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

Abstract

In connection with the assessment of the safety of genetically-engineered microorganisms released to natural environments (2), data concerning the frequency of plasmid transmission have been accumulated under various environmental conditions. The frequency of plasmid transfer is conventionally defined as the number of transconjugants divided by the number of donors after a fixed incubation period (S). However, the increase in the number of transconjugants is a result of not only plasmid transfer but also transconjugant growth. It must be noted that this may lead to overestimation of plasmid transmissibility, particularly when the growth rate is high, relative to the 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.If the law of mass action is applied to the process of conjugal plasmid transfer from donor to recipient cells, the increase of transconjugant concentration (NT) with mating time (t) in a continuous culture at a dilution rate (D) is written as dNT/dt = kptNDNR + /TNT -L>NT (1) where kpt denotes the rate constant for plasmid transfer; ND and NR are the concentrations of donor and recipient cells; /T is the specific growth rate of transconjugants. At a steady state,

Determination of the mechanism of retrotransfer by mechanistic mathematical modeling

“…The dynamics of plasmid transfer on surfaces and the adequacy of the model under these conditions was investigated by a method in which thin agar slides were used (24). The model has also been used for estimating the net rate of plasmid transfer in batch cultures (26) and multistage continuous cultures (30) and for studying the conditions for the establishment and maintenance of plasmids in bacterial populations (25). A model for mobilizable plasmids hitchhiking with a conjugative plasmid has been developed (12); in this model, mobilization of a nonconjugative plasmid is only possible when a conjugative plasmid is also present in the donor cell.…”