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。     

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.     

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.     

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.     

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.     

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%.     

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.     

Subcellular positioning of F plasmid mediated by dynamic localization of SopA and SopB

SopA, SopB proteins and the cis-acting sopC DNA region of F plasmid are essential for partitioning of the plasmid, ensuring proper subcellular positioning of the plasmid DNA molecules. We have analyzed by immunofluorescence microscopy the subcellular localization of SopA and SopB. The majority of SopB molecules formed foci, which localized frequently with F plasmid DNA molecules. The foci increased in number in proportion to the cell length. Interestingly, beside the foci formation, SopB formed a spiral structure that was dependent on SopA, which also formed a spiral structure, independent of the presence of SopB, and these two structures partially overlapped. On the basis of these results and previous biochemical studies together with our simulations, we propose a theoretical model named "the reaction-diffusion partitioning model", using reaction-diffusion equations that explain the dynamic subcellular localization of SopA and SopB proteins and the subcellular positioning of F plasmid. We hypothesized that sister copies of plasmid DNA compete with each other for sites at which SopB multimer is at the optimum concentration. The plasmid incompatibility mediated by the Sop system might be explained clearly by this hypothesis.     

Curing of four different plasmids in Yersinia pestis using plasmid incompatibility

Aims: Plasmids are critical for the pathogenicity of Yersinia pestis. In order to carry out a systematic investigation of their role in pathogenesis, we cured plasmids from Y. pestis. Methods and Results: Each plasmid’s replicon of Y. pestis was cloned into plasmid pEX18Gm containing a counter‐selectable sacB gene, and was then introduced into Y. pestis strain 201 by electroporation. Strains containing recombinant plasmids were cultivated under antibiotic selection. The resultant plasmid‐curing colonies, identified by specific polymerase chain reactions, were then cured off pEX18Gm under sucrose pressure. This method was used to successfully cure all four plasmids of Y. pestis, singly or in different combinations. Conclusions: Naturally evolving plasmids in Y. pestis are difficult to remove by conventional curing methods. We employed a method based on plasmid incompatibility to cure the plasmids from Y. pestis, which confirmed the efficacy of this method for curing plasmids with different types of replicons from one bacterium. Significance and Impact of the Study: There have been no reports on the curing of multiple plasmids by using replication mechanisms from one bacterium with this technique. In the present study, we were able to successfully apply this methodology to cure four plasmids from Y. pestis, confirming its feasibility.     

Sequence of the 68,869 bp IncP-1α plasmid pTB11 from a waste-water treatment plant reveals a highly conserved backbone, a Tn402-like integron and other transposable elements

To analyse the significance of conjugative broad-host-range IncP-1α plasmids for the spread of antibiotic resistance determinants in waste-water treatment plants we isolated and characterised five different IncP-1α plasmids from bacteria of activated sludge and the final effluents of a municipal waste-water treatment plant. These plasmids mediate resistance to ampicillin, cefaclor, cefuroxime, gentamicin, kanamycin, spectinomycin, streptomycin, tetracycline, tobramycin, and trimethoprim. The complete 68,869 bp DNA-sequence of the IncP-1α plasmid pTB11 was determined. The pTB11 backbone modules for replication (Rep), mating pair formation (Trb), multimer resolution (Mrs), post-segregational killing (Psk), conjugative DNA-transfer (Tra), plasmid control (Ctl), and stable maintenance and inheritance (KilA, KilE, and KilC) are highly conserved as compared to the ‘Birmingham’ IncP-1α plasmids. In contrast to the ‘Birmingham’ plasmids pTB11 carries an insert of a Tn402-derivative integrating a class 1 integron in the intergenic region between the multimer resolution operon parCBA and the post-segregational killing operon parDE. The integron comprises the resistance gene cassettes oxa2 (β-lactamase), aacA4 (aminoglycoside-6′N-acetyltransferase), and aadA1 (aminoglycoside-3′-adenylyltransferase) and a complete tniABQR transposition module. Integron-specific sequences were also identified on other IncP-1α plasmids analysed in this work. In contrast to the ‘Birmingham’ plasmids the pTB11 tetracycline resistance module carries a pecM- and a pncA-like gene downstream of the tetracycline resistance gene tetA and contains an insertion of the new insertion sequence element ISTB11. The transposable elements IS21 and Tn1 which disrupted, respectively, orf7 and klcB on the ‘Birmingham’ plasmids are not present on pTB11. Identification of IncP-1α plasmids in bacteria of the waste-water treatment plant’s final effluents indicates that bacteria carrying these kind of plasmids are released into the environment.     

