origin of mutations under selection: the adaptive mutation

ANRV286-MI60-21 ARI 16 August 2006 15:39

Origin of Mutations UnderSelection: The AdaptiveMutation ControversyJohn R. Roth,1 Elisabeth Kugelberg,1Andrew B. Reams,1 Eric Kofoid,1and Dan I. Andersson2

1Microbiology Section, Division of Biological Sciences, University of California,Davis, California 95616; email: [email protected], [email protected],[email protected], [email protected] of Medical Biochemistry and Microbiology, Uppsala University,Uppsala SE-751 23, Sweden; email: [email protected]

Annu. Rev. Microbiol. 2006. 60:477–501

First published online as a Review inAdvance on June 8, 2006

The Annual Review of Microbiology is onlineat micro.annualreviews.org

This article’s doi:10.1146/annurev.micro.60.080805.142045

Copyright c© 2006 by Annual Reviews.All rights reserved

0066-4227/06/1013-0477$20.00

Key Wordsnatural selection, cancer, genetic adaptation, gene amplification,gene duplication, stationary-phase mutagenesis

AbstractGrowth under selection causes new genotypes to predominate ina population. It is difficult to determine whether selection stimu-lates formation of new mutations or merely allows faster growthof mutants that arise independent of selection. In the practice ofmicrobial genetics, selection is used to detect and enumerate pre-existing mutants; stringent conditions prevent growth of the parentand allow only the pre-existing mutants to grow. Used in this way,selection detects rare mutations that cause large, easily observablephenotypic changes. In natural populations, selection is imposed ongrowing cells and can detect the more common mutations that causesmall growth improvements. As slighly improved clones expand, theycan acquire additional mutational improvements. Selected sequen-tial clonal expansions have huge power to produce new genotypesand have been suggested to underlie tumor progression. We sug-gest that the adaptive mutation controversy has persisted becausethe distinction between these two uses of selection has not beenappreciated.

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ContentsPERSPECTIVES . . . . . . . . . . . . . . . . . . . 479GENERAL INTRODUCTION TO

A 150-YEAR-OLDCONTROVERSY . . . . . . . . . . . . . . . 479

THE CAIRNS SYSTEM REOPENSTHE DEBATE . . . . . . . . . . . . . . . . . . 479The Basic System . . . . . . . . . . . . . . . . 479Detailed Structure of the Strain . . . 480The Reversion Experiment . . . . . . . 481

MODELS THAT ASSUMESTRESS-INDUCEDMUTAGENESIS OFNONGROWING CELLS . . . . . . . 482Functional Direction of

Mutagenesis . . . . . . . . . . . . . . . . . . . 482Generally Hypermutable State . . . . 482Positional Direction

of Hypermutability . . . . . . . . . . . . 482THE AMPLIFICATION MODEL:

GROWTH AND SELECTIONREPLACE MUTAGENESIS. . . . . 483

CHOOSING A MODEL: THECASE FOR AMPLIFICATION . . 485

AREAS OF DISCUSSION . . . . . . . . . . 487Leakiness of the lac Mutation . . . . . 487Role of Recombination . . . . . . . . . . . 488The Question of Mixed Clones and

Proposal of Two Pathways ofAdaptive Mutation . . . . . . . . . . . . 488

NEW ISSUES IN ADAPTIVEMUTATION . . . . . . . . . . . . . . . . . . . . 488Origins of Duplications and

Process of Amplification . . . . . . . 488Role of RpoS and Ppk . . . . . . . . . . . . 489

OTHER SYSTEMS THAT SEEMTO SHOW STRESS-INDUCEDMUTAGENESIS . . . . . . . . . . . . . . . . 489Deletions that Remove a Mu

Prophage and Fuse the

Arabinose Promoter to a lacZGene . . . . . . . . . . . . . . . . . . . . . . . . . 489

Evolved β-Galactosidase . . . . . . . . . . 490Accumulation of Mutants

in Aging Colonies . . . . . . . . . . . . . 490Loss of Fitness in Stationary-Phase

Cultures . . . . . . . . . . . . . . . . . . . . . . 490Selection for Loss of Kanamycin

Resistance in Stationary-PhaseCells . . . . . . . . . . . . . . . . . . . . . . . . . . 491

Selection for Loss of RepressorFunction in NongrowingPopulations . . . . . . . . . . . . . . . . . . . 491

