Plasmid Transfer Rates Research Articles

Quantification of the effect of substrate concentration on the conjugal transfer rate of the TOL plasmid in short-term batch mating experiments.

Batch mating experiments with Pseudomonas putida PAW 1 (TOL) as a donor and Pseudomonas aeruginosa PAO 1162 as a recipient strain were performed to quantify the effect of the substrate concentration in the mating medium on the observed plasmid transfer rate coefficient. The impact of the substrate concentration in the mating medium was highly correlated with the growth history of the donor strain. When the donor strain was harvested in exponential growth phase, no impact was observed; when the donor strain was taken from the stationary phase, however, a strong impact of the substrate concentration was measured: a 10-fold reduction in the substrate concentration decreased the observed plasmid transfer rate by 55%.

Effect of parental growth on dynamics of conjugative plasmid transfer in the pea spermosphere

Plasmid transfer rates for the conjugative plasmid R388::Tn1721 from Pseudomonas cepacia (donor) to Pseudomonas fluorescens (recipient) on agar media, in broth, and in microcosms containing sterile or nonsterile soil, in the presence or absence of germinating pea seeds, were determined. Donors, recipients, and transconjugants were enumerated on selective media after 1 day on agar or in broth culture and over a 7-day period in soil or pea spermosphere microcosms. Donor and recipient growth rates and plasmid transfer rate constants [(gamma), where (gamma) = transconjugants (middot) (donors (middot) recipients)(sup-1) (middot) h(sup-1)] were calculated for three initial parental densities (10(sup4), 10(sup6), or 10(sup8) CFU/g or ml) in each system. For all initial density levels, values of (gamma) in agar and broth matings were higher than those in soil or in the pea spermosphere-rhizosphere microcosms. Values of (gamma) were not influenced by the pea spermosphere or by sterile or nonsterile conditions of the soil. However, (gamma) values in microcosm experiments were inversely related to initial parental density and were directly related to donor growth rates. Values of (gamma) averaged 4 x 10(sup-10), 4 x 10(sup-12), and 3 x 10(sup-14) when initial donor and recipient cell densities were 10(sup4), 10(sup6), and 10(sup8) CFU/g, respectively. These results suggest that the plasmid transfer rate constant is independent of parental cell density only when parental growth is not limited. In a resource-limited environment, intra- or interspecific competition may reduce the transfer rate by limiting parental growth.

The specific growth rate of Pseudomonas putida PAW1 influences the conjugal transfer rate of the TOL plasmid.

The kinetics of the conjugal transfer of a TOL plasmid were investigated by using Pseudomonas putida PAW1 as the donor strain and P. aeruginosa PAO 1162 as the recipient strain. Short-term batch mating experiments were performed in a nonselective medium, while the evolution of the different cell types was determined by selective plating techniques. The experimental data were analyzed by using a mass action model that describes plasmid transfer kinetics. This method allowed analysis of the mating experiments by a single intrinsic kinetic parameter for conjugal plasmid transfer. Further results indicated that the specific growth rate of the donor strain antecedent to the mating experiment had a strong impact on the measured intrinsic plasmid transfer rate coefficient, which ranged from 1 x 10(-14) to 5 x 10(-13) ml per cell per min. Preliminary analysis suggested that the transfer rates of the TOL plasmid are large enough to maintain the TOL plasmid in a dense microbial community without selective pressures.

Plasmid dynamics in a model soil column

AbstractContinuous‐flow column reactors were used to study the dynamics of plasmid exchange in a structured, thermodynamically open system containing either Enterobacter cloacae or Pseudomonas cepacia, both carrying the transmissible recombinant plasmid R388::Tn1721. Plasmid transfer rates were higher in vermiculite and sterile soil columns supplied with nutrient solution than those in sterile and non‐sterile soil columns without input of nutrient solution. For both species, donor and recipient strains took about 5 days to reach their maximum densities in effluents from the columns supplied with nutrient solution. After about 8 day s the donor and transconjugant populations of P. cepacia in the effluent solution decreased exponentially, whereas E. cloacae donor, recipient and transconjugant strains maintained steady‐state concentrations. The difference between plasmid stability in the two species may have significant consequences in terms of releasing plasmid‐bearing genetically modified microorganisms into the natural environment. The plasmid is persistent in E. cloacae in non‐sterile soil even though its transfer to the marked recipient in non‐sterile soil was minimal.

