Spontaneously Arising mutL Mutators in Evolving Escherichia coli Populations Are the Result of Changes in Repeat Length

Over the course of thousands of generations of growth in a glucose-limitedenvironment, 3 of 12 experimental populations of Escherichiacoli spontaneously and independently evolved greatly increasedmutation rates. In two of the populations, the mutations responsiblefor this increased mutation rate lie in the same region of themismatch repair gene mutL. In this region, a 6-bp repeat ispresent in three copies in the gene of the wild-type ancestorof the experimental populations but is present in four copiesin one of the experimental populations and two copies in theother. These in-frame mutations either add or delete the aminoacid sequence LA in the MutL protein. We determined that thereplacement of the wild-type sequence with either of these mutationswas sufficient to increase the mutation rate of the wild-typestrain to a level comparable to that of the mutator strains.Complementation of strains bearing the mutator mutations withwild-type copies of either mutL or the mismatch repair geneuvrD rescued the wild-type mutation rate. The position of themutator mutations—in the region of MutL known as the ATPlid—suggests a possible deficiency in MutL's ATPase activityas the cause of the mutator phenotype. The similarity of thetwo mutator mutations (despite the independent evolutionaryhistories of the populations that gave rise to them) leads toa discussion of the potential adaptive role of DNA repeats.

INTRODUCTION

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Introduction

Mutation is a process under the control of complex systems ofcellular machinery. The components of these systems are encodedby genes, and mutations in these genes can thus alter the typeor rate of future mutations. In particular, mutators are mutationsthat impair the fidelity of DNA replication or the efficacyof DNA repair, resulting in an increase in the mutation rate.In an evolving population, mutators arise through spontaneousmutation and potentially present a target for selection foradjustments of the mutation rate of the population (reviewedin reference 48). While long-term selection is generally predictedto favor decreased mutation rates and thus select against thefixation of mutator mutations (17, 18, 20, 21, 28), there issome theoretical evidence that frequent bouts of strong selection,such as those experienced by pathogenic microbes, might resultin long-term selection for increased mutation rates in asexualpopulations (16, 21, 25, 56). Over shorter time scales, mutatoralleles can become transiently common, and can even rise tofixation, in asexual populations by hitchhiking with one ormore linked beneficial mutations (36, 45, 49, 52).

Experimental evolution studies with microbial populations presentparticularly good opportunities to observe the change in frequencyof mutator alleles (6, 7, 12, 32, 35, 36, 45, 49). For example,work in a variety of experimental systems has established thatwhen an increase in the frequency of mutators does occur itis exclusively as a result of hitchhiking with linked beneficialmutations and not as a result of direct selection on the mutatorallele itself (7, 32, 36, 45). A particularly well-studied systememploys 12 replicate populations of Escherichia coli which haveevolved in a constant environment for over 20,000 generations(22, 24). Three of the populations independently fixed mutatorsin the first 10,000 generations of evolution and retained thesehigh mutation rates for thousands of subsequent generations(45, 49). There is no evidence that any long-term fitness advantageaccrued to any of the mutator strains after the mutator hadbeen fixed (8, 9). Since it seems that the mutator phenotypeprovides no advantage in terms of an increased rate of adaptationsubsequent to its fixation and since a high mutation rate iscertainly expected to carry a potent fitness penalty in theform of an increased supply of deleterious mutations, the followingquestion is raised: why does the mutator phenotype persist forthousands of generations in these populations in the face ofits likely selective cost?

A possible solution to the problem of the persistence of mutatorsin these populations is that the mutator phenotype might notbe easily revertible; for example, the original mutator phenotypemight have resulted from the deletion of genes coding for anintegral part of the DNA repair system. Initial attempts todetermine the precise mutations responsible for the mutatorphenotype in these populations (49) implicated the methyl-directedmismatch repair (MMR) system, the major component of postreplicationDNA error checking and repair in E. coli (19). Of the threemutator populations, the responsible mutation in one (designatedthe Ara⁺3 mutator population) was determined to consist of theinsertion of a single base pair in the MMR gene mutS (45); sucha mutation is, in principle, readily revertible. However, theexact nature of the mutator mutations from the other two populations(Ara^-2 and Ara^-4) has to this point remained unconfirmed.

