BACKGROUND
biological systems possess the ability to adapt to environmental changes, which can generate a variety of phenotypes and genotypes.
such emergence of phenotypic and genotypic diversity is considered a result of stochastically fixed genomic mutations during the process of evolution.
a question that arises here is whether the process of evolution allows arbitrary phenotypic changes or whether there are constraints that restrict possible variations in phenotypes  <cit> .
the pioneering studies by waddington  <cit> , which have been corroborated by several other studies, suggests the latter, i.e., constraints on evolutionary dynamics is ubiquitous.
one example of such evolutionary constraint is that the earliest embryo of various organisms shows a conserved morphological pattern called the phylotypic period, which is a constrained distribution of phenotype  <cit> .
here, the relationship between evolutionary constraints and phenotypic plasticity without genetic alteration has generated significant attention .
however, despite the recognized importance of characterizing evolutionary constraints, quantitative understanding of the process still remains unclear.
for this purpose, greater analysis is needed on phenotypic and genotypic changes in a variety of evolutionary courses.
laboratory evolution of bacteria is a powerful tool to trace phenotypic and genotypic changes in adaptive evolution in a quantitative manner.
recent advances in high-throughput sequencing have made it possible to identify and study fixed mutations in whole-genomic sequences during microbial adaptive evolution.
for example, several mutations were identified as beneficial in adaptively evolved escherichia coli  strains that used glycerol as the carbon source  <cit> .
other studies using laboratory evolution and genome resequencing have provided evidence that genomic mutations contribute to adaptive phenotypic changes against various environments, including several carbon sources , different temperatures  <cit> , and the presence of antibiotics  <cit> .
the advancements of genome-wide analysis in laboratory evolution open the door to integrate quantitative data of phenotypic and genotypic changes, which can shed light on the nature of evolutionary dynamics including quantitative understanding of evolutionary constraints.
in this study, we analyzed phenotypic and genotypic changes in the laboratory evolution of e. coli cells.
in the previous study of laboratory evolution under the ethanol stress condition  <cit> , we found that the overall gene expression changes before and after long-term cultivation were similar among independently evolved tolerant strains.
however, it is still unclear relationship between phenotypic change and genetic change during evolution.
in this study, first to further analyze the relationship in phenotypic changes in the independently evolved tolerant strain, we quantified time-series of expression changes.
the changes of metabolite concentrations were also quantified in the tolerant strains.
then, we assessed genotypic changes in the tolerant strains using high-throughput sequencers, to analyze the relationship between fixed mutations and phenotypic changes.
to quantitatively evaluate the effects of fixed mutation on the ethanol tolerance, we introduced all the identified mutations in the genome of the parent strain into one of the tolerant strains.
by integrating these phenotypic and genotypic data, we analyzed how the phenotype-genotype mapping is organized in the process of adaptive evolution.
RESULTS
time-series expression analysis in adaptive evolution under ethanol
we previously obtained  <dig> independently evolved ethanol tolerant e. coli strains  by culturing cells under 5 % ethanol stress for about  <dig> generations and found a significantly larger growth rate than the parent strains  <cit> .
here, we defined "ethanol tolerance" as a state with significantly higher growth rate under 5 % ethanol stress condition, and the term "strain" is used for the mixed population without single-colony isolation.
to elucidate the phenotypic changes that occurred during adaptive evolution, we first quantified the time-series of the expression changes by microarray analysis.
starting from frozen stocks obtained at  <dig> time points in laboratory evolution , cells were cultured under 5 % ethanol stress, and mrna samples were obtained in the exponential growth phase for microarray analysis .
the results of the time-series transcriptome analysis revealed that the expression changes during adaptive evolution were similar among tolerant strains.
for example, fig.
<dig> shows the expression changes of genes in the central metabolic pathway including glycolysis, the tricarboxylic acid  cycle, and the pentose phosphate pathway.
interestingly, common expression changes were not always monotonic  over time, but were rather synchronized complex expression changes on a much longer time-scale than the generation time observed.
additionally, a common and gradual up-regulation of genes involved in some amino acid biosynthesis pathways was also observed .
our previous work suggests that these pathways might contribute to ethanol tolerance  <cit> .fig.
<dig> time-series transcriptome analysis for adaptive evolution of e. coli to ethanol stress.
expression changes of representative genes in the central metabolic pathway including glycolysis, the pentose phosphate pathway, and tca cycle are shown.
in each inset, the horizontal axis shows time , and the vertical axis shows expression level .
expression levels of 0 h in each gene represent the ones of parent strain.
asterisks  indicate expression levels of parent strain obtained without adding ethanol as a reference.
the numbers of generations from  <dig> to 2000 h were strain a: <dig>  b: <dig>  c: <dig>  d: <dig>  e: <dig>  and f: <dig>  respectively.
abbreviations: 2pg, 2-phosphoglyceric acid; 3pg, 3-phosphoglycerate; accoa, acetyl-coa; αkg, α-ketoglutarate; bpg,  <dig> -bisphosphoglycerate; cit, citrate; e4p, erythrose4-phosphate; f6p, fructose 6-phosphate; fbp, fructose  <dig> -bisphosphate; gap, glyceraldehyde 3-phosphate; g6p, glucose 6-phosphate; oxa, oxaloacetate; pep, phosphoenolpyruvate; pyr, pyruvate; r5p, ribose 5-phosphate; s7p, sedoheptulose 7-phosphate; suc, succinate; x5p, xylulose 5-phosphate
figure 2a shows overall expression changes during the adaptive evolution of the six tolerant strains by principal component analysis .
the orbits in pca space, which represent expression profile changes, were similar except for strain c. the reason for this exception might be that strain c has an approximately  <dig> kbp region in the genome that was duplicated , and the expression levels of genes in this region were increased by this duplication.
the expression analysis also demonstrated that the overall expression changes between the parent and tolerant strains at the endpoint were similar .
these results indicated that even though these strains adapted to ethanol stress in independent cultures, the expression profiles converged into almost identical adapted states with similar orbits of expression changes.fig.
