Infectious plasmid resistance and efflux pump mediated resistance

Various bacterial plasmids can be eliminated from bacterial species cultured as pure or mixed bacterial cultures by non-mutagenic heterocyclic compounds at subinhibitory concentrations. For plasmid curing, the replication should be inhibited at three different levels simultaneously: the intracellular replication of plasmid DNA, partition and intercellular transconjugal transfer. The antiplasmid action of the compounds depends on the chemical structure. The targets for antiplasmid compounds were analysed in detail. It was found that amplified extrachromosomal DNA in the superhelical state binds more drug molecules than does the linear or open-circular form of the plasmid or the chromosome, without stereospecificity which leads to functional inactivation of the extrachromosomal genetic code. Plasmid elimination also occurs in ecosystems containing numerous bacterial species simultaneously, but the elimination of antibiotic resistance-encoding plasmids from all individual cells of the population is never complete. The medical significance of plasmid elimination in vitro is, it provides a method to isolate plasmid-free bacteria for biotechnology without any risk of mutations, and it opens up a new perspective in rational drug design against bacterial plasmids. Hypothetically, the combination of antiplasmid drugs and antibiotics may improve the effectivity of antibiotics against resistant bacteria; therefore, the results cannot be exploited until the curing efficiency reaches 100%. Inhibition of the conjugational transfer of antibiotic resistance plasmids can be exploited to reduce the spreading of these plasmids in ecosystems.     

黄芩甙对痢疾杆菌R质粒体外消除作用的实验研究

以携带R质粒的痢疾杆菌F_(13)株为靶细菌,以黄芩甙作为R质粒消除剂,进行R质粒体外消除试验。实验组R质粒的消除率为0.3%,对照组中SDS的消除率为0.6%,吖啶橙的消除率0.42%。琼脂糖凝胶电泳结果显示,消除子都丢失了相应的质粒带。     

铜绿假单胞菌PA16株粘附性、菌毛与质粒关系的研究

为探讨PA的质粒与粘附性及质粒与菌毛的关系,围绕PA16株的耐药性与质粒的关系、质粒与菌毛及粘附性的关系作了一系列的研究,结果表明PA16对所测的7种抗生素全部耐药,其MIC＞400 mg/L;PA16仅含有一种27.3 kb(18 Mu)的质粒.转化后此质粒也使JM109获得了对四环素的耐药性.消除此质粒后,PA16对四环素的耐药性消失.粘附试验证明PA16质粒消除株对尿道上皮细胞的粘附能力较野生株显著性减小(P＜0.05),同时,透射电镜照片显示PA16野生株表面有致密、纤细刚直的菌毛,而PA16质粒消除株表面几乎无菌毛可见.     

Plasmid stability in budding yeast populations: Steady-state growth with selection pressure

Plasmid gene product accumulation in a cell population depends on the fraction of plasmid-containing cells and the distribution of single-cell plasmid content. These important population properties have been related to plasmid replication regulation and kinetics and to plasmid segregation rules at the single-cell level using population balance mathematical models. Budding yeast populations are considered in detail because of the practical potential of yeast host-vector systems and because of the model complications introduced by the asymmetric division pattern observed for Saccharomyces cerevisiae at all but the largest growth rates. Solutions are presented for several different reasonable models of plasmid replication and segregation. The results offer potential for identification of important qualitative features of yeast plasmid replication and of model parameter values from average and segregated experimental data on yeast populations.     

Evidence for a chromosomally determined bacteriocin production byKlebsiella pneumoniae

Location of the genes responsible for pneumocin production inKlebsiella pneumoniae was examined by classical procedures. Conjugal intrageneric transfers, elimination experiments with various curing agents, high temperature and plasmid isolation procedures showed that this strain did not harbour any plasmid. Hence chromosomal location of the genetic determinants is suggested.     

细菌质粒的消除

利用化学消除剂或改变生长条件可以消除细菌中的质粒,除宿主菌的特性及其所含质粒分子量大小之外,消除率还与消除剂浓度,作用时间有关,嵌合染料适用于消除大肠杆菌中的质粒,十二烷基硫酸钠对具有性纤毛的细菌作用效果较好,适当提高培养温度可消除一些细菌中的质粒,胸腺嘧啶限量法仅适用于其营养缺陷菌株的质粒消除,利用原生质体的形成与再生及反复冻融菌体均可消除细菌中的质粒。     

Plasmid ColVBtrp maintenance in Erwinia carotovora.

