Evolution of copper tolerance
The artificial evolution experiment was performed to test if selection
on a population’s standing variation of strains allows rapid evolution
of copper tolerance (Fig. S1). A detailed description of the
experimental approach and the outcome (changes in density, growth rate,
pH, and Fv/Fm) is included in the Supplement (Fig. S3). Briefly, chronic
toxic effects developed over time, and the copper concentration had to
be modified to allow stable growth and maintain inhibition levels at
around 50%. Strain RO5AC served as a reference point between the two
experiments and was used to control for these treatment differences.
During the experiment, the mining population’s growth rate increased to
about 0.2 day-1 higher than RO5AC, while the reference
population grew slower than RO5AC throughout the experiment (Fig. S3).
The absolute growth rate increases in the mining population, from the
start to the end of the selection phase, was 3-fold higher than the
growth rate of the reference population (0.50 versus 0.15
day-1). In both populations, and the RO5AC mono-clonal
control, the EC50 values increased from around eight to ten µM copper
(Fig. 2), which was similar to the mining inlet strain VG1-2_81’s
initial EC50, but significantly higher than any strain in the reference
population (Fig. 1A). The response ranges to copper also expanded
relative to the control (Table 1), resulting in adapted populations
being tolerant to a wider range of copper concentrations beyond what was
lethal to all strains during acute 72-hrs exposure.
In the reference population and in the RO5AC strain, a large part of the
increase in copper tolerance (50-100%) was plastic and rapidly lost
once the toxic stress was relaxed (Fig. 2A and B). The PAM assay showed
that this tolerance was induced in RO5AC after only three days of
exposure to copper, whereas it took up to 21 days before it was attained
in the reference population (Fig. 3). In the reference population, four
of the copper-exposed experimental replicates grew poorly in ambient
media without excessive copper (Fig. S4), indicating that copper
tolerance carries a cost in terms of limited ability to reach high cell
densities in low copper environments. Collectively, these observations
show that at least some strains of the reference population acclimated
to toxic conditions via a plastic response to copper stress, and that
this effect was not accurately captured by the acute 72-h dose-response
experiment.