Ex Uno Plures: Clonal Reinforcement Drives Evolution of a Simple Microbial Community
[["Illuminating the laws that produce Darwin's “tangled bank” remains one of the great challenges of biology , one that requires understanding how differences among forms are selected for , and how interdependence among forms is generated .", 'In natural environments , meeting this challenge is complicated by the fact that selection pressures often vary widely over both space and time .', 'Laboratory evolution experiments , in particular those using microbes , offer an attractive alternative by which to study , under controlled conditions , both the dynamic interplay between genotype and phenotype as well as the interactions among phenotypes in simple communities .', 'Classical models of large asexually evolving populations led to the expectation that in simple environments complexity should be transient and limited in scope [1] , [2] .', 'Experimental evidence now suggests otherwise .', 'Multiple genotypes that arise from a single ancestral clone can coexist over evolutionary time; in other words , ex uno plures ( out of one many ) .', 'This phenomenon has been documented in spatially and temporally unstructured chemostats [3] , [4] , in temporally-structured batch cultures [5]–[9] and in spatially-structured microcosms [10] .', 'In each setting , the emergence and persistence of polymorphism in the absence of sexual recombination requires that cohabitants opportunistically exploit unoccupied niche space , and/or accept trade-offs between being a specialist and a generalist [11]–[13] .', 'In serial dilution batch culture , multiple growth parameters can come under selection [14]: different clones may arise having reduced lag time , increased maximum specific growth rate , or enhanced capacity to survive at high cell densities in the presence of low nutrients [15] .', 'Periodic changes in population density and nutrient levels may bring balancing selection to bear on these different phenotypes ( e . g . , [8] ) , especially if mutations are antagonistically pleiotropic [16] , [17] .', 'In spatially structured environments selection may favor mutants better adapted to particular regions or better able to colonize microhabitats formed at the boundaries between such regions .', 'By contrast , in continuous nutrient-limited environments , theory predicts that selection will favor clones better able to scavenge the limiting resource or more efficiently convert that resource to progeny [18] , [19] .', 'Ultimately , the outcome of the ‘evolutionary play’ in any of these ‘ecological theaters’ ( sensu [20] ) will depend on founder genotype , mutation rate , the complexity of genetic pathways leading to different adaptive strategies , as well as pleiotropy [21] and epistatic interactions [22] .', "Increasing evidence points to the possibility of not one , but three potential outcomes when asexual microbes evolve in simple environments: clonal succession , where a population is successively swept by clones of higher fitness arising in the dominant lineage [23] , [24]; clonal interference , where fixation of a single fittest clone is deferred because independent beneficial mutations arise in multiple , independent clones that compete with one another and reduce each other's fitness [25]–[27] , and what we propose to call clonal reinforcement , where the emergence of one genotype favors the emergence and persistence of other genotypes via cooperative interactions .", 'Because cooperation is now recognized to be at least as important as competition in driving biological innovation and in structuring communities [28] , [29] , we investigated the genetic and environmental factors that foster interdependence in an evolving lab population .', 'Previously [30] , we examined the transcriptomes of an experimentally evolved E . coli community in which three different strains that arose from a common ancestor stably coexisted in a simple , unstructured environment through metabolic cross-feeding [3] , [31] .', 'This system is unusual in that it clearly involves symbiotic interactions among ecotypes that co-evolved in the absence of spatial or temporal variation .', "Remarkably , the community's expression profile did not proportionately represent the sum of each strain's expression profile grown singly under evolutionary conditions , indicating that the whole was not the sum of its parts .", "Instead , expression data suggested that consumption of one ecotype's overflow metabolites by the others relieved feedback inhibition , adding a layer of complexity to the