Here we report the surprising finding that despite the completelyindependent evolutionary histories of all of the experimentalpopulations, the mutator mutations from populations Ara^-2 andAra^-4 are very similar: each is the result of an alterationin the copy number of the same 6-bp repeat in the MMR gene mutL.Previous work (45) had demonstrated the presence of these repeatalterations in the mutator populations but did not address thepossibility that they were responsible for the mutator phenotype.In this work, we show experimentally that these sequence alterationsare sufficient to produce a greatly increased mutation rate.In addition to casting serious doubt on the hypothesis thatthese mutators have managed to persist simply by virtue of beingnonrevertible, these results are intriguing for several otherreasons. First, these particular mutations are unusual in thatthey are rescued by complementation with wild-type copies ofeither mutL or uvrD, another component of the MMR system. Second,the repeat within which both of the mutations occur lies ina region of mutL that forms the lid of the ATP-binding pocketof the MutL protein. The ATP lid has been pointed out as possiblyplaying an important role in determining the function of membersof the GHKL superfamily of proteins to which MutL belongs (10).To our knowledge, no other similar mutations which alter thelength of the ATP lid (and thus, presumably, its conformation)have been described for mutL or any of its homologs. Finally,the fact that both mutations are independent alterations ofthe same repeat draws attention to the possible role of shortrepeats as mutational hotspots in loci involved in DNA replicationand repair (44).

MATERIALS AND METHODS

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Materials and Methods

Plasmids and strains. The plasmids and strains used in the study are listed in Table1.

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TABLE 1. Plasmids and strains used in this study

Experimental system. The experimental system used in the study has been describedpreviously (reviewed in reference 22). Briefly, an Ara^- cloneof E. coli strain B (REL606) was used to found 12 replicatepopulations: Ara^-1 through Ara^-6 (founded from REL606) and Ara⁺1through Ara⁺6 (founded from REL607, a spontaneous Ara⁺ revertantof REL606). These populations were propagated independentlyby daily 100-fold dilutions in 10 ml of minimal glucose medium,a transfer schedule which resulted in approximately seven generationsof growth per day. The experiment is still continuing and thestrains have experienced over 20,000 generations of growth inthese conditions. Each population was regularly sampled andfrozen for archival purposes, first at 100-generation intervals(for the first 2,000 generations) and then every 500 generationsthereafter. Mutation rates at several loci were measured inclones taken from each population at 10,000 generations; theclones from populations Ara⁺3, Ara^-2, and Ara^-4 were found tobe mutators (49). The rates of mutation of these mutators toseveral different phenotypes were tested; the magnitude of themutator effect was found to be consistent at approximately 100-foldhigher than that of the wild-type strain (49). All tests ofmutation rate described in the present paper were performedusing selection with the antibiotic nalidixic acid.

Spot plating. Since the magnitude of the mutator effect was so large, qualitativedifferences in mutation rate could be easily determined usinga spot plate assay, as described previously (45). Four replicatecultures of each strain were grown overnight in minimal medium;the next day, 100 µl of each culture was spotted ontoLuria-Bertani (LB) agar supplemented with 20 µg of nalidixicacid/ml. Expected distributions of the number of resistant coloniesfor wild-type and mutator strains were determined via computersimulation on the basis of an algorithm describing the distributionof mutants in a growing culture (31). Results from a particularassay were compared to the results of the simulation for classificationof the experimental strains as mutator or wild type. In allcases described here, the results of a spot-plating assay wereonly considered definitive when they could classify the testedstrain with more than 99.9% certainty.

Fluctuation tests. For more exact measurements of mutation rate, fluctuation testswere performed. The fluctuation test is a classical technique(30) which employs one of a variety of methods to transforman observed distribution of mutant counts into a precise estimateof the mutation rate. In the tests performed here, a numberof replicate liquid cultures for each strain were grown to maximumdensity in an appropriate volume of minimal medium. The entireculture was then plated onto LB agar supplemented with 20 µgof nalidixic acid/ml, and the number of resistant mutant coloniesvisible after 48 h was recorded for each plate. This distributionof mutant counts was analyzed with a computer program developedby one of the authors which implements a maximum-likelihoodestimation technique (51). Statistical-confidence limits forestimates produced with this technique were determined usinga maximum-likelihood estimation theory, which agrees closelywith the method suggested by Stewart (51).