Rate of plasmid transfer among Escherichia coli strains isolated from natural populations.

The conjugative plasmid R1 was introduced into ten strains of Escherichia coli isolated from natural populations. Spontaneous nalidixic-acid-resistant mutants of the ten strains served as recipients. The ten donor and recipient strains were mated in all combinations and the rate at which R1 transferred between the strains was determined. The rate of transfer ranged from 5.2 x 10(-11)-1.1 x 10(-18) ml per cell h-1, and averaged 1.3 x 10(-15) ml per cell h-1. The results of these experiments suggest that the rates of conjugative transfer are far too low for plasmids to be maintained as parasites in their host populations. Infectious transfer is insufficient; plasmids must confer a selective advantage to their host to be maintained.

Plasmid transfer in natural soil: a case by case study with nitrogen-fixingEnterobacter

In strains of nitrogen-fixing Enterobacter agglomerans, isolated from the rhizosphere of cereals, the nif genes are located on large plasmids. Plasmid pEA9 (200 kb) is self-transmissible between closely related strains. To collect data on possible uncontrolled gene spread, for planned releases of such bacteria, plasmid pEA9 was labelled with transposons (Tn1725 and Tn5) and used in mating experiments between homologous Enterobacter strains with soil as substrate. The soil was from a plot into which an actual release was being planned. In the majority of experiments it was not sterilized. Survival and plasmid transfer is described, as are variations in temperature, time, moisture, pH and soil packing. Further experiments were with or without added energy sources, and with or without plant roots. Under standard conditions (22°C, pH 5.2, 15.5% moisture, loose soil, 2 × 107 inoculated donor and recipient cells each per g soil, 3 days incubation) sterilized soil gave low rates of plasmid transfer (10−6 per donor) but non-sterilized soil gave none. Adding Luria broth or sucrose to non-sterilized soil elicited strong cell propagation, together with plasmid transfer (optimum after incubation for 1 day: 10−4 exconjugants per donor). No transfer could be registered in the presence of wheat seedling roots for periods up to 5 weeks.

Plasmid transfer in natural soil: a case by case study with nitrogen-fixing Enterobacter

In strains of nitrogen-fixing Enterobacter agglomerans, isolated from the rhizosphere of cereals, the nif genes are located on large plasmids. Plasmid pEA9 (200 kb) is self-transmissible between closely related strains. To collect data on possible uncontrolled gene spread, for planned releases of such bacteria, plasmid pEA9 was labelled with transposons (Tn1725 and Tn5) and used in mating experiments between homologous Enterobacter strains with soil as substrate. The soil was from a plot into which an actual release was being planned. In the majority of experiments it was not sterilized. Survival and plasmid transfer is described, as are variations in temperature, time, moisture, pH and soil packing. Further experiments were with or without added energy sources, and with or without plant roots. Under standard conditions (22°C, pH 5.2, 15.5% moisture, loose soil, 2 × 107 inoculated donor and recipient cells each per g soil, 3 days incubation) sterilized soil gave low rates of plasmid transfer (10−6 per donor) but non-sterilized soil gave none. Adding Luria broth or sucrose to non-sterilized soil elicited strong cell propagation, together with plasmid transfer (optimum after incubation for 1 day: 10−4 exconjugants per donor). No transfer could be registered in the presence of wheat seedling roots for periods up to 5 weeks.