evolved clones' interactions .", 'Using a candidate gene approach we identified two mutations in the ancestral strain that may have predisposed clonal reinforcement [30] .', 'To better understand how physiological performance and expression profile map onto genotype , and to identify the molecular basis of community interactions we sequenced the genomes of each of the three community members and their common ancestor .', 'We found unexpectedly high levels of genetic variation in the three-membered community as a whole and the numerically dominant strain in particular , as well as a strong mutational bias due to specific lesions in DNA repair .', 'The dominant community member is a hypermutator that excels at acquiring glucose , but does so at the expense of carbon excreted as overflow metabolites to which it has limited access; this trade-off effectively opens up new niches for other genotypes .', 'We discovered a set of adaptive mutations in this clone that have not been previously reported to co-occur in other E . coli evolution experiments , specifically those that enhance glucose acquisition and may serve to maintain redox balance , but may also increase net flux to overflow metabolites under aerobic conditions .', 'Because this community member releases growth substrates that are differentially accessible to other community members , and because it shares only one SNP mutation with these strains , we suggest that it acts as an engine generating biodiversity , creating new metabolic niches , but not necessarily the occupants themselves .'], ['In previous publications [3] , [30] , [31] we described certain features of a stable polymorphism that arose from a single clone during the course of 765 generations of aerobic , glucose-limited culture at 30°C ( D≈0 . 20 hr−1 ) ( Table 1 ) .', 'The four clones isolated from this population ( CV101 , C103 , CV115 and CV116 ) were originally distinguished by their differences in antibiotic resistance and colony morphology , and later shown to exhibit strain-specific rates of glucose uptake , residual metabolite concentrations and expression profiles .', 'In reconstruction experiments performed under evolutionary conditions CV101 , CV103 and CV116 were shown to stably coexist at frequencies of approximately 0 . 10 , 0 . 65 and 0 . 025 , respectively .', 'Because CV115 was not stably maintained , the interactions among CV101 , CV103 and CV116 were studied more intensively .', "Based on differences in glucose uptake and residual substrate concentrations , and the fact that strains' steady state frequencies could be predictably altered by exogenously increasing the concentrations of residual metabolites , we concluded that this community was sustained by positive density-dependent interactions in the form of cross-feeding , in which the numerically dominant strain CV103 best takes up the limiting resource glucose , but excretes acetate and glycerol ( and/or a closely-related compound , glycerol 3-phosphate ) ( J . Adams unpublished results ) .", 'These two overflow metabolites are then scavenged by CV101 and CV116 , respectively .', 'Transcriptional profiling of each clone in monoculture relative to the common ancestor , JA122 , revealed gene expression differences among the evolved isolates related to carbon metabolism and expression differences specific to CV103 consistent with activation of stress response pathways and loss of motility .', 'As summarized in Table 2 , 584 mutations ( 580 SNPs , two insertions and two deletions ) were identified among the four evolved clones .', 'This number is substantially higher than has been previously reported for laboratory evolution experiments of similar duration conducted under similar conditions [32] .', 'A majority of these mutations , 504 ( 86% ) , is in coding regions and 374 ( 64% ) are non-synonymous , while 428 ( 73% ) , are unique to CV103 .', 'Almost all ( 99 . 7% ( 578 ) ) SNPs are GC→TA transversions , suggesting defects in DNA repair among all of the evolved isolates .', 'Strong transversion bias has been noted in other evolution experiments , and is likely due to defective repair of oxidatively damaged G:C base-pairs [32] , [33] .', 'Phylogenetic analyses of the whole genome sequences [30] show that the dominant clone CV103 is part of a highly divergent lineage , while CV115 and CV116 are very closely related to one another , and share a common ancestor with CV101 ( Fig . 