Plasmid construction. Complete sequences of the mutL gene were obtained via PCR asdescribed previously (45). The total size of the PCR productwas 2.35 kb, which included the 1.85-kb mutL sequence as wellas the surrounding noncoding sequence. Amplifications from PS17,the Ara^-2 10,000-generation clone, and PS19, the Ara^-4 10,000-generationclone, were cloned into pGem T-Easy vector (Promega, Madison,Wis.) and transformed into chemically competent JM109 cells.Plasmid DNA was isolated using QIAprep Miniprep kits (Qiagen,Valencia, Calif.), and the NotI fragment containing the PCRproduct was isolated via gel extraction. This fragment was ligated(using T4 DNA ligase [Promega]) into the NotI sites of plasmidpKO3 (29), a suicide vector designed for allele replacementvia homologous recombination. The pKO3-mutL ligation constructwas transformed via electroporation into XL-1 Blue cells andthen isolated via a Miniprep procedure. Intact plasmid was transformedinto chemically competent REL606.

Gene replacement. Selection for gene replacement was as described previously (29);integration of the plasmid was selected for by growth on LBagar supplemented with 20 µg of chloramphenicol/ml at43°C, and excision and curing of the plasmid were selectedfor by growth on LB agar supplemented with 5% (wt/vol) sucrose.The nature of this procedure is such that a substantial proportionof the clones obtained at the final step retain their originalgenotype rather than inserting the target sequence, so all clonesmust be screened for the proper genotype. A total of 20 clones(10 with each plasmid) that had been put through this procedurewere tested by spot plating. Sequencing of strains displayingan increased mutation rate to test for proper integration wasperformed at the Nucleic Acid/Protein Core Research Facilityat the Children's Hospital of Pennsylvania.

Complementation testing. Strains constructed as described above were transformed withplasmids carrying fully functional, wild-type copies of uvrD(pGT26) (53) and mutL (pGW1842) (40). Techniques for measurementof mutation rates were unchanged except that media were supplementedwith 100 µg of ampicillin/ml and growth times were extendedto compensate for the cost to the strains of carrying the plasmids.

RESULTS

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Results

Sequence of the mutL mutations. The 6-bp repeat CTGGCG, beginning at position 213 of the E.coli B mutL gene, is present in three copies in REL606, theancestor of the experimental strains (Fig. 1). In previous work(45), alterations in the copy number of the repeat were foundin two experimental populations of E. coli which had evolveda mutator phenotype. In population Ara^-2, fixation of this mutatorphenotype took place approximately between generations 1500and 3500; an additional copy of the repeat was found in mutatorisolates from the Ara^-2 population both at the beginning ofthis time period and at the end. In population Ara^-4, whichfixed its mutator roughly between generations 6500 and 9000,the copy number of the repeat in mutator samples from both thebeginning and the end of this time period was reduced to two.

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FIG. 1. Alignment of a portion of the mutL sequence of the experimental system's wild-type ancestor, REL606, with the mutations found in the populations Ara^-2 and Ara^-4. Also shown is the amino acid sequence of the corresponding section of the MutL protein. The numbering given is for the REL606 nucleic acid sequence.

The region of mutL in which the CTGGCG repeat occurs codes forthe amino acid sequence LALALA in REL606, beginning at position68 of the MutL protein. As a result of the mutations describedabove, this sequence is lengthened to LALALALA in Ara^-2 andshortened to LALA in Ara^-4. This region of MutL comprises theC-terminal end of ${alpha}$ -helix B, according to the labeling schemeof Ban and Yang (3); other attempts at structural identificationinstead place at least part of this region in the disorderedloop connecting two ${alpha}$ -helices (for an example, see reference33). In either case, the repeat region lies at the N-terminalend of the relatively disordered structure that forms a lidover the ATP-binding pocket of MutL and that is thus referredto as the ATP lid (3).

