Mechanism of plasmid ColEl and pMB-9 mobilization by plasmid F'lac+ in Escherichia coli K-12]

The genetic control and mechanism of mobilization of the non-conjugative plasmids ColE1 and pMB-9 by the conjugative plasmids was orived to be recA-independent process in contrast to the mobilization of the chromosomal marker pro. Acridine orange and ethidium bromide curing data together with the results of electrophoretic analysis of plasmid DNA suggest that the plasmids F' lac+ and pMB-9 as well as F' lac+ and ColE1 remain autonomous after their contransfer to recipient cells. These data argue in favour of non-recombinational nature of the plasmid mobilization process. The possibility of transmission of a non-conjugative plasmid without transmission of a conjugative one from the donor strain carrying both plasmids was established. The results obtained are discussed with respect to the hypothesis on the effect of diffusible products encoded by the conjugative plasmid and required of the mobilization of the non-conjugative plasmid.     

Amelioration of the cost of conjugative plasmid carriage in Eschericha coli K12

Although plasmids can provide beneficial functions to their host bacteria, they might confer a physiological or energetic cost. This study examines how natural selection may reduce the cost of carrying conjugative plasmids with drug-resistance markers in the absence of antibiotic selection. We studied two plasmids, R1 and RP4, both of which carry multiple drug resistance genes and were shown to impose an initial fitness cost on Escherichia coli. To determine if and how the cost could be reduced, we subjected plasmid-containing bacteria to 1100 generations of evolution in batch cultures. Analysis of the evolved populations revealed that plasmid loss never occurred, but that the cost was reduced through genetic changes in both the plasmids and the bacteria. Changes in the plasmids were inferred by the demonstration that evolved plasmids no longer imposed a cost on their hosts when transferred to a plasmid-free clone of the ancestral E. coli. Changes in the bacteria were shown by the lowered cost when the ancestral plasmids were introduced into evolved bacteria that had been cured of their (evolved) plasmids. Additionally, changes in the bacteria were inferred because conjugative transfer rates of evolved R1 plasmids were lower in the evolved host than in the ancestral host. Our results suggest that once a conjugative bacterial plasmid has invaded a bacterial population it will remain even if the original selection is discontinued.     

The evolution of a conjugative plasmid and its ability to increase bacterial fitness

Conjugative plasmids are extra-chromosomal DNA elements that are capable of horizontal transmission and are found in many natural isolated bacteria. Although plasmids may carry beneficial genes to their bacterial host, they may also cause a fitness cost. In this work, we studied the evolution of the R1 plasmid and we found that, in spite of the R1 plasmid conferring an initial cost to its host, after 420 generations the cost disappeared in all five independent evolution experiments. In fact, in two of these five experiments evolved conjugative plasmids actually conferred a fitness advantage to their hosts. Furthermore, the relative fitness of the ancestral clone bearing one of the evolved plasmids is significantly higher than both the plasmid-free ancestral cells and the evolved cells carrying the evolved plasmid. Given that the R1 plasmid may spread among different species of enterobacteria, we wondered what the effect of the evolved plasmid would be inside Salmonella enterica cells. We found that the evolved plasmid is also able to dramatically increase the relative fitness of these cells. Our results suggest that even if general usage of antibiotics is halted, conjugative plasmids that have been selected with antibiotics in previous years can still persist among bacterial populations or even invade new strains.     

Stability of plasmid-bearing microorganisms in batch and continuous cultures

The stability of five microbial strains bearing a domestic and/or exotic plasmid was investigated in continuous culture to obtain basic information on the fate of genetically engineered microorganisms released in the natural environment.The three strains with an exotic plasmid were constructed by the conjugal or mobilized transfer of conjugative plasmid R100-1 and non-conjugative plasmid RSF2124. Plasmid loss occurred only at the declining growth phase of batch culture of the transconjugants; the ratio of plasmid-free cells was 40–50% at the end of the culture, independent of the strains, whereas the plasmid in the native host cells was maintained at almost 100% of stability.In continuous culture of the transconjugant cells, the population ratio of plasmid-free cells at the pseudo-steady state was between 5–80% depending on the strain. The plasmid-bearing cells were not washed out of the continuous fermentor for 43 generations but maintained their quasi-stable concentration with some degree of oscillation. Simultaneous loss and retransfer of the plasmid from and to its host cells is suggested for the explanation.     