1 ) .', 'Indeed , as CV115 and CV116 differ by only two SNPs , one a synonymous substitution ( gutQ , A221A ) , the other C-terminal ( ycaO G546V ) , and because only CV101 , CV103 and CV116 could be stably maintained at steady state , we restrict most of our discussion to these three clones .', 'Remarkably , the evolved clones all share only one SNP .', 'Thus , it is likely that the CV103 and CV101/CV115/CV116 lineages diverged from one another early in the experiment and that stable co-existence of more than one lineage was an early feature of the population .', 'In fact , previous work documented a variant similar to CV103 ( small colony , ampicillin-resistant ) had risen to appreciable frequency by generation 340 [3] .', 'This result is not unexpected , as subsequent to the observations of Helling et al . 1987 , a number of other reports appeared showing that multiple genotypes can coexist in nutrient-limited chemostats [34]–[38] .', 'An unanticipated finding in our experiments was the large number of SNPs that accumulated along the CV103 branch .', 'Fluctuation analyses performed on the ancestral strain , JA122 , the canonical E . coli strain K12 MG1655 , and the four co-evolved clones revealed that the founder strain , JA122 , is a mutator whose mutation rate is approximately thirty-fold higher than E . coli K12 MG1655 ( Table 1 ) .', 'This observation can be explained by a nonsense mutation in JA122 affecting the adenine glycosylase mismatch repair enzyme MutY ( L299* ) .', 'Defects in MutY are known to cause a GC→TA transversion bias [33] , [39] and have been observed by others in glucose limited chemostat experiments [40] , [41] .', "Evolved strains CV101 , CV115 and CV116 all had mutation rates similar to JA122 , while CV103's mutation rate was almost 10-fold higher again ( Table 1 ) .", 'This difference can be attributed to a second mutation resulting in an amino acid substitution ( A56D ) in the base-excision repair pathway DNA glycosylase , MutM .', 'Increased mutation rate has been shown to be selected for in microbial evolution experiments performed under a variety of conditions [33] , [41]–[45] where it can strongly influence evolutionary dynamics [46] .', 'The fitness of mutators need not decrease over time; Maharjan et al . [47] recently showed that mutators arising under glucose limitation gain fitness relative to their ancestor as a result of adaptive mutations in , for example , rpoS and mglD .', 'Mutator lineages persisted because most of the other mutations accumulating in their backgrounds were neutral in their effect on fitness , deleterious mutations being purged as the lineages evolved .', 'A similar mechanism may be at work in our experiment , with an appreciable number of mutations in CV103 being neutral .', 'Given the large number of SNPs , we sought to determine whether certain genes or transcription units carried multiple mutations , either because they had had been selected for or because their inactivation allowed neutral mutations to accumulate .', 'Overall , 52 transcription units and 37 individual genes contained more than one SNP ( Table S1 ) .', 'Twenty-five of the single/multiple gene hits occur exclusively in strain CV103 ( Table S1 ) .', 'Four of these ( eno , maeA , malG and ptsI ) are directly involved in glucose uptake/metabolism suggesting that they contribute to the superior glucose uptake kinetics of this clone .', 'Five CV103-specific substitutions affect flagellar synthesis: three SNPs occur in fliM , which encodes a flagellar motor switch protein , one occurs in fliF , which encodes the flagellar M ring protein , and one occurs in fliH , which encodes another flagellar biosynthesis protein .', 'These findings are consistent with the previously observed down-regulation of flagellar genes in CV103 and the fact this strain is non-motile [30] .', 'When all of the evolved isolates are considered , it is perhaps surprising that CV116 , thought to salvage excreted glycerol/glycerol phosphate from CV103 , has a number of mutations ( 10 ) in transcription units that are also mutated in CV103 ( Table S1 ) .', 'The same is not true for CV101 , the strain that salvages overflow acetate ( Table S1 ) .', 'While most of these SNPs are silent mutations , they could plausibly affect RNA stability or translation .', 'Of the non-silent changes , particularly noteworthy are four mutations ( three in CV103 and one in CV116 ) that affect NADH:ubiquinone oxidoreductase I , as well as additional mutations affecting genes involved in anaerobic formate production and electron transfer ( Table S1 ) .', 'Because both CV103 and CV116 show enhanced glucose uptake relative to CV101 and the ancestral strain ( Table 1 and [3] ) , these changes may be shared features of