Development of efficient suicide mechanisms for biological containment of bacteria

To optimize plasmid containment, we have systematically investigated the factors that limit the killing efficiency of a suicide system based on the relF gene from Escherichia coli controlled by inducible lac promoters and placed on plasmids. In induction experiments with this suicide system, killing efficiency was unaffected by temperature and growth medium; there was no requirement for great promoter strength or high plasmid copy number. We could demonstrate that the factors limiting killing were the mutation rate of the suicide function and the reduced growth rate caused by a basal level of expression of the suicide gene during normal growth, which can give a selective growth advantage to cells with mutated suicide functions. The capacity of the plasmid-carried killing system to contain the plasmid was tested in transformation, transduction, and conjugational mobilization. The rate of plasmid transfer detected in these experiments seemed too high to provide adequate biological containment. As expected from the induction experiments, plasmids that escaped containment in these transfer experiments turned out to be mutated in the suicide function. With lac-induced suicide as a test, the efficiency of the system was improved by tightening the repression of the suicide gene, thereby preventing selection of cells mutated in the killing function. Reduction of the mutational inactivation rate of the suicide system by duplication of the suicide function augmented the efficiency of the suicide dramatically. These results permit the construction of extremely efficient biological containment systems.

Estimating the rate of plasmid transfer: an end-point method

We describe a method for determining the rate parameter of conjugative plasmid transfer that is based on single estimates of donor, recipient and transconjugant densities and the growth rate in exponential phase of the mating culture. The formula for estimating the plasmid transfer rate, gamma, was derived from a mathematical model describing cell growth and plasmid transfer in batch culture. Computer simulations were used to explore the sensitivity of this method to the realities of bacterial life, such as growth rate differences, plasmid segregation and transitory derepression of pilus synthesis. As predicted by the theory, mating experiments with the plasmid R1 in Escherichia coli K12 demonstrated that the estimate gamma is unaffected by cell density, donor:recipient ratio and mating time. Unlike previous techniques, our method allows us to investigate the effect of environmental factors on plasmid transfer rates when these factors also influence population growth rates. To illustrate this, we examined the effect of temperature on the rate of plasmid transfer.

The Evolution of Plasmids Carrying Multiple Resistance Genes: The Role of Segregation, Transposition, and Homologous Recombination

We used a mathematical model and experiments with laboratory populations of Escherichia coli to examine conditions for the evolution of plasmids conferring resistance to multiple antibiotics. In our model and its experimental analogue, two different resistance genes are initially carried on separate plasmids, and recombination between the plasmids can generate a third element that carries both resistances. The environment is such that only cells carrying the two resistance genes can replicate. Our simulations predict that under these conditions, populations carrying a single plasmid with both resistance genes increase in frequency at a rate approximately equal to that at which the two-plasmid state decays by vegetative segregation. When the segregation rate is high, the pace of evolution is nearly independent of the rates of recombination and plasmid transfer or of whether the antibiotic is bacteriostatic or bacteriocidal. In the main, our experimental results are consistent with these predictions. When t...

Application of multi-stage continuous culture for estimating net rate of plasmid transfer.

In the present study, we demonstrate a method of multi-stage continuous culture for determining the net rate of plasmid transfer. This method allows us to estimate the relative contributions of plasmid transfer and growth to the increase in the number of transconjugants

Factors affecting conjugative transfer rates of plasmids in batch and continuous cultures

Factors affecting the rates of plasmid transfer were investigated using Escherichia coli LC102 bearing a conjugative plasmid R100-1 and E. coli DH1. The rate constant of transconjugant increase, k ti, was used for presenting the degree of plasmid transmissibility instead of the plasmid transfer efficiency (pte). The rate constant was defined as the specific rate of transconjugant increase (srti, the number of transconjugants per donor per h) divided by the recipient cell concentration. The k ti values ranged between 10 −10 and 10 −15 ml cells −1 h −1, when estimated under various conditions. Moderate liquid agitation had a favorable effect on k tf but agitation rates higher than 33 s −1 (intergrated shear force) greatly decreased the value of k ti. The transconjugant-forming activity of the cells growing in continuous culture did not significantly change with the dilution rate, except those growing at dilution rates less than 0.1 h −1. The rate constant k ti at temperatures of 10–15°C was as low as the detection limit (10 −15 ml cells −1 h −1).