Plasmid ColVBtrp maintenance in Erwinia carotovora cells was followed by measuring kinetics of elimination of plasmid genetic markers and loss of plasmid deoxyribonucleic acid. An E. carotovora mutant stably carrying plasmid ColVBtrp was isolated. Besides stable plasmid maintenance, the mutant showed altered sensitivity to male-specific phage MS2, sensitivity to drugs, and colony morphology.     

A segregated model for plasmid content and product synthesis in unstable binary fission recombinant organisms

Plasmid propagation in populations of unstable, binary fission recombinant organisms has been studied using a segregated, population balance mathematical model. Segregated models have the advantage of direct incorporation of basic information on mechanisms and kinetics of plasmid replication and segregation at the single-cell level. The distribution of cellular plasmid content and specific rates of plasmid gene expression have been obtained for several single-cell models of plasmid replication, partition, and gene expression. Plasmid replication kinetics during cell growth significantly influence the plasmid content distribution. In the case of transient growth of plasmid-containing and plasmid-free cells in partially selective medium, the degree of selection required for stable maintenance of plasmid-containing cells has been determined. Guidelines are presented for applicability of simpler, nonsegregated models and for evaluation of the parameters in these models based on single-cell mechanisms and associated parameters.     

Involvement of the cell envelope in plasmid maintenance: Plasmid curing during the regeneration of protoplasts

Observations are described that demonstrate elimination of certain plasmids in up to 80% of Staphylococcus aureus cells during the formation and regeneration of lysostaphin-induced protoplasts of these organisms. All of nine small (≤3 megadaltons (Mdal)) plasmids studied showed the protoplast-dependent elimination to a greater or lesser extent; none of three larger (≤15 Mdal) plasmids showed the effect. This difference in behavior was not due to molecular weight per se, as curing was not shown by one of the large plasmids with a deletion of two-thirds of its genome but was shown by a chimera consisting of a 3-Mdal plasmid with a 5.7-Mdal DNA insertion. The curing effect was not related to copy number, as all of the curable plasmids have substantially greater copy numbers than the noncurable ones. Physical loss of plasmid DNA from the protoplasts could not be demonstrated; replication of plasmids in protoplasts appeared normal; but most of the plasmid-positive regenerant colonies consisted of mixed populations of plasmid-positive and negative organisms with a very wide range of composition. On the basis of these observations, we conclude that plasmid elimination occurs during the several protoplast divisions that occur before cell wall regeneration is completed and that it is due to a disruption of the plasmid partition system as a consequence of removal of the cell wall. If so, then the noncurable plasmids must be partitioned by a mechanism that is different from that by which the curable ones are normally partitioned.     

Analysis of unstable recombinant Saccharomyces cerevisiae population growth in selective medium

Widely applied selection strategies for plasmid-containing cells in unstable recombinant populations are based upon synthesis in those cells of an essential, selection gene product. Regular partitioning of this gene product combined with asymmetric plasmid segregation produces plasmid-free cells which retain for some time the ability to grow in selective medium. This theory is elaborated here in terms of a segregated model for an unstable recombinant population which predicts population growth characteristics and composition based upon experimental data for stable strain growth kinetics, plasmid content, and selection gene product stability. Analytical solutions from this model are compared with an unsegregated phenomenological model to evaluate the effective specific growth rate of plasmid-free cells in selective medium. Model predictions have been validated using experimental growth kinetics and flow cytometry data for Saccharomyces cerevisiae D603 populations containing one of the plasmids YCpG1ARS1, YCpG1DeltaR8, YCpG1DeltaR88, YCpG1DeltaH103, YCpG1DeltaH200, pLGARS1, and pLGSD5. The recombinant strains investigated encompass a broad range of plasmid content (from one to 18 plasmids per cell) and probability alpha of plasmid loss at division (0.05 相似文献   

Elimination of plasmid-linked polyglutamate production by Bacillus subtilis (natto) with acridine orange

Treatment of Bacillus subtilis (natto) strains Asahikawa, F, and M with acridine orange resulted in the conversion of approximately 64.2% of the Asahikawa population, 22.4% of the F population, and 9.2% of the M population to polyglutamate-nonproducing colonies. Such curing is suggestive of the involvement of plasmid DNA. Samples of cleared lysates of both parental and their cured strains were subjected to agarose gel electrophoresis to determine the plasmid composition. Parental strains were found to possess a plasmid, but polyglutamate-nonproducing derivatives were missing the plasmid. The plasmid-linked polyglutamate production, which was originally isolated from B. subtilis (natto), could be transformed in B. subtilis.     