Reversion of Auxotrophs inMultiply Marked Strains ofBacteria and Yeast . . . . . . . . . . . . . 491

THEORETICAL COSTS ANDBENEFITS OF MUTATION . . . . 491What Selective Forces Dictate

Evolution of Mutation Rates inNatural Populations? . . . . . . . . . . 491

Evidence that Bacteria MinimizeMutation Rates . . . . . . . . . . . . . . . . 492

Mutators Are DemonstrablyAdvantageous in SomeSituations . . . . . . . . . . . . . . . . . . . . . 492

Mutagenesis Is Often Unnecessary 493Mutagenesis Is Often Futile . . . . . . . 493Temporary Increases in Mutation

Rates Are Still Costly . . . . . . . . . . 493Mutators Are Common Among

Natural Isolates . . . . . . . . . . . . . . . 493Mutators Sometimes Win in

Long-Term GrowthExperiments . . . . . . . . . . . . . . . . . . 494

Directing Mutagenesis . . . . . . . . . . . . 494BROADER IMPLICATIONS OF

ADAPTIVE MUTATION . . . . . . . 494THE LEGACY OF THE

CAIRNS SYSTEM. . . . . . . . . . . . . . . 495

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PERSPECTIVESHave mechanisms evolved that allow cells tomutagenize their own genomes in response toselective stress? This question has remainedunresolved since the time of Darwin, whosuggested that environment might stimulateincreases in variability (15). We know of nocompelling experimental evidence for stress-induced mutability in any system. Moreover,there are theoretical reasons to expect thattemporary increases in mutation rate are un-likely to be of long-term benefit, due to theircost in deleterious mutations. Nevertheless, insome systems, selection does increase the yieldof mutants. Under particular selective condi-tions, increases in general mutation rate canbe shown experimentally and theoretically tobe advantageous. We suggest that the appar-ent conflicts between these statements can beresolved without recourse to stress-inducedmutability.

The term adaptive mutation has been de-fined as the process by which stresses that arenot directly mutagenic activate mechanismsfor causing mutations, even in nongrowingcells (stress-induced or stationary-phase mu-tagenesis). This definition assumes a mecha-nism that we think is unlikely to exist. In or-der to consider all explanations of the relevantphenomena, adaptive mutation is defined hereas the process by which mutations arise underselective conditions, whether or not mutationrates increase or growth is required. The areadiscussed here has been reviewed previouslyfrom various points of view (26–28, 81, 82,84, 85).

GENERAL INTRODUCTION TOA 150-YEAR-OLDCONTROVERSYDarwin suggested that stress might gener-ate the variability upon which natural selec-tion operates (15). The classic experiments ofLuria & Delbruck (63) and Lederberg (59)demonstrated that some mutations arise with-out the influence of selective stress. However,

Deleteriousmutation: amutation thatdecreases organismfitness

Adaptive mutation:any process by whichfitter mutations ariseunder selectiveconditions; may ormay not requiremutagenesis orgrowth

the lethal selections they used could not havedetected mutations induced by selective con-ditions. Shapiro (88), Cairns et al. (13), andHall (40, 42) pointed out this deficiency anddescribed genetic systems in which selectiveconditions seemed to increase the mutationrate. The Cairns system has been analyzed inmost detail and remains controversial despitethis effort.

It is difficult to separate any possi-ble effect of selective conditions on muta-tion rate from its known effect on relativegrowth rates of parents and mutants. The ac-cepted method for measuring mutation rate(Luria-Delbruck fluctuation test) demandsthat the new mutants grow at the same rate asthe parent during the period of the test. To cir-cumvent this problem, the Cairns system (likeothers) places cells on solid medium and pre-vents growth by imposing a nonlethal stress;new mutants are scored as visible colonies. Ifthe frequency of new mutants increases in anongrowing parent population, then naturalselection is eliminated as a cause and one iscompelled to conclude that mutation rate hasincreased. If, however, even a subset of thepopulation grows, new opportunities for mu-tations are provided and pre-existing variantswith minimal growth ability can improve. Formany of the systems described here, mutantsappear in a population that is not obviouslygrowing, but in most cases, the evidence doesnot exclude growth of a subpopulation andstepwise improvement of a mutant during theselection period.