<dig> similarity among expression changes in the tolerant strains.
a changes in pca scores during adaptive evolution.
starting from the parent strain, changes in the expression profiles during adaptive evolution are plotted as orbits in the two-dimensional pca plane.
b an example of the correlation between expression changes that occurred in two tolerant strains.
the expression changes in strain e and f were plotted.
horizontal and vertical axes are log-transformed expression ratios with the parent strain, and each dot represents the expression change of the gene.
all possible combinations are shown in additional file 3: figure s2
metabolome analysis of ethanol tolerant strains
to further characterize the phenotypic changes that occurred in the tolerant strains, we measured metabolite concentration changes between parent and tolerant strains.
using capillary electrophoresis time-of-flight mass spectrometry , we quantified the intracellular concentrations of  <dig> metabolites .
the intracellular concentrations of some amino acids in the parent and tolerant strains are presented in fig.
3a.
these concentrations generally decreased in the tolerant strains, except for that of methionine.
the decrease was especially true for amino acids that originated from precursors in the tricarboxylic acid  cycle.
this suggests a change of metabolic state in the tca cycle in tolerant strains, a conclusion supported by the significant decrease in the expression of genes related to the tca cycle .
for example, glutamate acts as a major amino-group donor in amino acid biosynthesis, and thus a decrease in its concentration can cause a decrease in the concentration of other amino acids.
the decrease in amino acid concentration can activate the amino acid starvation response, which is consistent with the up-regulation of genes related to amino acid biosynthesis.
in contrast, the concentrations of metabolites in purine metabolism generally increased .
this concentration increase might be caused by the up-regulation of genes involved upstream of the purine biosynthesis pathway , by which phosphoribosyl pyrophosphate , the precursor for purine nucleotide synthesis produced from ribose-5-phosphate, is converted into inosine 5'-monophosphate .
no significant concentration change was observed for metabolites in pyrimidine metabolism.fig.
<dig> metabolome analysis of ethanol tolerant e. coli strains under ethanol stress conditions.
a concentration of amino acids in ethanol tolerant strains.
in each inset, the vertical axis shows the log-transformed absolute concentration .
the blue bar and red bars indicate the data of the parent strain and tolerant strains, respectively.
b correlation between metabolite concentration changes in strains e and f. horizontal and vertical axes represent log-transformed concentration ratios with the parent strain, and each dot represents the concentration change of the metabolites
the metabolome analysis also demonstrated similar changes in metabolite concentration among the tolerant strains, which resembles observations for gene expression changes.
the correlation of overall metabolite concentration changes between independently obtained tolerant strains indicated similar metabolite shifts .
both the transcriptome and metabolome analyses showed that phenotypic changes were similar among tolerant strains even though they were obtained from independent long-term cultivations.
genome resequencing analysis of ethanol tolerant strains
genotype changes in each tolerant strain were analyzed using two high-throughput sequencers, solid and illumina miseq .
in the resequencing analysis, we extracted genomic dna samples from the cell population at the end-point of the experimental evolution without single-colony isolation, to identify genotype changes that were fixed in the majority of tolerant cells and to avoid a fixation of minority mutations.
for point mutations, solid and illumina analyses identified  <dig> and  <dig> fixed mutations in all  <dig> tolerant strains, respectively, with  <dig> of these mutations being identified in both analyses.
the discrepancy  was checked by sanger sequencing, and it was confirmed that  <dig> were true positives and the other a false positive.
after screening indels by solid sequencing, we identified  <dig> small  and  <dig> large indels in all tolerant strains.
we verified these small and large indels by sanger sequencing, finding all were true positives.
finally, in strain c, the coverage of an approximately  <dig> kbp region was significantly higher than in other strains , which strongly suggested that the corresponding region duplicated during long-term cultivation.
the identified mutations at the end-point of experimental evolution are summarized in table  <dig>  the number of mutations in strain a was significantly higher than other strains .
this was likely due to a mutation leading to a stop codon in the muts coding region, which codes a mismatch repair protein.
it is known that disruption of muts significantly increases the mutation rate of e. coli cells  <cit> .
we confirmed that there were only  <dig> mutations in strain a at 1512 h  after commencing laboratory evolution and these did not include a mutation in muts.
this result suggested that after 1512 h, the mutation in the muts gene was fixed and resulted in a significant increase in the mutation rate.
the emergence of a strain with a significantly high mutation rate, or a “mutator,” is often observed in the laboratory evolution of microorganisms .table  <dig> list of identified mutations
hns
a
year
iscr
ilvg
insh,nmpc,essd,ybcs,rzpd,rzod,bord,ybcv,ybcw,nohb,tfad,ybcy
nage
hns
yeay
menc
rela
rpoc
rpoa
hns
proq
ispg
rpsd
yjha
hns
cspc
rela
yhcm
atpe
miab
cspc
wzxc
iscr
rela
abold-faced genes represent overlap among evolved strains
in contrast to the more than one hundred fixed mutations in strain a, the number of fixed mutations was relatively lower in the other strains.
as mentioned above, the phenotypic changes that occurred in independently evolved tolerant strains were similar, which might suggest mutations fixed in identical or related genes contributed to the changes.
we found that mutations were commonly fixed in rela, which codes guanosine tetraphosphate synthetase.
rela regulates a stringent response by producing guanosine tetraphosphate   <cit> .
the stringent response is widely observed in bacteria as a stress response in reaction to nutrient starvation  <cit>  or various environmental stresses  <cit> .
when e. coli face such stresses, growth-related activities including replication, transcription, and translation are tightly inhibited, which are triggered by the accumulation of ppgpp.