For this work, sequencing of the entire mutL gene was carriedout for PS17 and PS19, the Ara^-2 and Ara^-4 isolates from 10,000generations, to determine whether any subsequent mutations inmutL might have accumulated. Only one additional coding differencefrom the REL606 ancestor was found to have arisen at 10,000generations: Ara^-4 substituted valine for alanine at amino acidposition 606 (608 in the wild-type protein) at the extreme C-terminalend of the MutL protein. No differences in DNA sequence betweenREL606 and either of the 10,000-generation isolates were observedin the region extending 200 bp upstream of the beginning ofmutL or 300 bp downstream of the end.

Confirmation of the mutator mutation. To analyze the precise effects of the mutL sequence alterationsin an isogenic background with a low, wild-type mutation rate,the putative mutator mutL sequences from PS17 and PS19 wereinserted into REL606. The technique used to introduce the mutatorsequences was developed on the basis of homologous recombinationbetween the target strain and a suicide vector bearing the sequenceto be inserted (29); this ensures that no unwanted flankingmaterial is introduced along with the mutation of interest,as occurs with transduction-based methods. To increase the successrate of the replacement technique as well as eliminate the remotepossibility of introducing a nonhomologous sequence into mutL,the fragments which were inserted into the suicide vector includedlong stretches of a homologous sequence extending beyond theboundaries of the mutL gene both up- and downstream of the targetmutations.

Spot plating (see Materials and Methods) was used to verifythat the mutL sequence had been replaced successfully; severalof the clones were classified as mutators. Sequencing of thesemutator clones verified that the REL606 mutL repeat sequencehad been cleanly replaced by the appropriate mutation, verifyingthat the mutL repeat alterations were sufficient to producea mutator phenotype. As a control, a clone that had been classifiedas wild type by spot plating was sequenced and was confirmedto have retained the REL606 sequence. Since all successful clonesproduced by this technique are isogenic, further results describedbelow were all carried out using only two of the mutator clones,PS2348 (Ara^-2) and PS2350 (Ara^-4).

Measurement of mutation rates. After spot plating had been used to confirm the mutator statusof PS2348 and PS2350, measurements of their mutation rates wereobtained using fluctuation tests (see Materials and Methods).REL606 mutates from nalidixic acid sensitivity to resistanceat a rate of approximately 10^-10 per replication, while therates for the evolved Ara^-2 and Ara^-4 mutators are roughly 100times as high (49). Tests of the rate of mutation to nalidixicacid resistance performed with 10-fold replication yielded amutation rate of 8.51 x 10^-9 per replication for PS2348 and8.11 x 10^-9 per replication for PS2350 (Fig. 2), rates consistentwith a mutator phenotype. There is presently no reliable andwell-tested method for statistical comparison of fluctuationtest results, and it is therefore not possible to rigorouslytest these figures for similarity with those obtained for theoriginal Ara^-2 and Ara^-4 mutators. Instead, we note that 95%confidence intervals calculated as described above (see Materialsand Methods) for PS2348 and PS2350 comfortably overlap previouslyobtained estimates for PS17 and PS19 (49). A fuller treatmentof the relationship between the mutation rates measured hereand those measured previously is included in the Discussionsection.

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FIG. 2. Rate of mutation to nalidixic acid resistance determined by fluctuation test. The mutation rate is shown on a log scale. Strains PS2348 and PS2350 exhibited mutation rates consistent (as defined in Results) with those measured previously for PS17 and PS19, the strains from which the mutL mutations were obtained. The four strains derived from PS2348 and PS2350 and complemented with uvrD⁺ (PS2353 and PS2355) or mutL⁺ (PS2358 and PS2360) all showed drastically reduced mutation rates. Error bars represent 95% confidence intervals calculated as described in Materials and Methods. The dashed line represents the upper 95% confidence interval calculated previously for REL606, the wild-type ancestor of PS2348 and PS2350 (49); this line was used as the cutoff to determine whether complementation had restored the wild-type mutation rate.