THE CAIRNS SYSTEM REOPENSTHE DEBATE

The Basic System

A system was developed by John Cairns to ad-dress the shortfalls of previous evidence forselection-independent mutability (13). In thenew system, as modified by Cairns & Foster(11), Lac− cells with a revertible lac muta-tion are plated on lactose medium and selec-tion is made for mutants that regain a Lac+

www.annualreviews.org • Mutation Under Selection 479

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108

109

1010

Days on lactose

Num

ber

of r

ever

tant

col

onie

s

Num

ber of parent cells

Revertant colonies (Lac+)

Parent cells

10 2 3 4 5 6

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80

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40

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0

Figure 1Diagram of a reversion experiment. While revertant accumulation isoften linear, many experiments show an upward inflection (dotted line forrevertants). Similarly, growth of the lawn frequently escapes inhibition(dotted line for parents), especially toward the end of the selection period.Whether or not this occurs, observed growth does not provide sufficientdivisions to explain the revertant number without some gimmick.

phenotype. The selective conditions are notlethal and are adjusted to just barely preventgrowth of the plated population. About 100Lac+ colonies accumulate above the platedlawn of 108 cells over 6 days. Thus the con-ditions appear to eliminate natural selection(growth) and reveal revertants, which accu-mulate at a nearly linear rate with time. Thatis, fully Lac+ revertants seem to arise in anongrowing parent population. This is dia-grammed in Figure 1.

During unrestricted growth, the originalmutant gives rise to fully Lac+ revertants at arate of less than 10−8/cell/division (80). Underselection, the apparently nongrowing popula-tion generates over 100 revertant colonies inthe course of 6 days incubation. If this wholetime period allowed the entire population todivide just once, a 100-fold increase in muta-tion rate (to 10−6) would be required to ex-plain the revertants.

Detailed Structure of the StrainThe lac mutation under selection is a +1frameshift in a chimeric gene made by fus-

Chromosome

Iq lacl - lacZ

dinB++1 frameshift

lacZ∆

finO-

F’128

Figure 2Genotype of the Cairns strain. The course of thereversion experiment depends on having lac on theF′ plasmid. The dinB gene is 16 kb from lac;associated mutagenesis, but not lac reversion,depends on the presence of dinB at this position(cis to lac).

ing the lacI and lacZ coding sequences (11)(Figure 2). The hybrid gene is expressed froma mutant lacI promoter (Iq) that is not sub-ject to regulation. The frameshift mutationis within the lacI portion of the gene andpresents a large target (about 100 bp) withinwhich any compensating (−1) frameshift mu-tation can allow expression of the distal lacZportion of the gene. This is a reversion tar-get 10- to 100-fold larger than expected fora frameshift mutation within a sequence thatencodes critical regions of a protein. The mu-tant lac allele is carried by a conjugative plas-mid (F128) in a strain with a deletion of thechromosomal lac region. The transfer func-tions (tra) of this plasmid are constitutivelyexpressed in Escherichia coli owing to a naturalfinO mutation in the plasmid.

The mutant lac allele is substantially leaky,producing 1% to 2% of the β-galactosidaselevel found in revertant strains (3). This resid-ual activity is sufficient to allow the parentstrain to form colonies on standard minimallactose medium without any genetic change.To prevent its growth during a reversion ex-periment, the tester is plated with a 10-foldexcess of scavenger cells having a lac deletionmutation. The scavengers consume any resid-ual nutrients contaminating the medium andany carbon sources that leak from the tester

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strain or revertant colonies (e.g., galactose, ac-etate, lactate, succinate). The scavenger actsas a competitor, poising the lac tester cells onthe brink of growth. The stasis of the testerpopulation does not reflect matched rates ofgrowth and death (11). Growth ability can beconferred on a cell either by increasing thenumber of mutant gene copies or by correct-ing the +1 frameshift mutation.

The Reversion ExperimentRevertant colonies accumulate over thecourse of 6 days after the testers (108)are plated with scavengers (109) on lac-tose medium (Figure 1). The first revertantcolonies to appear (before day 2) are assumedto reflect mutant cells that arose during pre-growth, and colonies that appear later areattributed to processes occurring under selec-tion on the plate. In some experiments, rever-tant accumulation is linear with time, but inmany other experiments there is an upwardinflection.

The Lac revertants appearing late (days5 and 6) are not slow growers, becausethey form colonies within 2 days in re-construction experiments. Furthermore, therate of accumulation of Lac+ revertants andthe final level achieved are not subject toLuria-Delbruck fluctuation when parallel in-dependent cultures are tested. The observed,essentially Poisson, distribution of revertantnumber (11) is expected if reversion occurs onthe selection plate. This distribution is alsoconsistent with revertants being initiated bypre-existing cells carrying an unstable lac du-plication (see below).