thus, the mutations commonly fixed in rela may relax the stringent response caused by ethanol stress to recover growth activity.
the mutations in rela and spot, which codes an enzyme that plays a major role in ppgpp degradation, have been widely observed in the laboratory evolution of e. coli under various conditions, including glucose limitation  <cit>  and high temperature  <cit> .
here, relaxing the stringent response by mutating the rela and spot genes may increase fitness under stress.
furthermore, in strains a, b, c, d, and e, insertion sequence element  <dig>  was inserted into the promoter region of hns, which codes for a dna binding protein that has various effects on gene expression  <cit> .
however, no significant change in hns expression was observed in these strains.
except for rela and hns, no functional overlap among the mutations fixed in more than two tolerant strains was determined.
fitness contribution of fixed mutations
to evaluate the contribution of fixed mutations to the growth increase under ethanol stress, we introduced all identified mutations in strain f into the parent genome by site-directed mutagenesis  <cit> .
we selected strain f for this analysis because the number of is insertion was smallest among the tolerant strains.
in strain f, we identified  <dig> mutations, including  <dig> single nucleotide substitutions, one small deletion, and one insertion in the genome of strain f. the sequence of the 1199-bp insertion was identical to is <dig>  which disrupted the orf of cspc .
since the insertion of is <dig> into the same position of the parent genome was difficult experimentally, we constructed a cspc deletion strain.
figure  <dig> shows the growth rates of the constructed strains by site-directed mutagenesis measured under the ethanol stress condition.
here, the main purpose of the analysis was to introduce all identified mutations in strain f into the genome of the parent strain, instead of constructing mutant strains with all possible combinations of mutations.
thus, the combinations of  <dig>   <dig>  and  <dig> mutations are arbitrary chosen to optimize the speed to construct the strain with  <dig> mutations.
the results in fig.
<dig> demonstrated that the fixed mutation in rela significantly contributed to the growth rate increase .
however, other mutations had no significant effect on the growth rate, and even when all fixed mutations in strain f were introduced into the parent genome, the observed growth rate of the mutated strain was significantly smaller than that of strain f under the ethanol stress condition.fig.
<dig> growth rates of site-directed mutants with ethanol stress.
all mutations identified in strain f were introduced back to the parent strain.
for each mutant, the names of the mutated genes are shown.
among the mutations identified in strain f, the mutations for cspc and miab correspond to is insertion and 88 bp deletion, respectively, while other mutations were snps.
error bars indicate standard deviations calculated from three independent cultures.
different asterisks  indicate significant differences , which were determined by dunnett’s test compared with parent strain
timing of fixed mutations
to further evaluate the contribution of the fixed mutations on ethanol tolerance, we analyzed the relationship between the growth increase under ethanol stress and the timing of mutation-fixation events in long-term cultivation in strain f. to identify the timing, genomic dna samples obtained from cell populations that had heterogeneous genotypes at  <dig> different time points were applied to sanger sequencing.
therefore, in some cases, the peak signals in the sanger sequencing revealed mixed populations, i.e., cells with and without a specific mutation coexisted in the population.
figure 5a shows the time the mutations in strain f emerged.
the increased growth rate did not always correlate with fixation events.
more importantly, although at 576 h after inoculation no mutation was fixed in the majority of the cell population, the growth rate under ethanol stress significantly increased.
some cells at 576 h had mutations in the rela and cspc genes that may have contributed to the observed growth increase.
to confirm this possibility, we isolated  <dig> clones from the cell population at 576 h and analyzed fixed mutations in rela and cspc by sanger sequencing.
among the  <dig> clones,   <dig> had both rela and cspc mutations,   <dig> had the cspc mutation only, and  the other  <dig> clones had no mutation.
to evaluate the effect of these mutations on the population at 576 h, we randomly selected  <dig> clones from the groups , , and , and measured the growth rates of clones with and without mutations under the ethanol stress condition.
as shown in fig.
5b, clones with or without rela and cspc mutations showed significantly larger growth rates than parent strain , and there was no significant growth difference between clones.
it should be noted that, the average growth rates of clones corresponding to groups , , and  were significantly higher than the clone in which  <dig> identified mutations were introduced .
these results suggested that, the effect of these mutations into the parent genome was smaller than the increase of growth rate in the cultivation from  <dig> to 576 h, even though in some clones no mutations was suggested to be fixed on the genome.fig.
<dig> time-series analysis of the mutation fixation.
a timing of mutation fixation events in strain f. to identify the timing of mutation fixation, genomic dna samples obtained at  <dig> different time points  were applied to sanger sequencing.
for each of the  <dig> identified mutations, the results of the sanger sequencing is presented as a solid or dotted line.
the solid line indicates that the mutation was fixed in the population at the corresponding time point, while the dashed line indicates the case of two peak signals, which indicates polymorphism in the population with and without the mutation.
for example, in cells at 576 h after inoculation, only some have mutations in rela and cspc.
b specific growth rates of cloned e. coli cells with and without cspc and rela mutations under ethanol stress condition.
each bar represents the specific growth rate of an isolated clone, which were obtained from strain f cell populations at 576 h. " + " and "-" mean with and without the corresponding mutation, respectively.
blue, green, and red bars represent the growth rates of clones without mutations, that with cspc mutation only, and with cspc and rela mutations, respectively.
error bars indicate standard deviations calculated from three independent cultures
the results in fig.
5a and 5b suggested the possibility of clonal interference.
the result in fig.
5b suggested that cspc mutation was fixed on a cell with rela mutation, while the result in fig.