Evaluation of a method to measure conjugal transfer of recombinant DNA in soil slurries

Release of recombinant microbes into the environment necessitates an evaluation of their ability to transfer genetic material. The present report evaluates a method to detect conjugal DNA plasmid transfer in soil slurries under various environmental conditions. DonorPseudomonas cepacia containing pR388::Tn1721 andP. cepacia recipient cultures were coincubated in soil slurries containing autoclaved or natural soil and treated with one or more of 14 experimental conditions. Conjugal mating frequency (transconjugants per initial donor) ranged from 4.8×10−1 to 1.9×10−7. Highest numbers of transconjugants, 1.5×107 colony forming units/ml soil slurry, were observed following incubation at 35°C with an enriched nutrient supplement added to the soil. Low numbers of transconjugants, 103 colony forming units/ml soil slurry, were observed when mating pairs were subjected to low nutrient or pH stress even though initial donor and recipient populations were maintained at high levels. This test system provides a simple way to estimate effects of changing environmental factors on plasmid transfer rates and on the survival of recombinant microorganisms. By use of soil collected from sites proposed to receive genetically engineered microorganisms, preliminary risk assessments can be obtained regarding the potential for gene transfer and microorganism survival with this soil slurry test system.

Influence of soil variables on in situ plasmid transfer from Escherichia coli to Rhizobium fredii.

A model system was established to determine whether intergeneric plasmid transfer occurs in soil and how various soil variables affect the rate of plasmid transfer. The donor bacterium, Escherichia coli HB101 carrying plasmid pBLK1-2 (pRK2073::Tn5), and the recipient bacterium, Rhizobium fredii USDA 201, were inoculated into a sterile Adelphia fine-sandy-loam soil. Transconjugants were enumerated by direct plating on antibiotic-amended HM [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; 2-(N-morpholino) ethanesulfonic acid] salts medium. Randomly chosen transconjugants were verified by serological typing and Southern hybridization with a Tn5 gene probe. The maximum transfer frequency was observed after 5 days of incubation (1.8 x 10(-4) per recipient). The influences of clay (0 to 50% addition), organic matter (0 to 15% addition), soil pH (4.3 to 7.25), soil moisture (2 to 40%), and soil incubation temperature (5 to 40 degrees C) on plasmid transfer were examined. Maximum transfer frequencies were noted at a clay addition of 15%, an organic matter addition of 5%, a soil pH of 7.25, a soil moisture content of 8%, and a soil incubation temperature of 28 degrees C. These results indicate that intergeneric plasmid transfer may occur in soil and that soil variables may significantly affect the rate of transfer.

Predictive model of conjugative plasmid transfer in the rhizosphere and phyllosphere.

A computer simulation model was used to predict the dynamics of survival and conjugation of Pseudomonas cepacia (carrying the transmissible recombinant plasmid R388:Tn1721) with a nonrecombinant recipient strain in simple rhizosphere and phyllosphere microcosms. Plasmid transfer rates were derived for a mass action model, and donor and recipient survival were modeled as exponential growth and decay processes or both. Rate parameters were derived from laboratory studies in which donor and recipient strains were incubated in test tubes with a peat-vermiculite solution or on excised radish or bean leaves in petri dishes. The model predicted donor, recipient, and transconjugant populations in hourly time steps. It was tested in a microcosm planted with radish seeds and inoculated with donor and recipient strains and on leaf surfaces of radish and bean plants also growing in microcosms. Bacteria were periodically enumerated on selective media over 7 to 14 days. When donor and recipient populations were 10(6) to 10(8) CFU/g (wet weight) of plant or soil, transconjugant populations of about 10(1) to 10(4) were observed after 1 day. An initial rapid increase and a subsequent decline in numbers of transconjugants in the rhizosphere and on leaf surfaces were correctly predicted.