The lac sequence changes found in laterevertants tend to be −1 frameshift muta-tions in homopolymeric base runs (31, 83).In the absence of selection, this type occursbut other mutations are more common (e.g.,−4, +2, extended deletions or +1 frameshiftmutations outside base runs). This has beentaken as evidence that mutations arise un-der selection by a mechanism that is quali-tatively different from that operating during

SOS: a gene systeminduced by DNAdamage that includesenzymes involved inDNA repair

nonselective growth. The observed shift isconsistent with a fivefold increase in muta-tion rate if the added mutations are all −1frameshift mutations in base runs.

Early hints of the complexity of this ex-periment were the following prerequisites fora full yield of Lac+ revertants (additional re-quirements are discussed below).

! Residual function of the mutant lac al-lele (3).

! Recombination ability (RecA, RecBC)(11, 45).

! Location of the lac mutation on an F′

plasmid (35, 38, 39, 75). With lac in thechromosome, roughly 100-fold-fewerrevertants are seen (79, 92).

! Expression of conjugative transfer func-tions (tra). Revertant yield is reduced byrepression of the plasmid tra operon,which encodes functions needed forconjugative DNA transfer (35). Rever-sion is essentially blocked by elimina-tion of TraI (32, 74), a plasmid-encodedprotein that nicks DNA and initiatestransfer replication. Transfer per se doesnot seem to be essential to reversion(32).

! A functional dinB gene (encoding anSOS-induced error-prone DNA poly-merase) must be located close to the lacgene in the plasmid for a full mutantyield (66, 91).

An additional property of the system hasproved the most divisive. Revertants appear-ing under selection show an average 20-foldincrease in the probability of carrying un-selected associated mutations (79, 102) andthus have been mutagenized. This mutagene-sis is unevenly distributed in that 10% to 20%of Lac+ revertant cells show a 200-fold in-crease, while the majority (80% to 90%) arisewith no obvious mutagenesis (79). The starvednonrevertant lawn shows about a fourfold in-crease in associated mutations (8). All generalmutagenesis is eliminated by removal of thedinB gene from its position near lac on the F′

plasmid—at least a 50-fold reduction (92); re-vertant yield drops only 4-fold without dinB


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Stress-inducedmutagenesis: thehypothesis thatorganisms possessmechanisms toincrease theirgeneral mutationrate in response toexternal stress

Hypermutablestate (HMS): amodel whereby stressincreases the generalmutation rate

(66). Several models have been developed toexplain the above observations.

MODELS THAT ASSUMESTRESS-INDUCEDMUTAGENESIS OFNONGROWING CELLS

Functional Direction of Mutagenesis

This early model proposes that nongrowingcells make only mutations that improve theirfitness (11, 13, 29). Two mechanisms for im-plementing this (16, 95) were eliminated byexperiment (29).

! Essence: Cells sense the problem posedby selective conditions and preferen-tially alter sites in their own genomethat can rectify the difficulty.

! Main evidence: Under selection, thenumber of revertants (Lac+) increasesmore than the frequency of unselectedmutant types sought in the lawn ofstarved bacteria.

Generally Hypermutable StateA model proposed by Hall (41) suggests thatstress induces a general (undirected, genome-wide) hypermutable state (HMS) in a subset(0.1%) of the nongrowing starved population.Cells in the subpopulation ultimately die oflethal mutations unless they first acquire a lacreversion event, which relieves the stress andterminates the HMS. Mutagenesis appears di-rected to lac because only Lac+ revertants sur-vive hypermutagenesis. To explain 102 rever-tants arising from 105 cells, the model requiresa 105-fold increase in mutation rate for cellsin the HMS (86).

! Essence: Stress induces intensegenome-wide mutagenesis in a subpop-ulation (105 of 108 cells) and only lac+

revertants survive.! Main evidence: The model predicts that

Lac+ revertants should often carry asso-ciated unselected nonlethal mutations.The predicted mutations were found

(102) and were fivefold more frequentamong Lac+ revertants than in thestarved lawn or about 20-fold more fre-quent than in an unselected population(8, 79). The associated mutations arecaused by the SOS-induced error-proneDinB polymerase (66).