5a suggested rela mutation rose to prominence before cspc mutation.
these results might suggest that, in the evolutionary dynamics of these populations, there were multiple clones with different beneficial mutations coexisting and competing in the environment, i.e., clonal interference  <cit> .
stability of ethanol tolerance
we reasoned that if the observed ethanol tolerance is due to phenotypic plasticity without genetic alteration, then the phenotype of the ethanol tolerance would likely be unstable when the environment changes.
we therefore cultivated cells with ethanol tolerance in an ethanol-stress free environment for  <dig> generations.
two cell populations were used: strain f and the cell population obtained at 576 h in the cultivation of strain f. after cultivation in the non-stress condition, we measured the growth rate under 5 % ethanol stress to evaluate the stability of the ethanol tolerance.
ethanol tolerance was stably maintained even after  <dig> generations , which suggests that the observed phenotypic changes in the tolerant strains were stably memorized and passed on to progeny cells.
growth evaluation of clones in the population of the ethanol tolerant strains
in this study, to analyze the characteristics of the majority of cell population, we measured mixed population without single colony isolation when we performed transcriptome, metabolome, and genome resequencing analysis.
to evaluate the effect of clonal interference in the population of the ethanol tolerant strains, we isolated clones from the end-point population of strains e and f. then, we quantified the growth rate of these clones under the ethanol stress condition as shown in additional file 11: figure s <dig>  the result demonstrated that there were no difference between isolated clones and mixed population, suggesting that the effect of clonal interference is negligible in the population of ethanol tolerant strain we obtained.
discussion
transcriptome and metabolome analyses revealed that phenotypic changes that occurred in ethanol tolerant strains were similar among independently evolved strains.
gene expression changes over time were found to exhibit high similarity among tolerant strains, which included non-monotonic expression changes with time scales much longer than the generation time.
the observed phenotypic convergence to similar orbits clearly demonstrates the existence of evolutionary constraints in the adaptive evolution dynamics.
using high-throughput sequencers, we identified fixed mutations in the tolerant strains.
one tolerant strain had a significantly higher number of fixed mutations than the others, probably due to disruption of muts, which is involved in the mismatch repair mechanism.
for the other tolerant strains, the number of fixed mutations was less than  <dig>  we found that these mutations were commonly observed in the rela gene, which is involved in stringent response via ppgpp production, suggesting that the stringent response triggered by the ethanol stress was relaxed by these mutations in the tolerant strains and therefore did not diminish growth activity as would otherwise be expected.
these mutations could be regarded as candidate beneficial mutations for ethanol tolerance.
the observed synchronized slow expression changes might suggest the existence of deterministic slow dynamics of adaptive evolution, and can at the same time be difficult to explain by phenotypic changes caused by a small number of mutations.
in fact, by analyzing the timing of the mutation fixation, we demonstrated that the increase in growth rate of strain f did not correlate with mutation fixation events, and e. coli clones without any identified beneficial mutation grew significantly faster than the parent strain under ethanol stress .
furthermore, to evaluate the contribution of identified mutations to phenotype, we introduced these mutations into the genome of the parent strain of strain f and then quantified the change in ethanol tolerance.
the results showed that the observed ethanol tolerance in strain f could not be reproduced by introducing the identified mutations .
importantly, the ethanol tolerance was maintained after cultivation of  <dig> generations under conditions without ethanol stress, which indicated that the phenotype of ethanol tolerance in these strains was somehow stably maintained, although the contribution of fixed mutations to the tolerance was obscure.
one possible explanation of these results is that there are unidentified contributions of genetic alteration to the observed phenotypic changes.
in our genome resequencing analysis, the genomic regions with low read coverage  were excluded from the mutation identification procedure.
such low coverage regions cover  <dig>  base pairs  in total.
most of the low coverage regions have identical or nearly identical sequences to the other regions of the genome.
in fact,  <dig>  out of  <dig>   base pairs in the low coverage regions correspond to the coding region of rrna, trna, and ribosomal proteins, which have several nearly identical sequences on the e. coli genome.
for such regions, the identification of mutations is difficult by using the short-read sequencing technology.
thus, we cannot exclude that the possibility that there were some mutations in such regions, which were failed to be identified but contributed to the observed ethanol tolerance.
another possibility is that the growth increase observed in the adaptive evolution experiment under ethanol stress was due to phenotypic plasticity without genetic alteration, and that this plasticity could be stably memorized in the intracellular state and be inherited by progeny cells, a phenomenon called trans-generational plasticity  <cit> .
several studies demonstrated the existence of trans-generational plasticity in eukaryotic organisms .
it has recently been shown in bacteria, that trans-generational transmission of cellular components allows responses to environmental change with a memory  <cit> .
however, in such cases, an adapted state is generally maintained only for several generations.
in contrast, that ethanol tolerance was maintained in tens of generations in the present study suggests machinery for stable information inheritance.
similar epigenetic memory was also suggested to play a role in the evolution of antibiotic resistance in e. coli  <cit> .
in e. coli cells, genome methylation patterns are known to act as epigenetic memory that controls the expression profile  <cit> , as well as the binding of histone-like proteins, such as h-ns and fis, to genomic dna  <cit> .
these epigenetic mechanisms might contribute to the observed non-genetic memory and should be considered in future studies.
CONCLUSIONS
we analyzed phenotypic and genotypic changes of e. coli cells that occurred during adaptive evolution to ethanol stress and found that the evolutionary orbits of phenotype among independent culture series were similar, indicating the existence of evolutionary constraints.
the relationship between genetic mutations and phenotypic changes were complex, which might suggest that part of the phenotypic changes were due to contribution of phenotypic plasticity without genetic alterations.
the detailed analysis of phenotypic and genotypic changes in this study provides a better understanding of the nature of adaptive evolution, including non-genetic contributions to adaptive phenotypic changes.

