Results
Evolution of compensatory mutations. In the ancestor to our
evolution experiment, mutations that inactivate the LacI repressor, and
lead to constitutive expression of the lac operon, confer a
fitness cost of approximately 2.2% during growth in glucose (Quan et
al. 2012). To determine whether this cost is compensated during
evolution in environments containing both glucose and lactose resources,
and, if so, whether this compensation differed depending on the
presentation of the two resources, we reverted evolved lacI-mutations in nine clones isolated from populations selected in lactose
alone (Lac, three populations), long-term switching of glucose and
lactose (G_L, one population), or daily switching of glucose and
lactose (G/L, five populations). We found that the cost of lacI -
in glucose was significantly changed only in evolved clone G/L4 where
the mutation had become beneficial (Fig. 2; Dunnett’s Test, P< 0.001). This result indicates the presence of sign
epistasis, in that the lacI- mutation changed from being
deleterious in the ancestor to being beneficial in the evolved
background. No other difference in evolved lacI- costs was
detectable among other evolved clones (χ 2 = 8.68, df
= 9, P = 0.467).
Effect of environment on compensatory mutations. Although the
cost of lacI- was significantly changed in only one clone when
compared to the ancestor, there may be changes in costs apparent when
grouping clones based on their evolution environment. To test this, we
compared the effect of the lacI- mutation on fitness in glucose
of each evolution environment group (strains evolved in Lac, G/L, or
G_L). We expect clones evolved in the presence of fluctuations of
glucose and lactose (i.e., G/L and G_L selection environments) to have
a reduced fitness cost of the lacI- mutation in glucose because
compensation for the original cost would provide an advantage. By
contrast, selection for compensatory mutations was expected to be
reduced or absent in the lactose only environment. We found no
significant difference in cost of the lacI- mutation measured in
the glucose environment among clones isolated from different evolution
environments whether or not the outlier clone, G/L4, was included (ANOVA
with G/L4: F2,106 = 2.65, P = 0.75; without G/L4:
F2,100 = 1.29, P = 0.28).
Pleiotropic effect of lacI- compensation. Given that compensation
for the cost of constitutive lac expression evidently can occur,
one explanation for the low frequency at which it does occur is that it
imposes a correlated cost in lactose. For example, it might be that
compensation to constitutive expression of the lac operon
involves a reduction in the maximum level of lac expression,
perhaps reducing fitness in lactose and thereby causing compensation to
be selectively disfavored. To test this possibility, we examined the
fitness effect of lacI - mutations across glucose and lactose
environments. To determine if there was any trend of a lower cost oflacI - in glucose corresponding to a lower benefit in lactose, we
determined the relationship between the fitness effect of lacI -
mutations in glucose and lactose across all strains. We found a
marginally significant positive correlation between fitness in the two
environments, indicating that a low cost of the lacI- mutation in
glucose was, if anything, associated with an increased benefit in
lactose (Fig. 3; Spearman’s rank correlation, rho = 0.72, P =
0.02). The significance of the correlation is dependent on the G/L4
evolved clone that compensated for the lacI- mutations cost in
glucose. When that clone was omitted from the analysis, the correlation
was no longer significant, though was still positive (rho = 0.63,P = 0.07). Focusing on the G/L4 clone revealed that thelacI - mutation is beneficial in glucose and its effect in lactose
is significantly higher than in three of the evolved clones tested, as
well as the ancestor (G_L1, G/L1, and G/L2; Dunnett’s test, P< 0.05). Together, these results indicate that there is no
trade-off with fitness in lactose that limits selection for compensation
of lacI - costs in glucose.
Mechanisms of lowered costs. To determine if lac operon
expression is associated with changes in the fitness effects of thelacI - mutation, we measured lac operon expression in
glucose using a reporter that is controlled by the promoter region,
Plac, that drives expression of the lacoperon (Fig. 4). Expression of the lac operon contributes to the
cost of constitutive expression, so we expected a negative relationship,
such that clones that had higher lac operon expression would have
lower fitness in the glucose environment (i.e., a higher cost) (Dekel
and Alon 2005, Stoebel et al. 2008). In fact, there was no correlation
(Fig. 5A; Spearman’s rank correlation, rho = 0.12, P = 0.78).
This is especially surprising because half of the evolved strains had
significantly higher lac expression in glucose than the ancestorlacI -, so that an effect of lac expression on fitness
could have been detected (Dunnett’s test: G_L1, G_L3, G/L4, Lac3,
Lac4, Lac6 P < 0.05). That increased expression was not
associated with any fitness cost might indicate the action of
compensation to some portion of the cost that would otherwise be
associated with increased lac expression. Alternatively, there
could be a limit to the cost associated with constitutive lacoperon expression (Eames and Kortemme 2012), although the model most
analogous to the situation prevailing in our experiments predicts
exponentially increasing costs with increasing expression (Dekel and
Alon 2005).
All clones except G/L4 had similar lacI- associated fitness costs
when compared to the ancestor, but clones varied when it came to
differences in expression compared to the lacI - ancestor. G/L4
had equal lowest lac expression in glucose (Dunnett’s test,P < 0.05; except for G/L3, P = 0.90; and G/L6,P = 0.46), and all G/L clones had lower expression in glucose
when compared to all Lac clones. Together these results indicate that
there was some differential evolution of lac expression based on
environment, but that consequences do not consistently map to fitness
effects: clones with similar expression levels in glucose have differentlacI- fitness effects (G/L4 compared to G/L3 and G/L6), and
clones with similar lacI- fitness effects have different
expression (G/L compared to Lac clones). Evidently, reduced lacoperon expression cannot explain all of the decreased costs in glucose
and alleviation of the cost is dependent on other mutations in the
evolved background.
Finally, our reporter strains allow us to address a trade-off betweenlac expression and fitness related to compensation. If
compensation of costs due to constitutive lac operon expression
in glucose reduce the benefit of high expression in the lactose
environment, compensation might impose a net cost and not be selected.
To test this, we compared fitness and expression effects of lacI-mutations in lactose. We expected that if higher lac expression
was selected due to increasing fitness in lactose, there would be a
positive correlation between lac expression and fitness. In fact,
although all evolved clones had increased lac expression, changes
were not correlated with fitness (Fig. 5B; Spearman correlation, rho =
-0.18, P = 0.64). Moreover, although the lacI- mutation
conferred one of the biggest benefits when added to the G/L4 clone,lac expression was not significantly different in this clone
compared to other evolved clones (Dunnett’s test, P> 0.05). These results suggest that the benefit of higher
expression depends on the genetic background in which it occurs and that
most of the changes in maximum lac expression are not caused bylacI - itself but by differences in the broader genetic
background.