an adaptive response to rapid glucose consumption and concomitant overproduction of NADH ( discussed below ) .', 'Because we previously observed significant differences in gene expression among evolved isolates grown in monoculture [30] , we sought to identify SNPs that could explain these differences .', 'Because the great majority of these transcriptional differences were manifest in CV103 , we focused on genes that could directly or indirectly regulate loci differentially expressed in this strain and on the 428 genes associated with a SNP in CV103 .', 'The intersection of these two gene lists yielded three global regulators that could explain the majority of transcriptional differences between CV103 and the other evolved isolates: rpoD , csrA and sdiA .', 'RpoD , the housekeeping sigma factor , controls transcription of over 2 , 300 genes .', 'The ancestor of the four evolved isolates , JA122 , has an amber nonsense mutation ( E26* , GAG→TAG ) in rpoD that likely leads to reduced translation of RpoD , but not necessarily its complete absence , as JA122 also carries the supE44 amber suppressor .', 'CV103 has an additional silent mutation in rpoD ( T459T , ACC→ACA ) that does not affect protein sequence but may affect translation , as the mutant codon ACA is 30% less common than the wild-type codon ACC [48] .', 'The regulatory protein CsrA favors gluconeogenesis and glycogen synthesis over glycolysis .', 'While the CV103 mutation in csrA is also silent ( G27G , GGC→GGA ) , the resulting codon change is from one that is common ( f\u200a=\u200a0 . 40 ) to one that is rare ( f\u200a=\u200a0 . 09 ) .', 'Finally , sdiA encodes an N-acylhomoserine-L-lactone receptor that functions in quorum sensing .', 'In CV103 , a SNP is located 27 base-pairs upstream of the sdiA transcriptional start site in a region that encodes a small RNA , RNA0-361 .', 'The function of this sRNA is unknown , but it has been repeatedly identified in screens for small RNAs that interact with the global RNA chaperone , Hfq [49] , [50] , in which CV103 has a missense mutation ( see below ) .', '( Fig . 2 )', 'In earlier work we showed that all evolved isolates were better able to scavenge limiting glucose than their common ancestor [31] , and that strain CV103 had a significantly higher rate of glucose uptake than the other evolved isolates [3] .', 'We later attributed these observations to increased expression of LamB glycoporin in all four strains relative to JA122 [51] , and significantly higher transcription of lamB in CV103 relative to CV101 , CV115 and CV116 [30] .', 'In other studies of adaptation under glucose limitation elevated lamB expression has repeatedly been tied to mutations that affect the regulation of the Mal operon , specifically mutations that affect expression of the lamB regulators Mlc and/or MalT [35] , [51] , [52] .', 'While we previously found that CV101 , CV115 and CV116 share a mutation in malT that could up-regulate lamB expression , this mutation did not occur in CV103 [30] , the strain that best scavenges limiting glucose .', 'Whole genome sequencing uncovered two other mutations in CV103 that help explain higher transcript levels of lamB mRNA and , by extension , its superior ability to scavenge limiting glucose .', 'The first mutation affects the gene for MalK , a negative regulator of MalT [53] .', 'Null mutations in malK promote constitutive mal/lamB gene expression [54] , [55] , presumably by disrupting the MalK-MalT interaction .', 'The malK SNP in CV103 causes an amino acid change ( D297E ) in the C-terminal portion of the protein that contains an important part of the MalT interaction domain .', 'Replacement of aa 297 is known to diminish the ability of MalK to inhibit MalT [56] , and thus the D297E SNP likely leads to increased lamB transcription during glucose-limited growth [57]–[60] .', 'The second CV103 mutation that may influence lamB expression occurs in the gene for the RNA chaperone Hfq .', 'Hfq is a global regulator that facilitates binding of small regulatory RNAs ( sRNAs ) to mRNAs and by doing so affects translation and/or degradation of those transcripts [61] , [62] .', 'Details are scant as to how most of these sRNAs regulate their targets and how Hfq enhances or attenuates their activity , but the profound effect these interactions can have on key cellular processes is being increasingly recognized [61] , [63] .', 'Hfq interacts with the mRNA of the E . coli stationary phase sigma factor , RpoS , and is required for efficient translation of RpoS mRNA [64] .', 'Reduced RpoS expression has been extensively