 biological systems possess the ability to adapt to environmental changes, which can generate a variety of phenotypes and genotypes. such emergence of phenotypic and genotypic diversity is considered a result of stochastically fixed genomic mutations during the process of evolution. a question that arises here is whether the process of evolution allows arbitrary phenotypic changes or whether there are constraints that restrict possible variations in phenotypes. the pioneering studies by waddington, which have been corroborated by several other studies, suggests the latter, i.e., constraints on evolutionary dynamics is ubiquitous. one example of such evolutionary constraint is that the earliest embryo of various organisms shows a conserved morphological pattern called the phylotypic period, which is a constrained distribution of phenotype. here, the relationship between evolutionary constraints and phenotypic plasticity without genetic alteration has generated significant attention. however, despite the recognized importance of characterizing evolutionary constraints, quantitative understanding of the process still remains unclear. for this purpose, greater analysis is needed on phenotypic and genotypic changes in a variety of evolutionary courses. laboratory evolution of bacteria is a powerful tool to trace phenotypic and genotypic changes in adaptive evolution in a quantitative manner. recent advances in high-throughput sequencing have made it possible to identify and study fixed mutations in whole-genomic sequences during microbial adaptive evolution. for example, several mutations were identified as beneficial in adaptively evolved escherichia coli strains that used glycerol as the carbon source. other studies using laboratory evolution and genome resequencing have provided evidence that genomic mutations contribute to adaptive phenotypic changes against various environments, including several carbon sources, different temperatures, and the presence of antibiotics. the advancements of genome-wide analysis in laboratory evolution open the door to integrate quantitative data of phenotypic and genotypic changes, which can shed light on the nature of evolutionary dynamics including quantitative understanding of evolutionary constraints. in this study, we analyzed phenotypic and genotypic changes in the laboratory evolution of e. coli cells. in the previous study of laboratory evolution under the ethanol stress condition, we found that the overall gene expression changes before and after long-term cultivation were similar among independently evolved tolerant strains. however, it is still unclear relationship between phenotypic change and genetic change during evolution. in this study, first to further analyze the relationship in phenotypic changes in the independently evolved tolerant strain, we quantified time-series of expression changes. the changes of metabolite concentrations were also quantified in the tolerant strains. then, we assessed genotypic changes in the tolerant strains using high-throughput sequencers, to analyze the relationship between fixed mutations and phenotypic changes. to quantitatively evaluate the effects of fixed mutation on the ethanol tolerance, we introduced all the identified mutations in the genome of the parent strain into one of the tolerant strains. by integrating these phenotypic and genotypic data, we analyzed how the phenotype-genotype mapping is organized in the process of adaptive evolution.  time-series expression analysis in adaptive evolution under ethanol we previously obtained independently evolved ethanol tolerant e. coli strains by culturing cells under 5 % ethanol stress for about generations and found a significantly larger growth rate than the parent strains. here, we defined "ethanol tolerance" as a state with significantly higher growth rate under 5 % ethanol stress condition, and the term "strain" is used for the mixed population without single-colony isolation. to elucidate the phenotypic changes that occurred during adaptive evolution, we first quantified the time-series of the expression changes by microarray analysis. starting from frozen stocks obtained at time points in laboratory evolution, cells were cultured under 5 % ethanol stress, and mrna samples were obtained in the exponential growth phase for microarray analysis. the results of the time-series transcriptome analysis revealed that the expression changes during adaptive evolution were similar among tolerant strains. for example, fig.  shows the expression changes of genes in the central metabolic pathway including glycolysis, the tricarboxylic acid cycle, and the pentose phosphate pathway. interestingly, common expression changes were not always monotonic over time, but were rather synchronized complex expression changes on a much longer time-scale than the generation time observed. additionally, a common and gradual up-regulation of genes involved in some amino acid biosynthesis pathways was also observed. our previous work suggests that these pathways might contribute to ethanol tolerance.fig. time-series transcriptome analysis for adaptive evolution of e. coli to ethanol stress. expression changes of representative genes in the central metabolic pathway including glycolysis, the pentose phosphate pathway, and tca cycle are shown. in each inset, the horizontal axis shows time, and the vertical axis shows expression level. expression levels of 0 h in each gene represent the ones of parent strain. asterisks indicate expression levels of parent strain obtained without adding ethanol as a reference. the numbers of generations from to 2000 h were strain a: b: c: d: e: and f: respectively. abbreviations: 2pg, 2-phosphoglyceric acid; 3pg, 3-phosphoglycerate; accoa, acetyl-coa; αkg, α-ketoglutarate; bpg, -bisphosphoglycerate; cit, citrate; e4p, erythrose4-phosphate; f6p, fructose 6-phosphate; fbp, fructose -bisphosphate; gap, glyceraldehyde 3-phosphate; g6p, glucose 6-phosphate; oxa, oxaloacetate; pep, phosphoenolpyruvate; pyr, pyruvate; r5p, ribose 5-phosphate; s7p, sedoheptulose 7-phosphate; suc, succinate; x5p, xylulose 5-phosphate figure 2a shows overall expression changes during the adaptive evolution of the six tolerant strains by principal component analysis. the orbits in pca space, which represent expression profile changes, were similar except for strain c. the reason for this exception might be that strain c has an approximately kbp region in the genome that was duplicated, and the expression levels of genes in this region were increased by this duplication. the expression analysis also demonstrated that the overall expression changes between the parent and tolerant strains at the endpoint were similar. these results indicated that even though these strains adapted to ethanol stress in independent cultures, the expression profiles converged into almost identical adapted states with similar orbits of expression changes.fig. similarity among expression changes in the tolerant strains. a changes in pca scores during adaptive evolution. starting from the parent strain, changes in the expression profiles during adaptive evolution are plotted as orbits in the two-dimensional pca plane. b an example of the correlation between expression changes that occurred in two tolerant strains. the expression changes in strain e and f were plotted. horizontal and vertical axes are log-transformed expression ratios with the parent strain, and each dot represents the expression change