Complementation by mutL⁺ and uvrD⁺. Previous work (49) has shown that the high mutation rate ofPS17 and PS19 is partially or fully restored to the wild-typelevel by transformation with a high-copy plasmid expressinga wild-type copy of either mutL or uvrD but not any other memberof the MMR system (including mutS and mutH) or other well-knownmutator genes (including mutT, dnaE, and dnaQ). Phenotypic assaysand sequence analysis have provided no evidence for any deficiencyin uvrD itself in these strains (45). To determine whether uvrD'sability to rescue the wild-type phenotype is in fact linkeddirectly to the mutL sequence alterations, we transformed PS2348and PS2350 with plasmids bearing mutL and uvrD. Fluctuationtests for nalidixic acid resistance were performed on theseplasmid-bearing strains with eight replicates per strain; theresults are shown in Fig. 2.

For all four plasmid-bearing strains, a large and consistentdecrease in mutation rate was observed relative to those ofPS2348 and PS2350. In every case, the mutation rate estimateof the complemented strain fell below the upper 95% confidenceinterval of REL606; this is the standard that was used previously,in the absence of robust statistical procedures, to indicaterescue of the wild-type rate by complementation (49). Thus,it is clear that the mutL mutations described above are sufficientto create a mutator phenotype which can be rescued by eithermutL⁺ or uvrD⁺.

DISCUSSION

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Discussion

In an experimental system consisting of 12 replicate populationsof E. coli evolving independently in a constant environment,3 of the populations spontaneously fixed mutators in the first10,000 generations of evolution (49). The mutation responsiblefor the mutator phenotype in one of the populations, Ara⁺3,has been determined to be an insertion in the MMR gene mutS(45). In this work, we show experimentally that the mutatormutations in the other two populations, Ara^-2 and Ara^-4, arealterations in copy number of the same 6-bp repeat in the MMRgene mutL; in Ara^-2, an additional copy is added, while in Ara^-4,a copy of the repeat is deleted. The introduction of eitherof these mutations into a wild-type strain is sufficient toincrease the mutation rate to a level comparable to that ofthe mutators. The wild-type, low mutation rate can be restoredin strains carrying these mutator mutations by complementationwith wild-type copies of either mutL or uvrD.

Origin versus persistence in evolving populations. Experimental studies have shown that when mutators fix in evolvingasexual populations, they do so not as the result of directselection on the mutator phenotype but instead only by hitchhikingwith linked beneficial mutations (36, 45). By virtue of theirincreased mutation rates, mutators are more likely than wild-typeindividuals to acquire these beneficial mutations; thus, theystand an increased chance of hitchhiking to high frequency.This relationship is illustrated by experimental manipulationswhich boost the proportion of mutators in bacterial populations;a mutator subpopulation only a fraction of the size of the wild-typesubpopulation increases in frequency at the expense of the wildtype (7, 12, 57). The importance of hitchhiking to the evolutionof mutation rates is also borne out by theory, which indicatesthat indirect selection can be a powerful force for the fixationof mutator alleles in asexual populations exposed to strongselection (39, 52, 54).

While this combined body of experimental and theoretical workhas made it clear that mutators of large effect can be fixedin rapidly evolving asexual populations, such short-term selectionis likely to increase the mutation rate to a level higher thanthe optimum for long-term evolution (reviewed in reference 48).The increased load of deleterious mutations that a high mutationrate brings with it represents a potent selective disadvantage,one which should in the long run outweigh the advantage thatcomes from an increased supply of beneficial mutations. Thisdisparity becomes especially pronounced in populations in whichthe initial selective pressure that resulted in fixation ofthe mutator alleles in the first place is relaxed and the rateor magnitude of beneficial mutation decreases; when this happens,the mutator allele retains its full cost while losing some ofits advantage.

It is thus somewhat difficult to explain why the mutators describedhere continued to persist for thousands of generations aftertheir initial fixation and out to at least 20,000 generationsfrom the start of the experiment (A. C. Shaver and P. D. Sniegowski,unpublished data). In the experimental populations used in thisstudy, the bulk of the increase in fitness occurred within thefirst 3,000 generations; subsequently, adaptation seems to haveslowed almost to a halt as, presumably, those beneficial mutationsmost likely to arise or those of largest fitness effect hadalready fixed and only very unlikely or ones of very minor effectwere left (24). If wild-type revertants could arise on the mutatorbackground in these highly evolved populations, they would nolonger suffer the increased deleterious load of the mutatorsand would therefore be predicted eventually to increase in frequency.The data presented here and in previous work (45) are importantbecause they show that the simple answer—that the natureof the mutator mutations is such that they simply cannot revert—canbe ruled out for all three populations. Thus, some other explanationmust be found for the persistence of these mutators.