Enhanced plasmid maintenance in a CSTR upon square-wave oscillations in the dilution rate

Summary The effects of forced square-wave perturbations in the dilution rate on plasmid maintenance and gene expression of a population ofEscherichia coli K12 strain carrying the vector plasmid pBR322 grown in a chemostat with a non-selective medium were studied. It was observed that in the control experiments, where the dilution rates were kept constant, the percentage of plasmid-containing cells decreased after a period of time. Eventually, the culture was displaced by the plasmid-free cells. However, when the cells were exposed to forced oscillations in the dilution rate, the reactor culture was able to maintain a mixed population of plasmid-free and plasmid-containing cells for a longer period of time. The above observation seems to be independent of the source of the host cells. That is, the same results were obtained when the plasmid-free cells were generated from the culture itself due to defective partitioning of the plasmids or introduced externally.     

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.     

Chromosomally integrated conjugative plasmids are common in antibiotic-resistant Haemophilus influenzae.

Twenty-three highly antibiotic-resistant strains of Haemophilus influenzae and two of Haemophilus parainfluenzae without detectable large plasmids were examined for conjugative transfer of their resistance to H. influenzae strain Rd or to other strains. Very inefficient transfer was observed for 18 H. influenzae strains and 1 H. parainfluenzae strain. All H. influenzae transcipients carried a large plasmid, and they were in turn efficient donors of their resistances in standard conjugation crosses with isogenic recipients. This was not seen for the H. parainfluenzae transcipients. It is concluded that most of the original antibiotic-resistant cultures carried an integrated conjugative R plasmid which had been excised in a few cells in each population. It was these cells which transferred resistance in the primary crosses.     

A model for growth of Saccharomyces cerevisiae containing a recombinant plasmid in selective media

A major problem in the use of plasmids as recombinant vectors is the problem of plasmid-free cell generation from plasmid shedding and subsequent growth. A common technique for controlling the population of plasmidfree cells is the use of selective media against these cells using an auxotrophic host and a plasmid that has the ability to produced the essential metabolite. A distributed model describing the growth of Saccharomyces cerevisiae containing a recombinant plasmid in selective media was developed. The model allows for growth and production of a metabolite by the plasmid-carrying strain and growth of the plasmid-free cells on resulting metabolite concentrations. Through a determination of system constants and numerical solution to the equations, experimental batch and continuous culture results for cell concentration transients could be simulated by the model. The results indicated that despite selective pressure, plasmid-free cell growth was significant.     

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   

Stability of pBR322-derived plasmids

The stability of pBR322-derived plasmids was studied during growth of their Escherichia coli host in the absence of antibiotics. Plasmid pBR322, as well as its delta rom and delta bla derivatives, were lost from their host within 60 generations, but a number of delta tet derivatives were quite stable under the same conditions. An evaluation of the data indicated that primary plasmid loss due to random partitioning corresponds to the generation of a plasmid-free cell about every 10(4) divisions (probability P0; = "intrinsic" instability). Secondary loss of plasmid-carrying cells resulted from a growth advantage of the plasmid-free cells when bacteria die, perhaps due to unrepaired lethal damage in the DNA, under conditions of stationary incubation (= "apparent" instability). This cell death also occurred in the absence of plasmids but was accelerated by the presence of extra plasmid DNA in the cell and further accelerated by a functional tet gene. This was the reason for the differential apparent stabilities of delta bla and delta tet plasmids. There was no indication that an accumulation of plasmid multimers contributed to the plasmid instability, as has been suggested in the literature. The value of P0 = 10(-4) is 14 orders of magnitude greater than expected under the assumption of a random (Poisson) distribution of plasmid copy numbers in a population of cells.     

Mobilization of R387 Inc K conjugative plasmid by the conjugative plasmid R64 Inc I

Plasmid aggregate (R387, R64) was constructed in E. coli K12 strain. Plasmid R387 Inc K was stimulated to conjugational transfer by plasmid R64 Inc I. This stimulation was caused neither by recombination between both plasmids nor by trans-complementation of R387 conjugational systems by gene(s) product(s) of R64 plasmid. The observed phenomenon resembled rather mobilization of nonconjugative plasmids by conjugative ones. As in mobilization, the observed increase in R387 transfer frequency could take place only when both interacting plasmids were present in donor cells. Moreover, the entry exclusion system functioning in recipient cells, toward stimulating R64 plasmid affected strongly the conjugational transfer of stimulated R387 plasmid. Analogous phenomenon was observed during mobilization of nonconjugative plasmids by conjugative ones.     