of the gene. all possible combinations are shown in additional file 3: figure s2 metabolome analysis of ethanol tolerant strains to further characterize the phenotypic changes that occurred in the tolerant strains, we measured metabolite concentration changes between parent and tolerant strains. using capillary electrophoresis time-of-flight mass spectrometry, we quantified the intracellular concentrations of metabolites. the intracellular concentrations of some amino acids in the parent and tolerant strains are presented in fig. 3a. these concentrations generally decreased in the tolerant strains, except for that of methionine. the decrease was especially true for amino acids that originated from precursors in the tricarboxylic acid cycle. this suggests a change of metabolic state in the tca cycle in tolerant strains, a conclusion supported by the significant decrease in the expression of genes related to the tca cycle. for example, glutamate acts as a major amino-group donor in amino acid biosynthesis, and thus a decrease in its concentration can cause a decrease in the concentration of other amino acids. the decrease in amino acid concentration can activate the amino acid starvation response, which is consistent with the up-regulation of genes related to amino acid biosynthesis. in contrast, the concentrations of metabolites in purine metabolism generally increased. this concentration increase might be caused by the up-regulation of genes involved upstream of the purine biosynthesis pathway, by which phosphoribosyl pyrophosphate, the precursor for purine nucleotide synthesis produced from ribose-5-phosphate, is converted into inosine 5'-monophosphate. no significant concentration change was observed for metabolites in pyrimidine metabolism.fig. metabolome analysis of ethanol tolerant e. coli strains under ethanol stress conditions. a concentration of amino acids in ethanol tolerant strains. in each inset, the vertical axis shows the log-transformed absolute concentration. the blue bar and red bars indicate the data of the parent strain and tolerant strains, respectively. b correlation between metabolite concentration changes in strains e and f. horizontal and vertical axes represent log-transformed concentration ratios with the parent strain, and each dot represents the concentration change of the metabolites the metabolome analysis also demonstrated similar changes in metabolite concentration among the tolerant strains, which resembles observations for gene expression changes. the correlation of overall metabolite concentration changes between independently obtained tolerant strains indicated similar metabolite shifts. both the transcriptome and metabolome analyses showed that phenotypic changes were similar among tolerant strains even though they were obtained from independent long-term cultivations. genome resequencing analysis of ethanol tolerant strains genotype changes in each tolerant strain were analyzed using two high-throughput sequencers, solid and illumina miseq. in the resequencing analysis, we extracted genomic dna samples from the cell population at the end-point of the experimental evolution without single-colony isolation, to identify genotype changes that were fixed in the majority of tolerant cells and to avoid a fixation of minority mutations. for point mutations, solid and illumina analyses identified and fixed mutations in all tolerant strains, respectively, with of these mutations being identified in both analyses. the discrepancy was checked by sanger sequencing, and it was confirmed that were true positives and the other a false positive. after screening indels by solid sequencing, we identified small and large indels in all tolerant strains. we verified these small and large indels by sanger sequencing, finding all were true positives. finally, in strain c, the coverage of an approximately kbp region was significantly higher than in other strains, which strongly suggested that the corresponding region duplicated during long-term cultivation. the identified mutations at the end-point of experimental evolution are summarized in table  the number of mutations in strain a was significantly higher than other strains. this was likely due to a mutation leading to a stop codon in the muts coding region, which codes a mismatch repair protein. it is known that disruption of muts significantly increases the mutation rate of e. coli cells. we confirmed that there were only mutations in strain a at 1512 h after commencing laboratory evolution and these did not include a mutation in muts. this result suggested that after 1512 h, the mutation in the muts gene was fixed and resulted in a significant increase in the mutation rate. the emergence of a strain with a significantly high mutation rate, or a “mutator,” is often observed in the laboratory evolution of microorganisms.table list of identified mutations hns a year iscr ilvg insh,nmpc,essd,ybcs,rzpd,rzod,bord,ybcv,ybcw,nohb,tfad,ybcy nage hns yeay menc rela rpoc rpoa hns proq ispg rpsd yjha hns cspc rela yhcm atpe miab cspc wzxc iscr rela abold-faced genes represent overlap among evolved strains in contrast to the more than one hundred fixed mutations in strain a, the number of fixed mutations was relatively lower in the other strains. as mentioned above, the phenotypic changes that occurred in independently evolved tolerant strains were similar, which might suggest mutations fixed in identical or related genes contributed to the changes. we found that mutations were commonly fixed in rela, which codes guanosine tetraphosphate synthetase. rela regulates a stringent response by producing guanosine tetraphosphate. the stringent response is widely observed in bacteria as a stress response in reaction to nutrient starvation or various environmental stresses. when e. coli face such stresses, growth-related activities including replication, transcription, and translation are tightly inhibited, which are triggered by the accumulation of ppgpp. thus, the mutations commonly fixed in rela may relax the stringent response caused by ethanol stress to recover growth activity. the mutations in rela and spot, which codes an enzyme that plays a major role in ppgpp degradation, have been widely observed in the laboratory evolution of e. coli under various conditions, including glucose limitation and high temperature. here, relaxing the stringent response by mutating the rela and spot genes may increase fitness under stress. furthermore, in strains a, b, c, d, and e, insertion sequence element was inserted into the promoter region of hns, which codes for a dna binding protein that has various effects on gene expression. however, no significant change in hns expression was observed in these strains. except for rela and hns, no functional overlap among the mutations fixed in more than two tolerant strains was determined. fitness contribution of fixed mutations to evaluate the contribution of fixed mutations to the growth increase under ethanol stress, we introduced all identified mutations in strain f into the parent genome by site-directed mutagenesis. we selected strain f for this analysis because the number of is insertion was smallest among the tolerant strains. in strain f, we identified mutations, including single nucleotide substitutions, one small deletion, and one insertion in the genome of strain f. the sequence of the 1199-bp insertion was identical to is which disrupted the orf of cspc. since the insertion of is into the same