Pleiotropic effects of adaptation to a single carbon source for growth on alternative substrates

Abstract

It is frequently assumed that populations of genetically modified microorganisms will perform their intended function and then disappear from the environment due to inherent fitness disadvantages resulting from their genetic alteration. However, modified organisms used in bioremediation can be expected to adapt evolutionarily to growth on the anthropogenic substrate that they are intended to degrade. If such adaptation results in improved competitiveness for alternative, naturally occurring substrates, then this will increase the likelihood that the modified organisms will persist in the environment. In this study, bacteria capable of degrading the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) were used to test the effects of evolutionary adaptation to one substrate on fitness during growth on an alternative substrate. Twenty lineages of bacteria were allowed to evolve under abundant resource conditions on either 2,4-D or succinate as their sole carbon source. The competitiveness of each evolved line was then measured relative to that of its ancestor for growth on both substrates. Only three derived lines showed a clear drop in fitness on the alternative substrate after demonstrable adaptation to their selective substrate, while five derived lines showed significant simultaneous increases in fitness on both their selective and alternative substrates. These data demonstrate that adaptation to an anthropogenic substrate can pleiotropically increase competitiveness for an alternative natural substrate and therefore increase the likelihood that a genetically modified organism will persist in the environment.

FIG. 1

Derivation of bacterial strains. Horizontal lines indicate periods of evolution, with the selective regimen and sole carbon source indicated above and below, respectively. Each horizontal line represents two independently evolved replicate populations. F and S represent the fast and slow ancestral strains, respectively. For stage I-derived strains, the first letter (F or S) indicates the ancestor and the second letter (B or C) indicates whether stage I evolution occurred in batch (B) or chemostat (C) cultures. The letter r indicates streptomycin resistance (see Materials and Methods). The first two letters for stage II evolution strains indicate their stage I-proximate ancestor, and the third letter (B) indicates stage II evolution in batch culture.

FIG. 2

Performance changes of stage I lines. (a) FB lines; (b) SB lines. Mean SRC values are shown, along with 95% confidence intervals, for both the selective substrate (succinate, left column) and the alternative substrate (2,4-D, right column). *, see Table 2.

FIG. 3

Performance changes of stage II F-derived lines. (a) FBB lines; (b) FCB lines. Mean SRC values are shown, along with 95% confidence intervals, for both the selective substrate (2,4-D, left column) and the alternative substrate (succinate, right column).

FIG. 4

Performance changes of stage II S-derived lines. (a) SBB lines; (b) SCB lines. Mean SRC values are shown, along with 95% confidence intervals, for both the selective substrate (2,4-D, left column) and the alternative substrate (succinate, right column).