studied as a key adaptation to continuous glucose-limitation [40] , [65] , [66] , and Hfq has recently been identified as an alternative mutational target under glucose limitation in rpoS+ strains [67] , [68] .', 'In these isolates , a missense mutation in Hfq enhances glucose uptake via PtsG , increases levels of LamB , and apparently reduces the amount of functional RpoS , resulting in lower biomass yield .', 'The hfq mutation in CV103 , which results in a Q52H substitution , could have similar effects: CV103 has increased uptake of the glucose analog α-MG ( Table 1 ) , which occurs exclusively via PtsG , increased LamB gene expression , and decreased biomass yield relative to the other evolved clones [3] , [30] .', 'Interestingly , the genomic context of the Hfq mutation in CV103 differs from those characterized by others in which defects in Hfq have evolved in an rpoS deficient background [67] , [68] .', 'CV103 and our other evolved isolates share an ancestral rpoS ( Am ) mutation which likely allows some RpoS translation .', '( Fig . 2 )', 'After entry into the periplasm , glucose is actively transported across the inner membrane using the glucose-specific sugar phosphotransferase system ( PTS ) .', 'Mutations that upregulate an alternative high-affinity glucose transporter , the galactose transporter MglBAC , are frequently observed following prolonged growth under glucose limitation [35] , [51] , [52]; in this regard , our system is no exception .', 'In fact , the only SNP shared by all of the evolved isolates is in the operator sequence for the MglD repressor ( mglO , C→A , +3 bp relative to the end of mglD ) , consistent with increased expression of the mglBAC transcription unit [30] , [35] .', 'Because the rate of glucose uptake in CV103 exceeds that of the other clones , we looked for mutations that might affect expression or activity of other inner-membrane glucose transporters .', 'CV103 has two mutations in ptsI , which encodes enzyme I of the E . coli sugar phosphotransferase system .', 'This protein is active in its dimeric form and participates in glucose uptake by accepting a phosphate group from phophoenolpyruvate ( PEP ) and passing it via phosphocarrier protein Hpr to sugar-specific enzymes such as PtsG , which then use phosphate to “charge” incoming sugars ( Fig . 2 and 3 ) [69] , [70] .', 'The mutations in ptsI result in two amino acid substitutions ( A328S , M518I ) , both of which occur in the C-terminal region responsible for binding PEP and dimerization .', 'These may be reasonably expected to alter PtsI activity [71] , [72]; aa 328 is close to two residues that are part of the PEP binding site ( R332 and D335 ) , while aa 518 is close to C502 , which is required for phosphotransfer [73] , [74] .', 'When ∼PO4 is not passed along by PtsI , there can be regulatory consequences: unphosphorylated PtsG can bind the transcriptional regulator Mlc and prevent it from negatively regulating expression of its targets which include ptsG itself , manXYZ and malT [75]–[77] .', 'Thus , these mutations could also explain the up-regulation of manXYZ in CV103 , and lead to higher expression of the LamB glycoporin though derepression of malT transcription [78] .', 'PtsI has also been shown to interact directly with other phospho-enzymes , notably acetate kinase ( ackA ) , thus mutations in this protein may also affect acetate excretion [79] .', 'Although glucose transport is increased in all evolved isolates relative to their common ancestor , and especially so in CV103 , expression of many glycolytic genes is decreased ( Fig . 4 ) .', 'This trend has also been observed in yeast evolution experiments carried out under glucose limitation , and may reflect the selective advantage of an energy conservation strategy under low nutrient conditions [80] .', 'Also , the hypermutator lineage that gave rise to CV103 accumulated a number of mutations predicted to impact conversions in glycolysis , fermentation , and the TCA cycle .', "Certain of these mutations likely underlie CV103's superior glucose uptake kinetics , but some may also favor excretion of and restricted access to overflow metabolites , which opens up new niches for other genotypes .", 'In CV103 , transcript levels of pfkB , which encodes PfkII , a secondary enzyme that converts fructose-6-phosphate into fructose-1 , 6 , -bisphosphate , is more highly expressed in CV103 than in the other strains; CV103 also carries a missense mutation in this gene ( Q201H ) ( Fig . 