position of the parent genome was difficult experimentally, we constructed a cspc deletion strain. figure  shows the growth rates of the constructed strains by site-directed mutagenesis measured under the ethanol stress condition. here, the main purpose of the analysis was to introduce all identified mutations in strain f into the genome of the parent strain, instead of constructing mutant strains with all possible combinations of mutations. thus, the combinations of and mutations are arbitrary chosen to optimize the speed to construct the strain with mutations. the results in fig.  demonstrated that the fixed mutation in rela significantly contributed to the growth rate increase. however, other mutations had no significant effect on the growth rate, and even when all fixed mutations in strain f were introduced into the parent genome, the observed growth rate of the mutated strain was significantly smaller than that of strain f under the ethanol stress condition.fig. growth rates of site-directed mutants with ethanol stress. all mutations identified in strain f were introduced back to the parent strain. for each mutant, the names of the mutated genes are shown. among the mutations identified in strain f, the mutations for cspc and miab correspond to is insertion and 88 bp deletion, respectively, while other mutations were snps. error bars indicate standard deviations calculated from three independent cultures. different asterisks indicate significant differences, which were determined by dunnett’s test compared with parent strain timing of fixed mutations to further evaluate the contribution of the fixed mutations on ethanol tolerance, we analyzed the relationship between the growth increase under ethanol stress and the timing of mutation-fixation events in long-term cultivation in strain f. to identify the timing, genomic dna samples obtained from cell populations that had heterogeneous genotypes at different time points were applied to sanger sequencing. therefore, in some cases, the peak signals in the sanger sequencing revealed mixed populations, i.e., cells with and without a specific mutation coexisted in the population. figure 5a shows the time the mutations in strain f emerged. the increased growth rate did not always correlate with fixation events. more importantly, although at 576 h after inoculation no mutation was fixed in the majority of the cell population, the growth rate under ethanol stress significantly increased. some cells at 576 h had mutations in the rela and cspc genes that may have contributed to the observed growth increase. to confirm this possibility, we isolated clones from the cell population at 576 h and analyzed fixed mutations in rela and cspc by sanger sequencing. among the clones, had both rela and cspc mutations, had the cspc mutation only, and the other clones had no mutation. to evaluate the effect of these mutations on the population at 576 h, we randomly selected clones from the groups,, and, and measured the growth rates of clones with and without mutations under the ethanol stress condition. as shown in fig. 5b, clones with or without rela and cspc mutations showed significantly larger growth rates than parent strain, and there was no significant growth difference between clones. it should be noted that, the average growth rates of clones corresponding to groups,, and were significantly higher than the clone in which identified mutations were introduced. these results suggested that, the effect of these mutations into the parent genome was smaller than the increase of growth rate in the cultivation from to 576 h, even though in some clones no mutations was suggested to be fixed on the genome.fig. time-series analysis of the mutation fixation. a timing of mutation fixation events in strain f. to identify the timing of mutation fixation, genomic dna samples obtained at different time points were applied to sanger sequencing. for each of the identified mutations, the results of the sanger sequencing is presented as a solid or dotted line. the solid line indicates that the mutation was fixed in the population at the corresponding time point, while the dashed line indicates the case of two peak signals, which indicates polymorphism in the population with and without the mutation. for example, in cells at 576 h after inoculation, only some have mutations in rela and cspc. b specific growth rates of cloned e. coli cells with and without cspc and rela mutations under ethanol stress condition. each bar represents the specific growth rate of an isolated clone, which were obtained from strain f cell populations at 576 h. " + " and "-" mean with and without the corresponding mutation, respectively. blue, green, and red bars represent the growth rates of clones without mutations, that with cspc mutation only, and with cspc and rela mutations, respectively. error bars indicate standard deviations calculated from three independent cultures the results in fig. 5a and 5b suggested the possibility of clonal interference. the result in fig. 5b suggested that cspc mutation was fixed on a cell with rela mutation, while the result in fig. 5a suggested rela mutation rose to prominence before cspc mutation. these results might suggest that, in the evolutionary dynamics of these populations, there were multiple clones with different beneficial mutations coexisting and competing in the environment, i.e., clonal interference. stability of ethanol tolerance we reasoned that if the observed ethanol tolerance is due to phenotypic plasticity without genetic alteration, then the phenotype of the ethanol tolerance would likely be unstable when the environment changes. we therefore cultivated cells with ethanol tolerance in an ethanol-stress free environment for generations. two cell populations were used: strain f and the cell population obtained at 576 h in the cultivation of strain f. after cultivation in the non-stress condition, we measured the growth rate under 5 % ethanol stress to evaluate the stability of the ethanol tolerance. ethanol tolerance was stably maintained even after generations, which suggests that the observed phenotypic changes in the tolerant strains were stably memorized and passed on to progeny cells. growth evaluation of clones in the population of the ethanol tolerant strains in this study, to analyze the characteristics of the majority of cell population, we measured mixed population without single colony isolation when we performed transcriptome, metabolome, and genome resequencing analysis. to evaluate the effect of clonal interference in the population of the ethanol tolerant strains, we isolated clones from the end-point population of strains e and f. then, we quantified the growth rate of these clones under the ethanol stress condition as shown in additional file 11: figure s the result demonstrated that there were no difference between isolated clones and mixed population, suggesting that the effect of clonal interference is negligible in the population of ethanol tolerant strain we obtained. discussion transcriptome and metabolome analyses revealed that phenotypic changes that occurred in ethanol tolerant strains were similar among independently evolved strains. gene expression changes over time were found to exhibit high similarity among tolerant strains, which included non-monotonic expression changes with time scales much longer than the generation time. the observed phenotypic convergence to similar orbits clearly demonstrates the