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 pLV1 0 1 6由快生型大豆根瘤菌 (Rhizobiumfredii)QB1 1 31向R .frediilux3的水平转移及pLV1 0 1 6由QB1 1 31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 pLV1 0 1 6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 .     

萘降解质粒ND1.860和NAH7的DNA同源性研究

通过DNA:DNA杂交技术,用NAH7质粒的全部EcoR Ⅰ片段或ECOR Ⅰ A片段作为~(32)P标记的DNA探针,研究了萘降解质粒ND1.860和NAH7之间的DNA同源性。在ND1.860的9个HindⅢ片段中,5个与NAH7有同源性,其中3个与NAH7的编码萘降解途径的EcoR Ⅰ A片段有同源性。     

Relation between Cyclopropane Fatty Acid Formation and Saturation of Phospholipid Species in Escherichia coli K-12

Alterations in the degrees of saturation of phosphatidylethanolamine, phosphatidylglycerol and cardiolipin of Escherichia coli K–12 were determined after raising or lowering the growth temperature during the exponential growth phase. After raising the growth temperature from 17 to 42°C, the cells continued to grow with increasing degrees of saturation of the three phospholipids. cis-9,10-Methylenehexadecanoic acid increased only in phosphatidylethanolamine. During growth after lowering the growth temperature from 42 to 17°C, no increase was found in cyclopropane fatty acid content of phosphatidylethanolamine, in which cis-vaccenic acid increased. Significance of cyclopropane fatty acid formation in phospholipids was discussed.     

一种简便的适用于酵母双杂交系统的酵母质粒提取方法

目的:建立一种适用于酵母双杂交系统的简便快捷的酵母质粒提取方法。方法:以酿酒酵母为供试材料,用玻璃珠振荡法破除酵母细胞壁,提取酵母总DNA,最后通过电转化大肠杆菌DH10B获得目的质粒。结果:粗提得到的质粒可直接转化DH10B,作为模板用于PCR分析及酵母双杂交后续的序列分析等,大大降低了工作量。结论:该方法简便快捷,经济实用,降低了成本,提高了效率,可以作为一种实验室酵母质粒提取方法。     

带有sop基因稳定表达载体的构建

F质粒的第五个EcoRⅠ片段mini-F具有质粒的分配功能。其EcoRⅠ-BamHⅠ片段含有oriS、ccd、repD和sop基因(sopA,B和C)。该片段与pBR322重组,得到质粒pDMC32。pDMC32经SmaⅠ酶切,T4连接酶连接,得到衍生质粒pDMC311,消除了oriS、ccd和repD片段。MI32(pDMC32)和MI311(pDMC311),在液体限磷基础培养基中培养100代,质粒保持率分别为93％和100％。而对照MIR322(pBR322)培养55代时,质粒保持率仅为10％。带有色氨酸启动子质粒pDR720与pBR322重组,得到质粒pDMC40。pDMC40再与mini-F的sop基因重组,得到带有sop基因的稳定表达质粒pDMC48。MI48(pDMC48)在液体限磷基础培养基中培养100代,质粒保持率为100％。     

Osa protein encoded by plasmid pSa is located at the inner membrane but does not inhibit membrane association of VirB and VirD virulence proteins in Agrobacterium tumefaciens

Abstract The osa gene of IncW plasmid pSa encodes a 21-kDa protein that completely abolishes the oncogenic activity encoded by virulence genes in Agrobacterium tumefaciens. osa is the last gene of a four-gene operon in pSa, the expression of which appears to be highly regulated since the Osa protein is absent when either pSa or the osa operon is present in the Agrobacterium cell. When the osa gene alone or together with upstream genes within the operon are expressed under the control of a constitutive promoter, Osa protein is produced, enabling us to determine its subcellular location. Immunoblot analyses located Osa protein at the inner membrane of both A. tumefaciens and Escherichia coli . Because Osa inhibits oncogenicity of A. tumefaciens , and because alterations of the products of the virB and virD genes affect oncogenicity, studies were conducted to determine if there are changes in their specific association with the membranes in the presence Osa. Immunoblot analyses of VirB2, VirB3, VirB4, VirB9, and VirD4 in the presence and absence of Osa revealed no differences between the two treatments in these Vir protein associations with the membranes. These results indicate that both virB and virD gene products are produced in the presence of Osa; that they appear unaffected in their association with the membranes; and that Osa is associated with the inner membrane, where VirB2, VirB4, and VirD4 proteins are also located.