There are three possible reasons why wild-type revertants havefailed to increase in frequency in these populations. First,the rate of adaptation in the experimental populations, evenafter thousands of generations of evolution, may remain highenough for the mutator phenotype to retain its advantage overthe wild type. Second, the magnitude of selection in favor ofwild-type revertants on the basis of their decreased geneticloads may be too small to have an effect over the time scalesurveyed. Finally, it is possible that further mutations mighthave accumulated in the mutator populations which make it impossiblefor wild-type revertants to arise, although this possibilityis not supported by data from the 10,000-generation isolates(49).

Of possible relevance to the question of the persistence ofmutators in these experimental populations is the observationthat while the mutation rates observed for the two mutator strainsconstructed on the ancestral background were broadly consistentwith previously reported measurements (49) for 10,000-generationmutator isolates from the same populations, the mutation ratesof the constructed strains do seem to have been lower than thoseof the 10,000-generation strains. As mentioned in Results, theabsence of reliable procedures for statistical hypothesis testingwith mutation rate estimates makes it impossible at this timeto rule out experimental error as an explanation for the apparentdecrease. However, the possible further evolution of mutationrates in the time between the first appearance of the mutatorsand 10,000 generations might also explain the difference. Thesearch for such long-term but relatively slight changes in mutationrates subsequent to mutator fixation is the subject of furtherwork currently in progress.

Relevance to structural studies of MutL. The protein MutL is a member of the GHKL superfamily of ATPasesand kinases; this grouping is defined by its members' uniqueATP-binding pocket, which is covered in the ATP-bound conformationby a loop structure known as the ATP lid (10). The structuresand positions of the ATP lid seem to differ greatly betweenGHKL members, and there is ample evidence that the lid playsa potentially important role in determining the activity ofthe ATP-binding site (2, 5, 10, 33).

Mutations have previously been located in or near the ATP lidregion of MutL or its homologs (1, 37, 41-43, 46, 47). In almostevery case, these are missense mutations rather than insertionsor deletions; an exception is a 1-codon deletion in human MLH1(41). To our knowledge, no other mutations have been describedwhich alter the length and therefore presumably the conformationof the ATP lid.

Complementation by uvrD. MutL's crucial role in DNA mismatch repair is to mediate betweenthe detection of a base pair mismatch by MutS and the nicking,unwinding, excision, and recopying of the mismatched regionby other repair proteins. In E. coli, MutL has been shown tointeract physically with MutS (13); the endonuclease MutH (3,15); and DNA helicase II, the gene product of uvrD (14, 58);MutL also binds DNA, particularly single-stranded DNA (2, 4).Most of these interactions are dependent on MutL's ATPase activity,which has been shown to be specifically required for mismatchrepair both in E. coli (50) and in Saccharomyces cerevisiae(55). MutL's position at the center of a network of interactingrepair proteins makes it conceivable that the effect of specificmutL mutations might be rescued by complementation with otherMMR genes. In fact, some mutL mutators in E. coli seem to bepartially or fully complemented by mutS⁺ or mutH⁺ (1). However,we are aware of no previously reported mutL mutator which isrescued by complementation with uvrD⁺ (for an example, see reference38).

The mutators described in this work showed an equivalent decreasein mutation rate when complemented with either mutL⁺ or uvrD⁺.Direct interaction with DNA helicase II, the uvrD gene product,occurs in the C-terminal region of MutL (14), while the mutationsdescribed here were located at the extreme N terminus of theMutL protein, indicating that impairment of ATPase functionwas more likely to be the cause of the mutator phenotype thanspecific deficiencies in MutL-DNA helicase II interaction. SinceATPase function is important for many of MutL's protein-proteininteractions, it is striking that only uvrD was able to rescuethe mutator phenotype.