4 ) .', 'It is unclear whether this mutation is beneficial , as PfkII is thought to be responsible for less 5% of the phosphofructokinase activity in E . coli [81] .', 'However , PfkII can also use tagatose-6-phosphate as a substrate [82] .', 'CV103 has a surprising number of differences compared to the other clones in the expression and sequence of other genes in the galactitol/tagatose-6-P glycolytic pathway , including downregulation of gatZY ( encoding tagatose-1 , 6-bisphosphate aldolase 2 ) and gatABC ( galactitol PTS permease ) as well as a mutation in the gene for GatY ( G49* ) ( Table S2 ) .', 'As in a number of other K12-derived strains , the gat operon is likely constitutively expressed in JA122 due to IS3E element insertion in the galactitol regulator ( GatR ) gene [83] .', 'Interestingly , increased expression of gat genes has been observed in experiments where E . coli has been evolved under lactulose and/or methyl-galactoside limitation [12] , [84] , [85] .', 'Enolase , responsible for the conversion of 2-phosphoglycerate into phosphoenolpyruvate ( PEP ) , has two mutations in CV103 , one silent and one missense ( L60L , A37S ) ( Fig . 4 and Table S2 ) .', 'Aside from the regulatory role played by its product , PEP , enolase participates in the degradation of certain RNAs as part of the degradosome [86] .', 'In particular , enolase is needed to degrade ptsG mRNA when intracellular levels of G-6-P are high ( i . e . , during phosphosugar stress ) [86] , [87] .', 'This interaction also involves the sRNA SgrS , Hfq and Pnp ( polynucleotide phosphorylase ) , in which CV103 also has a substitution ( P104Q ) [50] , [87]–[89] ( Table S2 ) .', 'ptsG mRNA is not degraded when Hfq is mutated [90] , thus in CV103 the combined action of mutations in enolase , Pnp and Hfq may increase longevity of ptsG transcripts .', 'Pyruvate is a major metabolite that sits at the branch point between glycolysis , the TCA cycle and fermentation ( Fig . 4 ) .', 'Measurements of intracellular pyruvate indicated that all of the evolved isolates had significantly less intracellular pyruvate than their common ancestor under glucose-limited conditions , although no significant differences in pyruvate concentration could be detected among the evolved strains ( Table S3 ) .', 'The primary route for oxidation of pyruvate during aerobic growth is pyruvate dehydrogenase ( PDH ) , a three-enzyme complex that catalyzes the conversion of pyruvate into acetyl-CoA and contributes to the redox burden by transferring electrons to NAD+ ( Fig . 4 and 5 ) .', 'Protein profiling of CV103 suggested the presence of a mutation in one of the three PDH enzymes , lpd , severe enough to eliminate the corresponding spot on a 2D gel [51] .', 'Whole genome sequencing confirmed a missense mutation in the CV103 lpd gene that results in an amino acid substitution ( F76L ) in the N-terminal portion of the translated protein ( Table S2 ) .', 'Because this substitution occurs in the FAD binding domain , it likely affects electron transfer from the reduced co-factor FADH2 to NAD+ .', 'PDH specific activity in CV103 was previously shown to be 2–3 fold lower than that in the other strains , indicating that this mutation does indeed have a negative effect [51] .', 'Loss of lpd activity can lead to phenotypic changes consistent with many of the unique characteristics of CV103 .', 'In a screen for E . coli knockouts with extended lifespans , lpd null mutants were identified that had extended survival compared to wild-type E . coli K-12 MG1655 [91] .', 'This enhanced survival was accompanied by reduced growth rate , reduced stationary phase cell density , reduced oxygen consumption , reduced respiration and increased accumulation of extracellular acetate , many of which phenotypes are exhibited by CV103 ( Table 1 , [3] ) .', 'Given that CV103 rapidly consumes glucose while excreting acetate , it seems unusual that a mutation affecting the conversion of pyruvate into acetyl-CoA would be retained in this strain .', 'An lpd knockout can still grow on glucose and produce acetate , but it does so more slowly by using an alternate route to acetate , pyruvate oxidase ( PoxB ) [92] .', 'In an effort to determine whether CV103 might be using an alternate enzymatic pathway to convert pyruvate into acetate , we compared gene expression and sequence data for the three alternative pyruvate oxidation pathways: pyruvate oxidase ( POX ) , pyruvate:flavodoxin oxidoreductase ( PFOR ) and pyruvate formate-lyase ( PFL ) ( Fig . 5 ) .', 'The POX pathway directly converts pyruvate into acetate without concomitant ATP generation ( Fig . 