Experimental and mathematical models of Escherichia coli plasmid transfer in vitro and in vivo.

Little is known about the factors that govern plasmid transfers in natural ecosystems such as the gut. The consistent finding by earlier workers that plasmid transfer in the normal gut can be detected only at very low rates, if at all, has given rise to numerous speculations concerning the presence in vivo of various inhibitors of plasmid transfer. Plasmids R1, R1drd-19, and pBR322 were studied in Escherichia coli K-12 and wild-type E. coli hosts in two experimental systems: (i) gnotobiotic mice carrying a synthetic indigenous microflora (F-strains) which resemble in their function the normal indigenous microflora of the mouse large intestine, and (ii) anaerobic continuous-flow cultures of indigenous large intestinal microflora of the mouse, which can simulate bacterial interactions observed in the mouse gut. Mathematical models were developed to estimate plasmid transfer rates as a measure of the "fertility," i.e., of the intrinsic ability to transfer the plasmid under the environmental conditions of the gut. The models also evaluate the effects of plasmid segregation, reduction of the growth rates of plasmid-bearing bacterial hosts, repression of transfer functions, competition for nutrients, and bacterial attachment to the wall of the gut or culture vessel. Some confidence in the validity of these mathematical models was gained because they were able to reproduce a number of known phenomena such as the repression of fertility of the R1 plasmid, as well as known differences in the transmission and mobilization of the plasmids studied. Interpretation of the data obtained permitted a number of conclusions, some of which were rather unexpected. (i) Fertility of plasmid-bearing E. coli in the normal intestine was not impaired. The observed low rates of plasmid transfer in the normal gut can be explained on quantitative grounds alone and do not require hypothetical inhibitory mechanisms. (ii) Conditions for long-term spread and maintenance throughout human or animal populations of a diversity of conjugative and nonconjugative plasmids may be optimal among E. coli strains of low fertility, as are found among wild-type strains. (iii) E. coli strains carrying plasmid pBR322 plus R1drd-19 were impaired in their ability to transfer R1drd-19, but strains carrying pBR322 were significantly better recipients of R1drd-19 than a plasmid-free recipient E. coli. (iv) Long-term coexistence of plasmid-bearing and plasmid-free E. coli, in spite of undiminished fertility, appeared to be due to a detrimental effect of the plasmid on the growth rate of its host bacterium, rather than due to high rates of plasmid segregation. (v) Mathematical analysis of experimental data published by earlier investigators is consistent with the conclusion that plasmid transfer occurs consistently in the human gut, but that the resulting transconjugant E. coli populations are too small to be detected regularly with the culture methods used by earlier investigators. It is concluded that the long-term interactions observed were often the consequences of minor differences in parameters such as growth rates, fertility, rates of segregation, etc., which were too small to be detected except by precise mathematical analysis of long-term experiments, but which were nevertheless decisive determinants of the ultimate fates of the plasmids and their hosts.

The spread of plasmids in model populations of Escherichia coli K12

Comparison of R100 with its derepressed derivative R100-1 showed that the capacity to repress tra function does not significantly affect the spread by retransfer of R100. F′ lac was used to investigate the contributions of growth and transfer to spread of a plasmid through a recipient population. Ability to transfer F′ lac was lost rapidly when donor cultures entered stationary phase, but aggregate-forming ability was lost much more slowly. Comparison of F′ lac tra + with F′ lac traH88, which is unable to retransfer from recipients, showed the importance of retransfer. We used a mathematical model to calculate the amount of retransfer needed to explain the rate of increase of F′ lac progeny. This showed that the lag between a cell receiving F′ lac and being able to retransfer it was a less important constraint on this rate of increase than the inherent rate of plasmid transfer by established donors.