含有pJB5JI的转移接合子7653R-197的竞争结瘤能力和共生固氮能力均高于7653R.pJB5JI不能恢复7653RD在紫云英上的结瘤能力.含有pJB5JI的7653RD可以在豌豆上结无效瘤,表明pJB5JI可以在7653R的染色体背景下表达其功能.对转移接合子中的质粒稳定性进行检测,结果表明pJB5JI在人工传代的情况下可以稳定存在,但经过共生之后发生了遗传分离,对转移接合子和出发菌株及分离菌株进行kan基因的PCR扩增,除了受体菌外其他菌株都可得到PCR产物,由此推测,pJB5JI可能部分或全部整合到了受体菌的染色体基因组中.     

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.     

接种S.fredii WGF03及突变体对大豆土壤微生物群落结构的影响

为了研究接种S.fredii WGF03及其exo D基因突变体对大豆结瘤及土壤的微生物群落影响,进一步了解exo D基因的功能,在大豆盛花期摘取每株大豆的根瘤并计数,利用PCR-DGGE电泳结合测序技术分析土壤的微生物群落。结果表明,大豆接种S.fredii WGF03后,根瘤数比不接种组增加191.67%。而大豆接种驻exo D突变体的根瘤数最少,比不接种组减少了16.67%。与空白相比,种植大豆后土壤细菌的种类和数量明显增加;接种不同根瘤菌后,细菌种类及细菌多样性也有变化;测序结果显示,土壤中细菌以Proteobacteria为主,占45.5%,土壤中土著根瘤菌为Bradyrhizobium。总之,S.fredii WGF03能够促进大豆结瘤,种植作物比接种根瘤菌对土壤细菌群落的影响更大。     

三叶草素基因（tfx）在快生型大豆根瘤菌（Sinorhizobium fredii）H12— …

将含有三叶草素基因的重组质粒ｐＴ２ＴＦＸＫ和ｐＴ２１Ｘ３Ｋ以接合转移的方式导入快生型大豆根瘤菌Ｈ１２－２,转移接合子Ｈ１２－２（ｐＸＫ）能产生三叶草素并具有抑菌活性;Ｈ１２－２（ｐ３Ｋ）表现出对三叶草素的抗性。抑菌谱试验结果表明：８２％的供试快生型大豆根瘤菌株对三叶草素敏感;所有供试慢生型大豆根瘤菌则表现抗性。稳定性检测结果表明,在共生与人工培养条件下,导入的ｐＸＫ和ｐ３Ｋ质粒均可在宿主菌中稳定存     

质粒介导重排子的发现、结构和功能

细菌耐药性已成为全球关注的重大公共卫生问题。接合转移是耐药质粒快速传播的重要方式,其中Ⅳ型菌毛在此过程中发挥着重要的作用。Ⅳ型菌毛具有黏附作用,能够使细菌黏附在宿主细胞和其他介质表面,帮助细菌形成生物被膜、细菌聚集体和微菌落,是细菌在液体中接合转移的重要因素。PilV蛋白是R64质粒Ⅳ型菌毛尖端黏附素,可以识别细菌细胞膜上的脂多糖(lipopolysaccharide, LPS)。重排子(shufflon)结构是多重DNA倒位系统,可使PilV蛋白多样化,进而影响细菌液体接合转移过程中的受体菌识别和接合转移频率。重排子结构最先发现存在于IncI1质粒R64,后陆续在IncI2、IncK、IncZ质粒以及伤寒沙门氏菌致病岛中被发现,其主要由A、B、C、D这4个片段及特异性重组位点sfx组成,受其下游重组酶基因rci的调控,不同质粒的重排子结构存在一定的差异。近期备受关注的可转移多黏菌素耐药基因mcr-1主要位于IncI2型质粒上,重排子可能是其快速传播的原因之一。本文综述了质粒介导重排子的发现、结构、功能和流行,期望基于对重排子更全面深入地了解,能够为耐药质粒的传播机制和控制策略研究提供...     

Frequent conjugative transfer accelerates adaptation of a broad-host-range plasmid to an unfavorable Pseudomonas putida host

IncP-1 plasmids are known to be promiscuous, but it is not understood if they are equally well adapted to various species within their host range. Moreover, little is known about their fate in bacterial communities. We determined if the IncP-1beta plasmid pB10 was unstable in some Proteobacteria, and whether plasmid stability was enhanced after long-term carriage in a single host and when regularly switched between isogenic hosts. Plasmid pB10 was found to be very unstable in Pseudomonas putida H2, and conferred a high cost (c. 20% decrease in fitness relative to the plasmid-free host). H2(pB10) was then evolved under conditions that selected for plasmid maintenance, with or without regular plasmid transfer (host-switching). When tested in the ancestral host, the evolved plasmids were more stable and their cost was significantly reduced (9% and 16% for plasmids from host-switched and nonswitched lineages, respectively). Our findings suggest that IncP-1 plasmids can rapidly adapt to an unfavorable host by improving their overall stability, and that regular conjugative transfer accelerates this process.     