5 ) .', 'Nevertheless , this pathway is active during growth on glucose and can substitute for PDH if highly expressed [93] .', 'In our system , expression of the gene for POX ( poxB ) is downregulated in all four evolved isolates relative to their common ancestor .', 'This is not unexpected , as other glycolytic genes are downregulated .', 'However , CV103 has a nonsense mutation at amino acid 14 of poxB ( E14* ) which , given its position at the extreme N-terminus , is likely to completely inactivate the protein .', 'While the ancestral supE44 amber suppressor might allow limited poxB translation to occur , it is unlikely that the POX pathway produces an appreciable amount of acetate in CV103 .', 'A second gene involved in acetate excretion , ydbK is expressed under both anaerobic and aerobic ( albeit at very low levels ) conditions ( Fig . 5 ) , [94] , [95] .', 'ydbK encodes PFOR , which catalyzes the conversion of pyruvate into acetyl-CoA with concomitant reduction of flavodoxin or ferredoxin [96] .', 'Interestingly , both of these reduced molecules can be used to re-activate oxygen sensitive PFL ( see below ) [94] .', 'CV103 carries a missense mutation of unknown effect ( R539L ) in ydbK; however , because this ORF was not represented on our expression array , its transcription level is unknown .', 'Finally , acetyl Co-A can also be produced by the cleavage of pyruvate by pyruvate formate-lyase ( PFL ) .', 'PFL activity is primarily associated with the pflB gene but is also encoded by tdcE , ybiW and pflD [97]–[99] .', 'PFL is oxygen sensitive and thus the primary route to acetyl-CoA under anaerobic conditions , but it is transcribed , active and useful during microaerobiosis [100]–[103] .', 'Moreover , production of PFL requires a smaller anabolic investment than production of PDH , and may thus be preferred under conditions of nutrient stress [104] .', 'Functional PFL requires both transcriptional ( regulated by FNR and ArcA/B ) and post-translational activation by the activating enzyme PflA or the alternate activator YfiD [103] , [105] .', 'Consistent with the downregulation of many glycolytic genes , transcript levels of both the primary PFL ( pflB ) and its activator ( pflA ) are also downregulated across all of the evolved strains relative to the ancestor JA122 .', 'The gene for pflB is unchanged , and pflA has a silent substitution in CV103 ( L205L ) .', 'By contrast , transcript levels of the three alternate PFLs ( tdcE , ybiW , pflD ) and their alternate activator yfiD are not altered in any of the evolved strains relative to JA122 , though CV103 has SNPs in the genes that encode two of the alternate PFLs: YbiW ( A469S ) and PflD ( G513G ) ( Fig . 5 ) .', 'Perturbations in pyruvate transformation are known to impact a key state variable in central metabolism: cellular redox balance .', 'NADH accumulates in cells with high glycolytic flux because it cannot be re-oxidized as fast as it is generated .', 'This increased redox ratio ( NADH/NAD+ ) creates a cellular response reminiscent of anaerobiosis , stimulating the cell to direct pyruvate toward overflow metabolites and leading to repression of TCA cycle genes such as isocitrate dehydrogenase ( icd ) and citrate synthase ( gltA ) [106] .', 'Given the relatively rapid uptake of glucose and higher rate of acetate production by CV103 , stress in the form of high redox ratio may have influenced the evolution of this strain .', 'NAD+ regeneration typically occurs downstream of pyruvate either via the TCA cycle or fermentation , but could also occur via the conversion of DHAP into glycerol ( see section on glycerol metabolism below ) .', '( Fig . 6 )', 'The TCA cycle consists of eight steps beginning with the conversion of acetyl CoA to citrate and ending with the conversion of malate to oxaloacetate .', 'Expression profiling of each evolved isolate showed that relative to their common ancestor , levels of transcripts for proteins involved in three of these steps ( α-ketoglutarate dehydrogenase , succinate dehydrogenase and fumarase ) were upregulated across all evolved strains .', 'At two other steps , aconitase ( acnB ) and isocitrate dehydrogenase ( icdA ) , transcript levels were elevated in three of four strains , but reduced in CV103 .', 'Whole-genome sequencing uncovered intergenic SNPs in CV103 that could impact flux at the icd branch point connecting the TCA cycle with the glyoxylate bypass .', 'In the ancestral strain , JA122 , the negative regulator of the glyoxylate bypass , iclR , has a promoter mutation that likely affects iclR negative autoregulation , leading to higher expression and concomitant repression of the glyoxylate shunt genes aceA , aceB and aceK .', 'AceK negatively impacts flux through isocitrate dehydrogenase by phosphorylation , diverting carbon through isocitrate lyase ( aceA ) and malate synthase ( aceB ) ( Fig . 6; [107] ) .', 'Thus , in the ancestor increased IclR can reasonably be expected both to decrease transcription of glyoxylate bypass enzymes and to prevent inactivation of Icd by AceK .', 'While CV101 and CV116 exhibit increased relative expression of icdA and acnB , CV103 shows decreased expression of both , perhaps owing to a C→A mutation that lies between t
