Discussion
Few studies have focused on the influence of compensatory mutations
during adaptation to a general environment rather than to a specific
genetic perturbation. Our results demonstrate that compensation to
deleterious consequences of an adaptive mutation is possible but is rare
among evolved clones in this experiment. Of the nine clones tested, only
one clone evolved a mechanism to alleviate the cost of constitutivelac operon expression in glucose (Fig. 2). No differences in
compensation were evident comparing groups of clones evolved in
different environments, including some that were and some that were not
expected to select for compensation.
Compensation of deleterious mutation effects has been observed in many
systems and contexts – including compensation of costs due to
antibiotic resistance, adaptation, and gene deletion (Levin et al. 2000;
Björkman et al. 1998; Björkman et al. 2000; Nagaev et al. 2001, Szamecz
et al. 2014; Zee et al. 2014, Blank et al. 2014, Knöppel et al. 2016). A
common theme is that compensation is readily selected, consistent with
their being many available genetic mechanisms. For example, one study
found 68% of single-gene deletion slow growth mutants could be
compensated to restore growth to near wild type within
~400 generations (Szamecz et al. 2014). Indeed,
compensation to the costs of the lacI- mutation apparent in
glucose containing environments was possible in our experiment, but
occurred in only one of the nine clones we examined. One explanation is
that compensation is limited not by mutational opportunity but by
selection for those mutations. Our populations evolved asexually so that
beneficial mutations of large effect outcompete those of smaller effect
(Gerrish and Lenski 1998). Thus, even if they arise, small benefit
compensatory mutations might be outcompeted until mutations of large
effect are exhausted. Our populations exhibit higher fitness gains at
early versus later time points (Satterwhite and Cooper 2015), however,
the latter gains may still be larger than the small
~2.2% cost of lacI - in glucose. Indeed, the G_L
populations steadily increased in glucose fitness up to 6000 generations
so compensatory mutations may not be sufficiently competitive to be
selected (Satterwhite and Cooper 2015). By contrast, G/L populations had
small or undetectable fitness change across glucose and lactose
environments after 4000 generations, which would have allowed selection
of small benefit mutations and may be why only a G/L clone was suggested
to have compensatory mutations (Satterwhite and Cooper 2015).
Another possible explanation for the rarity of compensation is that
multiple mutations are required to compensate the cost of lacI -
in glucose without affecting its benefit in lactose (Poon and Chao
2005b). Multiple compensatory mutations will take longer to fix and will
be rare because successful compensation can depend on the order in which
each required mutation occurs (Gong et al. 2013), the presence/absence
of other mutations in the genetic background (Shah et al. 2015; Lunzer
et al. 2010), and a clone’s fitness at each step relative to others in
the population which can subject an intermediate clone to being purged
by purifying selection (Gerrish and Lenski 1998). Replacing lacI -
with a functional LacI repressor in the G/L4 clone, in which
compensation did occur, significantly reduces fitness in glucose and
expression of the lac genes. In other words, this clone has
somehow rewired the lac network such that lac operon
expression is beneficial for growth in glucose. The basis of this
rewiring is unknown but might depend on multiple mutations as is the
case, for example, in the case of evolved citrate utilization selected
in a population started from the same ancestor as the one used here
(Blount et al. 2008, 2012).
We consider that LacY is the best candidate lac gene to be
involved in some new compensatory interaction. Not only is it the likely
source of the cost of constitutive lac expression (Eames and
Kortemme 2012), but it can also support glucose uptake, though, as
characterized, that requires a mutational change in the enzyme that did
not occur in clone G/L4 (Sahin-Tóth et al. 2001, Gram and Brooker 1992;
King and Wilson 1990). Although glucose transport is not likely to limit
growth at the concentration used in this experiment it is possible that
an alternative import mechanism could provide an advantage by reducing
dependence on the phosphotransferase system (Jahreis et al. 2008;
Ferenci 1996; Postma et al. 1993). This system is a common mutational
target in populations evolved from the same ancestor and selected in a
glucose environment, suggesting it is not optimized for growth in the
selective environment prevailing in our experiment (Woods et al. 2006).
Sequencing of a clone isolated from the G/L4 population identified two
genes that were not mutated in the other populations we examined here or
in lines that evolved only in glucose. Of these, one, sohB, is a
candidate for interacting with LacY. SohB is a peptidase active, along
with LacY, in the cell periplasm (Baird et al. 1991). Although the
function of SohB is not well characterized, it has been shown to be able
to compensate for loss of other peptidases involved in periplasm protein
recycling, suggesting a possibility of affecting LacY function.
An alternative mechanism of lacI- compensation is a change inlacI regulation. This could occur in either of two ways.
First, the 4 bp insertion frameshift mutation in lacI - of G/L4
results in a truncated 204 residue LacI repressor. Although
non-functional at the lac operator, it is possible that the
truncated LacI, which includes the DNA binding domain, interacts with
another region in the genome to cause some beneficial effect that is
lost when the ancestral lacI was restored (Platt et al. 1973).
Second, an operator site for a different gene may have evolved to
provide a benefit in the absence of LacI. The LacI repressor has
homology to other repressors in E. coli (Weickert and Adhya
1992), and studies have shown that as few as two mutations are needed to
increase the affinity of LacI to another operator (Lehming et al. 1987;
Lehming et al. 1990; Salinas et al. 2005; Daber and Lewis 2009). We
note, however, that the sequenced G/L4 clone does not have mutations in
recognized lacI family operator sites.
In summary, compensation was rare and did not occur based on specific
fluctuations of glucose and lactose. In the clone in which compensation
of lacI - did occur, lac operon expression was reduced but
not more so than other strains that had a similar cost in glucose
compared to the ancestor. This indicates that costs of constitutive
expression were overcome by epistatic interactions with other mutations
in the evolved background, and that the reduction in cost was not due
solely to a reduction in expression. Future studies will examine
potential long-term tradeoffs of compensation and what makes
compensation rare.
Acknowledgements. This work was supported by grant DEB-1253650
awarded to T.F.C. by the National Science Foundation and by grant
19-MAU-082 awarded to T.F.C. by the Royal Society of New Zealand Marsden
fund.
Data availability. T.F.C will make the strains constructed in
this study available to qualified recipients following completion of an
institutional material transfer agreement. The results of competition
experiments, summary input data, and analysis scripts that pertain to
the experiments and analyses reported in this paper have been deposited
at http://dx.doi.org/XX.XXX/ dryad.XXXX.