On the basis of these findings, the modelwas made more explicit and now proposesthat stress induces expression of the DinBpolymerase, which causes the genome-wideincrease in mutation rate and is responsiblefor the observed lac revertants. Mutagenesisis proposed to occur in nongrowing cells dur-ing unscheduled DNA replication initiatedby recombination events (33, 46). Mutationshave previously been shown to occur duringrecombination in yeast (77).

Positional Directionof HypermutabilityThis model is a hybrid of the previous twomodels (12, 27). It assumes that stress inducesDinB in nongrowing cells and mutagenizesany region in which recombinational repli-cation is occurring. Mutagenesis is directedto the F′ plasmid, whose conjugation transferfunctions (tra) produce DNA ends that stim-ulate intense recombination between plasmidsequences. Because only the plasmid is mu-tagenized, associated chromosomal mutationsare not expected. The observed lac revertantscan be explained by a 100-fold increase in mu-tation rate on the plasmid (rather than a global105-fold increase) because the whole popula-tion (108 cells) is affected rather than a sub-population (105). The genome-wide mutage-nesis seen in a minority of revertant clones(10%) is not considered central.

! Essence: Stress in nongrowing cells in-duces DinB, which preferentially muta-genizes the F′ plasmid. More broadly, allrecombination becomes mutagenic dur-ing stress, and the Cairns system pro-vides a sensitive test for this becauseits target lac region experiences intense

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recombination owing to its position onthe F′ plasmid.

! Main evidence: During starvation forlactose, a +1 frameshift mutation in tetAlocated on the F′ plasmid produces moreunselected revertants than does an iden-tical mutation located in the chromo-some (8, 25).

THE AMPLIFICATION MODEL:GROWTH AND SELECTIONREPLACE MUTAGENESISThis model assumes that mutations arise ina growing subpopulation of plated cells. Itproposes that rare cells with a lac duplicationare present in the plated population and ini-tiate slowly growing clones on the selectiveplate. This is possible because tester cells arepoised on the brink of growth (by scavengers)and extra lac copies upset this poise. Withinthese developing clones, selection favors com-mon cells with further increases in mutant laccopy number (higher amplification); these areformed by unequal recombination betweenrepeats (Figure 3).

Growth rate improves in cells of the colonythat gain more copies of the mutant lac al-lele. Ultimately a reversion event (frameshift)occurs in some lac copy; selection now holdsthe revertant allele and counterselects the re-maining mutant copies, which make the re-vertant allele subject to loss by segregation.Haploid lac+ cells appear and overgrow thecolony (Figure 4).

Reversion to Lac+ is made more likely be-cause more target lac alleles are added to eachdeveloping clone (more cells and more copiesper cell). This process requires no stress-induced mutagenesis and appears to directmutations to the precise base pairs that limitgrowth. This occurs because only the lac re-gion is selectively amplified and only the re-vertant allele is retained in the final product.

Development of a revertant clone is de-scribed in Figure 5. Note that the sequenceof events is basically a random walk withtwo boundary conditions or absorbing states.

Parent haploid

Duplication

Amplification

RecA

RecA RecA

RecA RecA

RecA?

Junction repeatRecombination event

Figure 3Recombination events involved in duplication and amplification.Duplications are formed by a rare exchange that can occur without RecA.Segregation and amplification events occur with much higher frequencyand are heavily RecA dependent.

Gene amplification:an increase in genecopy number,generally byaccretion of tandem,head-to-tail copies ofa genetic region

Whenever a duplication or amplification seg-regates to haploid, it is essentially removedfrom the walk. The process is initiated by alac duplication, and copy number increasesby further amplification. In the absence ofselection, the absorbing condition at the left(haploid lac−) limits the increase in copy num-ber. However, under selection, copy numberis driven higher when selection favors growthof cells with more mutant lac alleles. Once re-version occurs, all lac alleles (including lac+)are subject to loss by segregation. Instability oflac+ is minimized by removal of mutant copiesuntil the second boundary is crossed, leavingan irreversibly lac+ haploid cell.