Elimination of plasmid-linked polyglutamate production by Bacillus subtilis (natto) with acridine orange.

Treatment of Bacillus subtilis (natto) strains Asahikawa, F, and M with acridine orange resulted in the conversion of approximately 64.2% of the Asahikawa population, 22.4% of the F population, and 9.2% of the M population to polyglutamate-nonproducing colonies. Such curing is suggestive of the involvement of plasmid DNA. Samples of cleared lysates of both parental and their cured strains were subjected to agarose gel electrophoresis to determine the plasmid composition. Parental strains were found to possess a plasmid, but polyglutamate-nonproducing derivatives were missing the plasmid. The plasmid-linked polyglutamate production, which was originally isolated from B. subtilis (natto), could be transformed in B. subtilis.     

Sequential heat-curing of Tn5-Mob-sac labelled plasmids from Rhizobium to obtain derivatives with various combinations of plasmids and no plasmid

All four plasmids from Rhizobium leguminosarum bv. trifolii W14-2 were sequentially labelled with Tn 5 -Mob- sac , which codes for resistance to kanamycin and sensitivity to sucrose, and cured by exposing rhizobia to supra-optimal temperatures. This is the first report where a plasmid-less derivative of Rhizobium was obtained. No relationship was found between plasmid size and elimination. The efficiency of Hynes' selection system decreased when used to cure plasmids from rhizobial derivatives already lacking one plasmid or more. The authors' results suggest that previous reports of failure in curing Tn 5 -Mob- sac labelled plasmids may only be due to an insufficient number of colonies being screened.     

A mathematical model for plasmid replication and distribution in microbial populations

A mathematical model and a computer program for its implementation have been developed to predict the distribution of plasmid copy numbers in the individual cells of a microbial population. The kinetics of accumulation of plasmid-free cells. the copy number distribution within the population and the mean copy number can all be calculated using the computer program. The model has been shown to accurately predict these parameters for recombinant plasmids in yeast populations.     

Antagonism of the B subunit of DNA gyrase eliminates plasmids pBR322 and pMG110 from Escherichia coli.

The constructed plasmid pBR322 and the native plasmid pMG110 were eliminated (cured) from growing Escherichia coli cells by the antagonism of the B subunit of the bacterial enzyme DNA gyrase. The antagonism may be by the growth of cells (i) at semipermissive temperatures in a bacterial mutant containing a thermolabile gyrase B subunit or (ii) at semipermissive concentrations of coumermycin A1, an antibiotic that specifically inhibits the B subunit of DNA gyrase. The kinetics of plasmid elimination indicate that plasmid loss occurs too rapidly to be explained solely by the faster growth of that plasmid-free bacteria and, therefore, represents interference with plasmid maintenance.     

Filamentation promotes F'lac loss in Escherichia coli K12.

The stability of plasmid F'lac in Escherichia coli strain SP45 (a temperature conditional mutant which grows as spherical cells at 42 degrees C and as a rod at 30 degrees C) was studied. F'lac elimination was demonstrated when bacteria exposed to subinhibitory concentrations of various chemicals were induced to form filaments. No plasmid loss was found when spherical cells were subjected to the same treatments. Plasmid loss was also observed in dnaA46 and lexA41 mutants when cell filamentation was induced at 42 degrees C, but not when they were cultured at 30 degrees C. Nalidixic acid promoted F'lac elimination at 0.25 micrograms ml-1 in a recA13 mutant and at 1.5 micrograms ml-1 in the recA+ counterpart. A marked difference was found in the rate of F'lac elimination from thermosensitive DNA gyrase mutants [gyrA43(Ts) and gyrB41(Ts)] between rods and their spherical (rodA51) derivatives growing at semipermissive temperature (36.5 degrees C). Plasmids carrying the ccd segment of F in DNA gyrase mutants were lost after 2.5 generations from rods and after 6 generation from spherical cells. Plasmid segregation into non-viable minicell-like elements was found after induction of filaments. These data suggest that plasmid stability is correlated with cell shape and that curing is more easily achieved when bacteria can elongate normally.     