['Introduction', 'Results and Discussion', 'Materials and Methods']
['A major goal of genetics is to define the relationship between phenotype and genotype , while a major goal of ecology is to identify the rules that govern community assembly .', 'Achieving these goals by analyzing natural systems can be difficult , as selective pressures create dynamic fitness landscapes that vary in both space and time .', 'Laboratory experimental evolution offers the benefit of controlling variables that shape fitness landscapes , helping to achieve both goals .', 'We previously showed that a clonal population of E . coli experimentally evolved under continuous glucose limitation gives rise to a genetically diverse community consisting of one clone , CV103 , that best scavenges but incompletely utilizes the limiting resource , and others , CV101 and CV116 , that consume its overflow metabolites .', 'Because this community can be disassembled and reassembled , and involves cooperative interactions that are stable over time , its genetic diversity is sustained by clonal reinforcement rather than by clonal interference .', "To understand the genetic factors that produce this outcome , and to illuminate the community's underlying physiology , we sequenced the genomes of ancestral and evolved clones .", 'We identified ancestral mutations in intermediary metabolism that may have predisposed the evolution of metabolic interdependence .', 'Phylogenetic reconstruction indicates that the lineages that gave rise to this community diverged early , as CV103 shares only one Single Nucleotide Polymorphism with the other evolved clones .', "Underlying CV103's phenotype we identified a set of mutations that likely enhance glucose scavenging and maintain redox balance , but may do so at the expense of carbon excreted in overflow metabolites .", 'Because these overflow metabolites serve as growth substrates that are differentially accessible to the other community members , and because the scavenging lineage shares only one SNP with these other clones , we conclude that this lineage likely served as an “engine” generating diversity by creating new metabolic niches , but not the occupants themselves .']
["The variability of natural systems makes it difficult to deduce how organisms' genotypes manifest as phenotypes , and how communities of interacting organisms arise .", 'Using laboratory experimental evolution we can control this variation .', 'We previously showed that a population of E . coli that originated from a single clone and was cultured in the presence of a single limiting resource , evolves into a stable , three-membered community , wherein one clone excretes metabolites that the others utilize as carbon sources .', "To discern the genetic factors at work in producing this outcome and to illuminate the community's physiology , we sequenced the genomes of the ancestral and evolved clones .", 'We identified in the ancestor mutations that may have predisposed evolution of cross-feeding .', 'We found that the lineages which gave rise to the community diverged early on , and that the numerically dominant lineage that best scavenges limiting glucose does so as a result of adaptive mutations that enhance glucose uptake but favor fermentative over respiratory pathways , resulting in overflow metabolites .', 'Because this clone produces secondary resources that sustain other community members , and because it shares with them only one mutation , we conclude that it is an “engine” generating diversity by creating new niches , but not the occupants themselves .']
['genome evolution', 'population genetics', 'microbiology', 'escherichia coli', 'mutation', 'coevolution', 'evolutionary adaptation', 'bacterial pathogens', 'genetic polymorphism', 'medical microbiology', 'microbial pathogens', 'evolutionary genetics', 'microbial ecology', 'ecology', 'natural selection', 'evolutionary processes', 'genetics', 'biology and life sciences', 'genomics', 'evolutionary biology', 'computational biology']