Short Communication Survival of, and plasmid transfer between modi®ed Enterobacter cloacae strains under long-term starvation conditions in a continuous-¯ow system Li Sun 1 , Michael J. Bazin 1 and James M. Lynch 2, * 1 Microbiology Group, King's College London, Campden Hill Road, London W8 7AH, UK 2 School of Biological Sciences, University of Surrey, Guildford, Surrey GU2 5XH, UK *Author for correspondence: Fax: +44-1483-259728, E-mail: j.lynch@surrey.ac.uk Received 15 March 1999; accepted 29 May 1999 Keywords: Column reactors, genetically-engineered microorganisms (GEMs), nutrient starvation, plasmid transfer Summary Continuous-¯ow, packed-bed column reactors, which provide an experimental model of a soil pro®le, were used to investigate survival of, and plasmid transfer between, strains of Enterobacter cloacae. When columns, inoculated with nutrient-sucient donor and recipient strains, were provided with a minimal salts medium with no added carbon source, transconjugant cells appeared in their euents. During the ®rst few days of such experiments, the concentration of cells in the euent declined but then the donor population stabilized, while the recipient and transconjugant populations continued to decrease. The results indicate that the amount of nutrient required to maintain and transfer plasmids is very low. No transconjugants were observed in the euent from columns inoculated with pre-starved donor and recipient strains. Introduction Genetically modi®ed microorganisms (GMMs) have numerous potential applications in agriculture and pollution control. However, they will not be able to carry out their intended tasks if they cannot survive long enough in the receiving environment and, poten- tially, they may cause environmental problems if they persist for too long. There is increasing concern about the possible ecological eects of GMMs released to the natural environment. Most natural ecosystems are characterized by low concentrations of available sub- strates from organic matter and periods of starvation are probably experienced by most free-living bacteria. However, microbes survive starvation conditions in closed systems for considerable periods of time (Ku- rath & Morita 1983; Lynch 1990) and introduced bacteria persisted in their receiving environment with- out losing viability when starved (Liang et al. 1982). On the other hand, it has been reported that bacteria lose their viability very quickly under starvation conditions (Reeve et al. 1984). When GMMs are introduced into the natural environment, competition with indigenous micro¯ora for nutrient sources is inevitable. The soil is a thermodynamically open ecosystem as there is an input and output of both mass and energy resulting from the unidirectional ¯ow of solute through it. It is also a porous matrix and microorganisms grow both on the solid substrate and in the pores between the soil particles. We have used continuous-¯ow packed-bed column reactors, containing relatively inert packing material, to experimentally model these two physical characteristics of soil which signi®cantly aect the way in which microorganisms grow and interact; (e.g. Bazin et al. 1982; Theodorou et al. 1980; Sun et al. 1993; Pearce et al. 1998). The advantage of employing con- tinuous-¯ow packed bed reactors for such investigations is that they not only mimic the major properties of soil ecosystems but the eects of varying a single parameter can be tested while other parameters are ®xed. In a previous study, we demonstrated plasmid transfer at high rates from donor to recipient strains of Entero- bacter cloacae and Pseudomonas cepacia in continuous- ¯ow columns (Sun et al. 1993). The dynamics of plasmid transfer diered between the two species quite funda- mentally; in the P. cepacia columns, the plasmid disap- peared from the system exponentially while the same plasmid persisted in columns containing E. cloacae. We concluded that, as a result of transgeneric plasmid transfer, indigenous microorganisms with properties similar to that of E. cloacae could enhance the existence of an introduced plasmid even if it was unstable in its vector. However, our experiments were carried out under nutrient-sucient conditions and, as pointed out above, many soil ecosystems contain only low concen- trations of microbial substrates. Therefore, we carried out a series of experiments under conditions that more World Journal of Microbiology & Biotechnology 15: 639±642, 1999. 639 Ó 1999 Kluwer Academic Publishers. Printed in the Netherlands.