Plasmid involvement in the formation of a spontaneous bacteriophage insensitive mutant of Lactococcus lactis

Lactococcus lactis subsp. lactis biovar. diacetylactis DPC721 is a spontaneous bacteriophage insensitive mutant of strain DPC220, isolated after challenge with an industrial bacteriophage, phi D1. Plasmid analysis demonstrated that the bacteriophage insensitivity was associated with the absence of two native DPC220 plasmids (pAH82 and pAH33), and the presence of a novel plasmid (pAH90) in DPC721. The plasmids were transferred by conjugative mobilization to a plasmid free background where it was confirmed by restriction mapping that pAH90 is a co-integrate formed by the precise recombination of pAH82 and pAH33. The resistance phenotype encoded by pAH90 was also active against two bacteriophage homologous for the plasmid-free strain. Plasmid pAH90 was shown to encode at least two independent resistance mechanisms, including an adsorption-inhibition mechanism and a restriction and modification system. The adsorption-inhibition mechanism encoded by the co-integrate plasmid was specific for one of the phage used in this study.     

Adaptative potential of the Lactococcus lactis IL594 strain encoded in its 7 plasmids

The extrachromosomal gene pool plays a significant role both in evolution and in the environmental adaptation of bacteria. The L. lactis subsp. lactis IL594 strain contains seven plasmids, named pIL1 to pIL7, and is the parental strain of the plasmid-free L. lactis IL1403, which is one of the best characterized lactococcal strains of LAB. Complete nucleotide sequences of pIL1 (6,382 bp), pIL2 (8,277 bp), pIL3 (19,244 bp), pIL4 (48,979), pIL5 (23,395), pIL6 (28,435 bp) and pIL7 (28,546) were established and deposited in the generally accessible database (GeneBank). Nine highly homologous repB-containing replicons, belonging to the lactococcal theta-type replicons, have been identified on the seven plasmids. Moreover, a putative region involved in conjugative plasmid mobilization was found on four plasmids, through identification of the presence of mob genes and/or oriT sequences. Detailed bioinformatic analysis of the plasmid nucleotide sequences provided new insight into the repertoire of plasmid-encoded functions in L. lactis, and indicated that plasmid genes from IL594 strain can be important for L. lactis adaptation to specific environmental conditions (e.g. genes coding for proteins involved in DNA repair or cold shock response) as well as for technological processes (e.g. genes encoding citrate and lactose utilization, oligopeptide transport, restriction-modification system). Moreover, global gene analysis indicated cooperation between plasmid- and chromosome-encoded metabolic pathways.     

The Population Biology of Bacterial Plasmids: A PRIORI Conditions for the Existence of Mobilizable Nonconjugative Factors

A mathematical model for the population dynamics of nonconjugative plasmids that can be mobilized by conjugative factors is presented. In the analysis of the properties of this model, primary consideration is given to the conditions under which these nonself-transmissible extrachromosomal elements could become established and would be maintained in bacterial populations. The results of this analysis demonstrate the existence of conditions where, as a consequence of infectious transmission via mobilization, nonconjugative plasmids could become established and be maintained even when the bacteria carrying them have lower reproductive fitnesses than plasmid-free members of the population. However, these existence conditions are stringent and suggest therefore, that it is highly unlikely that plasmids of this type would become established and maintained without some direct selection favoring their carriage. The general implications of these results and limitations of the model are discussed. Brief consideration is also given to the implications of these theoretical findings to the problems of the spread of multiple antibiotic resistance plasmids (R-factors) and the risk of contaminating natural populations of bacteria with chimeric plasmids produced by work with recombinant DNA.     

Linear plasmids pWR1A and pWR1B of the yeast Wingea robertsiae are associated with a killer phenotype

Wingea robertsiae CBS6693 (synonym Debaryomyces robertsiae) was previously reported to harbor two cryptic linear plasmids, designated pWR1A (8.3 kb) and pWR1B (14.6 kb). Reexamination of a putative plasmid encoded killer phenotype involved UV-curing as well as a highly sensitive toxin assay. Killer activities of concentrated culture supernatants prepared from both, a plasmid carrying and a cured plasmid-free strain, were examined in liquid media. Supernatants collected from plasmid carrying strains subjected to cultures of the plasmid-free derivative had clear concentration-dependent inhibitory effects, whereas plasmid harboring cells were not affected. Incubation at 65 degrees C for 10 min totally destroyed the toxin. Since supernatants prepared from the plasmid-free strain did not possess such killer activity and the presence of the plasmids confered resistance, toxin as well as immunity functions appear plasmid encoded. Beyond this, chitin affinity chromatography and Western blot analysis proved plasmid specific expression and secretion of a protein displaying similarities to the alpha-subunit of the Kluyveromyces lactis killer toxin. The assay applied in this study will most probably allow disclosure of other hidden killer phenomena, which may have escaped detection by conventionally applied plate assays.     