A novel metabolic pathway for creatinine degradation in Pseudomonas putida 77

Abstract A simple and rapid method is described to determine the plasmid content of cyanobacteria. This procedure is a modification of the Eckhardt in-well lysis and agarose gel electrophoresis technique and can be used for both unicellular and filamentous cyanobacteria.     

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。     

Location of a second partitioning region (ParL) on the F plasmid

Abstract The leading region of the F plasmid has been found to extend the maintenance of the normally unstable plasmid vector pACYC184. This ability is due to effective partitioning of plasmid molecules at cell division. Cloning, deletion analysis and transposon mutagenesis have located the partitioning region (ParL) to be encoded within 63.65–64.11F. Complementation studies indicated that parL is a cis -acting locus.     

Investigation of subpopulation heterogeneity and plasmid stability in recombinant escherichia coli via a simple segregated model

Many microbial and cell cultures exhibit phenomena that can best be described using a segregated modeling approach. Heterogeneties are more marked in recombinant cell cultures because subpopulations, which often exhibit different growth and productivity characteristics, are more easily identified by selective markers. A simple segregated mathematical model that simulates the growth of recombinant Escherichia coli cells is developed. Subpopulations of different growth rate, plasmid replication rate, and plasmid segregation probability are explicitly considered. Results indicate that a third mechanism of plasmid instability, referred to here as a "downward selective pressure," is significant when describing plasmid loss in batch and chemostat cultures. Also, the model agrees well with experimental data from cultures under antibiotic selective pressure. Finally, model simulations of chemostat cultures reveal the importance of initial conditions on culture stability and the possible presence of nonrandom partitioning functions. (c) 1993 John Wiley & Sons, Inc.     

水稻内生链霉菌中的线型和环型质粒

植物内生放线菌的研究是一个近年来兴起的学科领域,在进一步探索和开发微生物资源方面,植物内生放线菌逐渐成为相关领域同行的关注热点.     

Incompatibility behavior of a symbiotic plasmid pMH7653Rb in <Emphasis Type="Italic">Mesorhizobium huakuii</Emphasis> 7653R

Mesorhizobium huakuii strain 7653R harbored two indigenous plasmids named pMH7653Ra and pMH7653Rb. The larger plasmid pMH7653Rb (symbiotic plasmid) was transferred to M. huakuii HN308SR harboring three plasmids: pMHHN308a, pMHHN308b and pMHHN308c, and HN3015SR harboring three plasmids: pMHHN3015a, pMHHN3015b and pMHHN3015c by tri-parent mating. Two stable indigenous plasmids, pMHHN308b and pMHHN308c of HN308SR, were co-eliminated due to the introduction of pMH7653Rb, and the transconjugant was named HN308SRN14. The results implied that pMH7653Rb and pMHHN308b, pMHHN308c were incompatible and might have been ascribed to the same incompatible group. The plasmid profiles of transconjugant HN3015SRN14 showed that the second largest plasmid pMHHN3015b of HN3015SR was cured due to the introduction of pMH7653Rb. The results also implied that pMH7653Rb and pMHHN3015b were incompatible. Results from plant nodulation tests showed that pMH7653Rb could only maintain the nodulation ability in transconjugant HN308SRN14 and its nodule number was more than that of wild strain HN308SR, but could not replace the nitrogen fixation effect of pMHHN308b and pMHHN308c. The plasmid cured mutant HN308SRN14D harboring only pMHHN308a formed null nodules that demonstrated pMHHN308a was relevant to nodulation ability. HN3015SRN14 harboring pMH7653Rb, pMHHN3015a and pMHHN3015c formed null nodules while HN3015SRN14D containing pMHHN3015a and pMHHN3015c lost the nodulation ability. The plasmid replication repC-like gene sequences were detected by a polymerase chain reaction from 7653R, HN308, HN3015, HN308SRN14 and HN3015SRN14. The repC gene sequence similarities of the strains tested attained 99%.     

水稻植物内生链霉菌中线型和环型质粒的检测

以广东番禺和五山地区水稻植株中分离到的内生链霉菌为对象,调查可能存在的内源质粒.利用脉冲电泳技术从8个菌株中检测到大小在60 kb～410 kb的线型质粒,其中4个菌株的线型质粒可能有保守的端粒复制基因.该结果与土壤链霉菌中检测到线型质粒和具有保守端粒复制基因的比例相似,表明水稻植物组织内部的独特环境不会造成链霉菌线型质粒的多样性分布产生大的变化.此外,从13个菌株中检测到6 kb～60 kb的环型质粒.