existence of evolutionary constraints in the adaptive evolution dynamics. using high-throughput sequencers, we identified fixed mutations in the tolerant strains. one tolerant strain had a significantly higher number of fixed mutations than the others, probably due to disruption of muts, which is involved in the mismatch repair mechanism. for the other tolerant strains, the number of fixed mutations was less than we found that these mutations were commonly observed in the rela gene, which is involved in stringent response via ppgpp production, suggesting that the stringent response triggered by the ethanol stress was relaxed by these mutations in the tolerant strains and therefore did not diminish growth activity as would otherwise be expected. these mutations could be regarded as candidate beneficial mutations for ethanol tolerance. the observed synchronized slow expression changes might suggest the existence of deterministic slow dynamics of adaptive evolution, and can at the same time be difficult to explain by phenotypic changes caused by a small number of mutations. in fact, by analyzing the timing of the mutation fixation, we demonstrated that the increase in growth rate of strain f did not correlate with mutation fixation events, and e. coli clones without any identified beneficial mutation grew significantly faster than the parent strain under ethanol stress. furthermore, to evaluate the contribution of identified mutations to phenotype, we introduced these mutations into the genome of the parent strain of strain f and then quantified the change in ethanol tolerance. the results showed that the observed ethanol tolerance in strain f could not be reproduced by introducing the identified mutations. importantly, the ethanol tolerance was maintained after cultivation of generations under conditions without ethanol stress, which indicated that the phenotype of ethanol tolerance in these strains was somehow stably maintained, although the contribution of fixed mutations to the tolerance was obscure. one possible explanation of these results is that there are unidentified contributions of genetic alteration to the observed phenotypic changes. in our genome resequencing analysis, the genomic regions with low read coverage were excluded from the mutation identification procedure. such low coverage regions cover base pairs in total. most of the low coverage regions have identical or nearly identical sequences to the other regions of the genome. in fact, out of base pairs in the low coverage regions correspond to the coding region of rrna, trna, and ribosomal proteins, which have several nearly identical sequences on the e. coli genome. for such regions, the identification of mutations is difficult by using the short-read sequencing technology. thus, we cannot exclude that the possibility that there were some mutations in such regions, which were failed to be identified but contributed to the observed ethanol tolerance. another possibility is that the growth increase observed in the adaptive evolution experiment under ethanol stress was due to phenotypic plasticity without genetic alteration, and that this plasticity could be stably memorized in the intracellular state and be inherited by progeny cells, a phenomenon called trans-generational plasticity. several studies demonstrated the existence of trans-generational plasticity in eukaryotic organisms. it has recently been shown in bacteria, that trans-generational transmission of cellular components allows responses to environmental change with a memory. however, in such cases, an adapted state is generally maintained only for several generations. in contrast, that ethanol tolerance was maintained in tens of generations in the present study suggests machinery for stable information inheritance. similar epigenetic memory was also suggested to play a role in the evolution of antibiotic resistance in e. coli. in e. coli cells, genome methylation patterns are known to act as epigenetic memory that controls the expression profile, as well as the binding of histone-like proteins, such as h-ns and fis, to genomic dna. these epigenetic mechanisms might contribute to the observed non-genetic memory and should be considered in future studies.  we analyzed phenotypic and genotypic changes of e. coli cells that occurred during adaptive evolution to ethanol stress and found that the evolutionary orbits of phenotype among independent culture series were similar, indicating the existence of evolutionary constraints. the relationship between genetic mutations and phenotypic changes were complex, which might suggest that part of the phenotypic changes were due to contribution of phenotypic plasticity without genetic alterations. the detailed analysis of phenotypic and genotypic changes in this study provides a better understanding of the nature of adaptive evolution, including non-genetic contributions to adaptive phenotypic changes. 
the results demonstrated that the convergence of adaptive phenotypic changes and diverse genotypic changes, which suggested that the phenotype–genotype mapping is complex. in this study, we analyzed phenotypic and genotypic changes in escherichia coli cells during adaptive evolution to ethanol stress. bacterial cells have a remarkable ability to adapt to environmental changes, a phenomenon known as adaptive evolution. during adaptive evolution, phenotype and genotype dynamically changes; however, the relationship between these changes and associated constraints is yet to be fully elucidated. phenotypic changes were quantified by transcriptome and metabolome analyses and were similar among independently evolved ethanol tolerant populations, which indicate the existence of evolutionary constraints in the dynamics of adaptive evolution. furthermore, the contribution of identified mutations in one of the tolerant strains was evaluated using site-directed mutagenesis. the integration of transcriptome and genome data provides a quantitative understanding of evolutionary constraints. the result demonstrated that the introduction of all identified mutations cannot fully explain the observed tolerance in the tolerant strain.
 bacterial cells have a remarkable ability to adapt to environmental changes, a phenomenon known as adaptive evolution. during adaptive evolution, phenotype and genotype dynamically changes; however, the relationship between these changes and associated constraints is yet to be fully elucidated.  in this study, we analyzed phenotypic and genotypic changes in escherichia coli cells during adaptive evolution to ethanol stress. phenotypic changes were quantified by transcriptome and metabolome analyses and were similar among independently evolved ethanol tolerant populations, which indicate the existence of evolutionary constraints in the dynamics of adaptive evolution. furthermore, the contribution of identified mutations in one of the tolerant strains was evaluated using site-directed mutagenesis. the result demonstrated that the introduction of all identified mutations cannot fully explain the observed tolerance in the tolerant strain.  the results demonstrated that the convergence of adaptive phenotypic changes and diverse genotypic changes, which suggested that the phenotype–genotype mapping is complex. the integration of transcriptome and genome data provides a quantitative understanding of evolutionary constraints. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. issue-copyright-statement© the author 2015 