In this process, selection detects commonsmall improvements in growth rate (due todosage increases) and divides the reversionprocess into many small steps, each of whichinitiates a clone in which the next event can oc-cur (Figure 6). The process of serial mutation


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++

++

108

106

n

n

Selection formore mutantalleles

Reversion to lac+

102

Improvinglactose use

Improvingstability of lac+

Selection forfewer mutantalleles

Figure 4Amplification model.Pre-existingduplications cellsgrow under selectionand growth isimproved by furtherlac amplification.Reversion requiresno mutagenesis butis made moreprobable by theincreased number oflac alleles (more cells,more copies per cell).

in nested clones is a powerful way of chang-ing genotype with small populations and lowmutation rates. This does not occur in the ab-sence of selection, when mutants grow at thesame rate as the parent. Without selection,mutation frequency increases only by additionof new mutations (Figure 6).

The general mutagenesis seen in 10% ofrevertants (79) is thought to occur in cloneswhose lac amplification includes the nearbydinB gene (91). About 20% of revertants (closeagreement to 10%, given the methods used)have a lac amplification that includes dinB, andthese cells are shown to have a higher muta-tion rate than cells whose amplifications in-clude only lac. This agrees with evidence thatgeneral mutagenesis by DinB (even of targetson an F′ plasmid) requires expression of DinB

from a high-copy plasmid (52, 64) in orderto overwhelm mismatch repair. General mu-tagenesis during reversion under selection isseen only when dinB is located on an F′ plas-mid cis to lac, and not when dinB and lac areon different conjugative plasmids or in cellswhose only dinB gene is in the chromosome.This position effect is expected if mutagenesisrequires coamplification of dinB and lac (91).

Thus the genome-wide mutagenesis seenduring the Cairns experiment (in 10% ofclones) seems to be an artifact reflecting thehappenstance that dinB is located only 16 kbfrom lac on the F′

128 plasmid and is occasion-ally coamplified with lac to provide enoughdinB copies to overwhelm mismatch repair.Removal of dinB eliminates associated muta-genesis but reduces revertant yield only aboutfourfold (66). In the absence of DinB, when nomutagenesis occurs, amplification alone in-creases revertant yield about 25-fold, basedon the number of plated cells, or about 104-fold, based on the number of plated duplica-tion cells. The minor increase in mutation ratecaused by DinB explains the altered spectrumof revertant sequence changes, but its effect onrevertant number is detectable only because itis imposed on cells with 10 to 100 copies ofthe target lac gene.

! Essence: Selection detects small pro-gressive growth improvements due tolac duplication and amplification. Re-vertant alleles (−1 frameshifts) arisein the growing cells within developingcolonies. Base changes are made moreprobable by an increase in the numberof mutational targets (more cells andmore lac copies per cell).

! Main evidence: (a) Each revertantcolony includes some unstable Lac+

cells (with a lac amplification) and somestable Lac+ cells (revertant segregants)that are clonally related (49) (Figure 7).This fulfills a prediction of the modelthat each colony contains evidence ofits history, including rare cells witha lac amplification. This evidence has

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One lac+ copy anddecreasing lac-

Boundarycondition

Boundarycondition

Reversionevent

Parent strain

Colony at t 1

Colony at t 2 Colony at t 3

Mature revertantP

erce

nt o

f po

pula

tion

Haploidlac-

Haploidlac+

Increasing lac-

copy number

100

50

0

Figure 5Population dynamics—a selection-biased random walk. The bottom of the figure presents the events as abounded random walk with two absorbing conditions. The frequency of intermediates in this walk (top) ispulled to the right by selection (improved growth on lactose) and by the absorbing condition at the right,in which the Lac+ phenotype becomes irreversible in haploid cells. The move to the right is opposed byspontaneous segregation and by the left-hand absorbing condition, in which cells become haploid Lac−

(with low probability or forming a new duplication).

been disputed, as discussed below. (b) ATn10 element placed near lac on the F′

plasmid reduces Lac revertant number(49). This is attributed to inhibition ofgrowth when the amplified region in-cludes both lac and the tetA gene; TetAprotein is known to be toxic when over-expressed (21). This experiment sug-gests that growth and amplification arenecessary precursors of reversion. (c) Aconstructed duplication of the mutantlac region in the parent tester causesan increase in revertant yield (91). Se-lection stimulates reversion (about 25-fold) in strains that lack dinB (66, 91).These results suggest that amplificationis sufficient to explain reversion, us-ing only the basal (uninduced) mutationrate. (d) Time for a colony to maturefrom miniscule to full-size is highly vari-able as expected for a multistep stochas-

tic process of growth improvement (49);this is not expected for one-step rever-sion from Lac− to Lac+ as proposed byall the other models. (e) This processhas been mathematically modeled andis numerically feasible with reasonableparameter values (73).