Repeats and local hypermutability. As described above, the general cost of an increased mutationrate stems from the greater load of deleterious mutations sufferedby a mutator. However, by increasing the mutability of onlycertain regions of DNA where the potential for beneficial mutationis high, some of the cost of a high mutation rate can be avoidedwhile the possible payoff is maximized. Such local hypermutabilityis displayed by contingency loci, specific areas in the genomesof pathogenic microorganisms which are involved in the interactionwith the host's immune system and are thus exposed to constantlyshifting selection (11, 34). A high rate of mutation is consistentlydesirable at these loci, since it assists the pathogen populationin outpacing the immune response of the host and so mechanismswhich elevate the mutation rate in such regions while preservinga low mutation rate in the rest of the genome are selected for.These mechanisms often involve DNA repeats, since repeats increasemutability both as a result of slipped-strand mispairing (26,27) and by presenting a target for homologous recombination.

Although it is certainly true that DNA repeats can be targetsof selection in specific instances, such as the contingencyloci described above, it is less clear whether they play a roleof more general adaptive significance. It has been suggested(44) that short repeats in genes involved in DNA repair or stressresponse are the targets of selection to increase variabilityin times of stress via slipped-strand mispairing. The mutationsdescribed here lend support to the more limited hypothesis thatalterations in such short repeats make up a disproportionatelylarge segment of the potential pool of mutators which can hitchhiketo high frequency. While a sample of three populations is certainlynot large enough to permit broad conclusions, the fact thattwo of the mutator mutations arose independently from the same18-bp region is unlikely to be a coincidence. However, it isnot necessary to conclude from these results that the repeatedsequence in this region was targeted by selection to increasethe region's mutability.

Any potential selection that would act to increase the mutabilityof the mutL locus would have no immediate effect but to increasethe chance of creating a mutL mutator, which in turn would haveno positive selective effect but to increase the rate of beneficialmutations at other loci. This "third-order" selection is, byits very nature, extremely weak—much weaker, for example,than the direct selective advantage provided by the pathogeniccontingency loci described above. An analysis of the repeatregion of mutL in a variety of bacterial species (Fig. 3) showsthat there is little evidence for the selective maintenanceof the repetitive nature of this region. Even in other strainsof E. coli and in very closely related taxa the copy numberof the repeat is decreased, and in more distantly related bacterialspecies any trace of the repeated sequence disappears completely.

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FIG. 3. Alignment of the mutL repeat sequence in the experimental E. coli B strain (REL606) with homologous sequences from mutL genes of other bacterial species. Positions at which the sequence of another species or strain is identical to that of E. coli B are indicated by periods. Only E. coli O157:H7 shares complete sequence identity with E. coli B within this stretch. E. coli K-12, the two Salmonella species, and Shigella flexneri all contain only two complete repeats. The sequences of more distantly related bacterial species, including many not shown here, were found to contain no trace of any repeated elements in this region. The E. coli B sequence obtained from REL606 and all other sequences shown here are available from GenBank. The numbering given is for the E. coli B nucleic acid sequence.

While a role for higher-order selection in the similarity ofthe mutations described in this study cannot be ruled out, analternative worthy of consideration is that the repeat in mutLis the product of genetic drift and its apparently increasedrate of mutation is simply an accidental result. The uncertaintyin this case is an example of the difficulty in studying complexphenomena such as the evolution of mutation rates, in whichthe causes of a selective event cannot always be inferred easilyfrom its consequences (48). Fortunately, the experimental systemdescribed here presents a unique opportunity for future researchto address the question of whether such higher-order selectioncan operate in evolving populations.

ACKNOWLEDGMENTS

This research was supported by a grant to P.D.S. from the NationalScience Foundation (DEB 9981518). A.C.S. was supported by apredoctoral fellowship from the Howard Hughes Medical Institute.

We thank R. E. Lenski for generous access to his strains, G.M. Church for providing us with the pKO3 plasmid, and two anonymousreviewers for helpful comments. Computer code written by P.J. Gerrish served as the basis for an early version of the fluctuationtest analysis program used in this work.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biology, University of Pennsylvania, 415 S. University Ave., Philadelphia, PA 19104. Phone: (215) 573-4085. Fax: (215) 898-8780. E-mail: paulsnie{at}sas.upenn.edu.

REFERENCES

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