Conjugal transfer of the 5-nitroimidazole resistance plasmid pIP417 from Bacteroides vulgatus BV-17: characterization and nucleotide sequence analysis of the mobilization region.

Three small 5-nitroimidazole (5-Ni) resistance plasmids (pIP417, pIP419, and pIP421) from Bacteroides clinical isolates are transferable by a conjugative process during homologous or heterologous matings. The mobilization properties of pIP417 originated from strain BV-17 of Bacteroides vulgatus were studied. The plasmid was successfully introduced by in vitro conjugation into different strains of Bacteroides and Prevotella species and could be transferred back from these various strains to a plasmid-free 5-Ni-sensitive Bacteroides fragilis strain, indicating that in vivo spread of the resistance gene may occur. The transfer of plasmid pIP417 harbored by the Tc(r) strain BF-2 of B. fragilis was stimulated by low concentrations of tetracycline or chlorotetracycline. This suggests a possible role for coresident conjugative transposons in the dissemination of 5-Ni resistance among gram-negative anaerobes. The nucleotide sequence of the 2.1-kb DNA mobilization region was determined. It contains a putative origin of transfer (oriT) in an A+T-rich-region, including three inverted repeats, and two integration host factor binding sites. The two identified mobilization genes (mobA and mobB) are organized in one operon and were both required for efficient transfer. Southern blotting indicated that the mobilization region of plasmid pIP417 is closely related to that of both the erythromycin resistance plasmid pBFTM1O and the 5-Ni resistance plasmid pIP419 but not to that of the 5-Ni resistance plasmid pIP421.     

Evidence for a chromosome-borne resistance transposon (Tn916) in Streptococcus faecalis that is capable of "conjugal" transfer in the absence of a conjugative plasmid

Streptococcus faecalis strain DS16 harbors the conjugative hemolysin-bacteriocin plasmid pAD1 (35 megadaltons) and the nonconjugative R-plasmid pAD2 determining resistance to streptomycin, kanamycin, and erythromycin; a tetracycline resistance (Tetr) determinant is located on the chromosome. When strain DS16 was mated (on membrane filters) with the plasmid-free strain JH2-2, Tetr transconjugants could be obtained at a frequency of about 10(-6) per recipient. Analyses of transconjugants showed that some contained the Tetr determinant linked to pAD1. Subsequent studies showed that the Tetr determinant was located on a 10-megaldalton transposon, designated Tn916, which could insert into two hemolysin plasmids: pAM gamma 1 and pOB1. In addition, derivatives of DS16 devoid of pAD1 were capable of transferring Tetr to recipient strains. Transconjugants (plasmid-free) from such matings could subsequently act as donors in the transfer of Tetr. Both transposition and transfer were found to be rec independent.     

Coexistence of incompatible plasmids in a bacterial population living under a feast and famine regime

A model is formulated to examine the possibility of (co)existence of plasmids of the same incompatibility and surface exclusion group in a bacterial population living under a feast-and-famine regime. The condition is given under which a growth rate decreasing plasmid can invade a bacterial population. It appears that in case only one plasmid type is present, the frequency of plasmid bearers will tend to a stable equilibrium if the food supply at each growth site gets exhausted and if both plasmid-free and plasmid-bearing bacteria need an equal quantity of food per cell division. If these two conditions are not satisfied, the frequency of plasmid-bearers might oscillate. Two plasmids will sometimes be able to coexist, but only if they follow different survival strategies; one with a high conjugational transfer rate and a lower fitness of its host, and the other with a low transfer rate and a higher host fitness. Coexistence of three plasmids of the same surface exclusion group is impossible.     

The population biology of bacterial plasmids: a hidden Markov model approach

Horizontal plasmid transfer plays a key role in bacterial adaptation. In harsh environments, bacterial populations adapt by sampling genetic material from a horizontal gene pool through self-transmissible plasmids, and that allows persistence of these mobile genetic elements. In the absence of selection for plasmid-encoded traits it is not well understood if and how plasmids persist in bacterial communities. Here we present three models of the dynamics of plasmid persistence in the absence of selection. The models consider plasmid loss (segregation), plasmid cost, conjugative plasmid transfer, and observation error. Also, we present a stochastic model in which the relative fitness of the plasmid-free cells was modeled as a random variable affected by an environmental process using a hidden Markov model (HMM). Extensive simulations showed that the estimates from the proposed model are nearly unbiased. Likelihood-ratio tests showed that the dynamics of plasmid persistence are strongly dependent on the host type. Accounting for stochasticity was necessary to explain four of seven time-series data sets, thus confirming that plasmid persistence needs to be understood as a stochastic process. This work can be viewed as a conceptual starting point under which new plasmid persistence hypotheses can be tested.