Adaptation of model genetically engineered microorganisms to lake water: growth rate enhancements and plasmid loss

Abstract

When a genetically engineered microorganism (GEM) is released into a natural ecosystem, its survival, and hence its potential environmental impact, depends on its genetic stability and potential for growth under highly oligotrophic conditions. In this study, we compared plasmid stability and potential for growth on low concentrations of organic nutrients of strains ofPseudomonas putida serving as model GEMs. Plasmid-free and plasmid-bearing (NAH7) prototrophic isogenic strains and two amino-acid auxotrophs, all containing antibiotic resistance markers, were held physically separate from but in chemical contact with lake water containing the natural bacterium-sized microbial populations. Cells were reisolated at intervals over a 2-month period to determine the percent retaining the plasmid and the specific growth rate on various media. Plasmid stability in lake water was strongly strain specific; the NAH7 plasmid was stably maintained by the prototrophic strain for the duration of the test but was lost within 24 h by both of the auxotrophs. Specific growth rates of reisolates, compared with those of the corresponding non-lake water-exposed strains (i.e., parental strains), were not different when measured in rich medium (Luria-Bertani broth). However, specific growth rates were 42, 55, and 63% higher in reisolates of auxotrophs and the plasmid-free prototroph, respectively, when measured in 10-fold-diluted medium after exposure of 15 days or longer to lake water. Moreover, lake water-exposed strains grew actively when reintroduced into sterile lake water (28to 33-fold increase in numbers over 7 days), while the corresponding unadapted parental strains exhibited no growth over the same period. Postrelease adaptations to oligotrophic environments tend to increase the fitness of GEMs for survival and establishment of populations in aquatic ecosystems. Such genetic changes might not be detectable by using standard laboratory characterizations of parental strains yet may be critical to assessments of a particular GEM's efficacy or the risks associated with its release into the environment.

Survival of allochthonous bacteria in aquatic systems: a biological approach

“…In contrast, other authors have not observed effects of plasmids on the survival of their hosts [55]. Using Pseudomonas putida as a model of a genetically engineered microorganism, post‐release adaptation to oligotrophic environments tends to increase their survival [64]. Only when the plasmid encodes proteins that confer resistance to environmental parameters that compromise survival (e.g., visible light) does their presence enhance the survival of the host strains [65].…”

FITNESS OF GENETICALLY MODIFIED MICROORGANISMS In situ (RIVER WATER)

“…Comparing with parental strains, RS1 was similar to their parents (PU21A, MAM2A), RS2 has a good fitness than their parents (PAO1A, MAM2A). Altered organisms would have reduced fitness for survival and growth in the environment, due in part to the increased metabolic load imposed by maintenance and expression of the foreign genes (Sobecky, et al, 1992). Release of such organisms, it was argued, would be inherently safe since they would either die off quickly or gradually be eliminated by their naturally occurring analogs (Sobecky et al, 1992 andRyder et al, 1994).…”

“…Altered organisms would have reduced fitness for survival and growth in the environment, due in part to the increased metabolic load imposed by maintenance and expression of the foreign genes (Sobecky, et al, 1992). Release of such organisms, it was argued, would be inherently safe since they would either die off quickly or gradually be eliminated by their naturally occurring analogs (Sobecky et al, 1992 andRyder et al, 1994). Results from other laboratories demonstrated that post-release mutational changes can increase the fitness of genetically altered organisms to level equal to or higher than those of the wildtype organisms from which they were derived (Sobecky, et al, 1992).…”

Construction and evaluation of a gfp-tagged <i>Salmonella</i> Typhimurium strain for environmental applications

“…Recombinant bacteria are known to lose foreign genes in the absence of selective pressure or under stress conditions (Sobecky et al, 1992). Autecological studies or studies dealing with the biological relationship between an individual species and its environment should accurately reflect the prevailing The insertion point of the mini-Tn5 transposon is selected at random, and may therefore involve the disruption of a chromosomal sequence.…”