Dynamics of plasmid transfer on surfaces

A protocol was developed to study the dynamics of growth and plasmid transfer in surface populations of bacteria. This method allows for quantitative estimates of cell population densities over time, as well as microscopic observations of colony growth and interactions. Using this 'surface slide system' (SSS), the dynamics of the plasmid R1 and its permanently derepressed mutant R1drd19 in surface cultures of Escherichia coli K12 was examined. In surface culture, the stationary-phase cell densities were constant over a wide range of initial cell density (= colony density) and comparable to those obtained in liquid culture. For high initial cell densities, where the cells formed a confluent layer at stationary phase, the kinetics of growth and plasmid transfer was similar to that obtained in liquid culture, and the relative yields of R1drd19 and R1 transconjugants were similar in the two habitats. In surface culture, however, R1drd19 transconjugant yield was profoundly affected, and R1 transfer to a lesser extent, by colony density. In contrast, liquid matings were virtually unaffected by initial cell density. The transfer advantage of the permanently depressed over the repressed plasmid was much less apparent for lower colony densities. I propose a hypothesis for plasmid transfer between colonies that explains these observations as a consequence of the geometry of the surface habitat and the effect of transitory derepression of the synthesis of pili.     

Effects of the Recipient Strain and Ultraviolet Irradiation on Transduction Kinetics of the Penicillinase Plasmid of Staphylococcus aureus

When the penicillinase plasmid of Staphylococcus aureus PS 81(P(81))(T(81)) was transferred to its cured derivative of PS 81(N(P))(T(81)), there was a fivefold increase in the transduction frequency of penicillinase plasmid markers after ultraviolet (UV) irradiation of the phage instead of the expected decrease typical for plasmid-borne markers. These results were independent of the transducing phage, the donor, and the method of curing the recipient and were also obtained with a cured derivative of PS 80(PI(80)). With PS 52, a naturally occurring penicillin-sensitive strain, and a cured transductant of PS 52 as the recipients, typical plasmid kinetics were observed. The plasmid location of penicillinase plasmid markers in transductants was confirmed by their instability in ethidium bromide (EB). In a cross between isogenic plasmids (PI(258)penZ cad x PI(258)penI asa ero), transductants were doubly selected for cadmium and erythromycin resistances. There was a twofold increase in transduction frequency after UV irradiation of the transducing phage and an increase in the proportion of recombinant type transductants. CsCl-EB density centrifugation revealed that plasmid deoxyribonucleic acid (DNA) was present in PS 81(P(81))(N(T)) and its cured derivative [PS 81(N(P))(N(T))], but not in PS 52. Sucrose gradient analysis of plasmid DNA showed that the penicillinase plasmid of PS 81(P(81))(N(T)) was larger than the plasmid in its cured derivative. Thus, the cured derivative contains plasmid DNA which appears to recombine with the incoming plasmid, causing the rise in transduction frequency noted after UV irradiation of transducing phage.     

重组类人胶原蛋白工程菌补料发酵过程建模

对产类人胶原蛋白的重组大肠杆菌Escherichia coli(E. Coli) 的批式和分批-补料培养动力学进行了研究。通过检测发酵过程的基质浓度、菌体量和产物浓度,建立了一组反映发酵的动力学模型,并考虑了非工程菌存在的影响,分析了细胞生长、底物消耗、基因工程产物生成的过程,结果显示该动力学模型可以很好的拟合发酵过程。     

水/乙醇混合溶剂中的阿奇霉素结晶动力学

利用粒数密度和粒度之间的关系判别晶体生长模型;采用间歇动态法,以粒数衡算方程、溶质质量守恒和McCabe定律为基础,利用Beer-Lambert定律,借助光学关联的方法,建立了包含透光率变量的伴有成核和晶体生长的动力学模型;通过在线测量溶液密度与透光率数据,采用非线性最小二乘法拟合得到了晶体成核和生长动力学经验方程,并以实时浓度为目标验证了动力学参数的准确性以及模型表达式的正确性。