CHOOSING A MODEL: THECASE FOR AMPLIFICATIONThe functional direction model explains thedata but has a mystical quality in requiringthat the target specificity of mutation be in-formed by the benevolence of its outcome.Like Intelligent Design and deus ex machinaliterary devices, it solves a complex problemby invoking a mysterious ad hoc mechanism.In principle, this model could explain all ofevolution without recourse to natural selec-tion. The mechanistically more conventional


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Freq

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Generations

Generations

Lac- to Lac+++ revertants

Lac- Lac--+ Lac-++ Lac+++

No selection

With selection

Figure 6Expansion of nested clones under selection. Without selection (orwithout growth) (top) a population grows and new revertants (to fullLac+) are added by rare mutations that can be detected in later tests.With selection (bottom), common new mutations (one shown) arise thatprovide a slight growth improvement and they increase in frequencybecause they grow faster than the parent. Within each expanded subcloneanother improving change can occur. Growth under selection divides theprocess of becoming fully Lac+ into small steps. Individually, these smallsteps would be hard to detect experimentally.

explanations offered by later models make thefunctional direction model less attractive(whether or not these alternatives provecorrect).

The HMS model is clever but implausible.It proposes that 100 revertants arise from 105

Haploid lac+

Duplication

Revertant colony

Amplification

Amplificationwith one lac+ allele

Figure 7Revertant colony. The amplification modelpredicts that each colony should includerepresentatives of each step in the history of theprocess, since individual improved cells growfaster but leave behind many unimproved sibs.

mutagenized cells and therefore requires thatstress increase the mutation rate 105-fold—a general mutagenesis more intense than hasever been demonstrated in resting cells (86).If realized genome wide, this mutagenesis isestimated to produce an average of five lethalmutations per cell. The observed 20-fold in-crease is insufficient to form even one rever-tant from 105 cells. Furthermore, 90% of re-vertants arise with no apparent mutagenesis(79). The observed general mutagenesis is in-sufficient and unnecessary to explain the re-vertants in the Cairns system. Its source is bet-ter explained by occasional coamplification ofdinB with lac.

The positional direction model is attrac-tive in that it explains much of the data usinga plausible mechanism and does not requireunrealistically intense mutagenesis. Establish-ment of this model would require demon-stration (to the satisfaction of all) that selec-tion causes an increase in the mutation rate(mutations per target copy) on the F′ ratherthan an increase in lac copy number by am-plification. This will be difficult to demon-strate because there is no generally accept-able way to measure mutation rates under

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selective conditions. The Cairns system wasdesigned to solve this problem but so farhas not eliminated growth and progres-sive phenotype improvement within subpop-ulations as suggested by the amplificationmodel.

! Several lines of evidence suggest to usthat the positional direction model isincorrect. Its main support is that dur-ing a lactose selection, a tetA frameshiftmutation reverts more frequently whenlocated on the F′ plasmid (25) than whenit is in the chromosome (8). However,enhanced reversion of tetA on F′ has notbeen shown to require starvation for lac-tose. This position effect may reflect in-creases in tetA dosage due to coampli-fication with lac. In addition, the tetAframeshift mutation (like lac) is leakyand may be subject to independent se-lective amplification in the presence oftetracycline; this amplification (like thatof lac) would work better on the plasmid.

! Positional direction proposes that stressmutagenizes any region in whichrecombination occurs. This occursgenome wide but is more easily de-tected on F′ where recombination is in-tense. Our measurements suggest that,contrary to this model, reversion rateof lac is not higher on the F′ thanin the chromosome during nonselec-tive growth (when rates can be mea-sured by a fluctuation test) (E. Sano, S.Maisnier-Patin & J. R. Roth, unpub-lished data). Moreover, the unselectedrate is not influenced by removing recA,by varying expression of the tra operon,by varying SOS induction, or by remov-ing dinB—any of which might be arguedto change in response to selection. Un-der selection on lactose, the apparent di-rection of mutation to the plasmid canbe explained by selective increases in laccopy number (the mutation target) pro-moted by recombination and growth.

! The DinB polymerase is significantlymutagenic only when its error pro-

duction exceeds the capacity of themismatch repair system. During non-selective growth, mutagenesis requiresoverproduction of DinB from a high-copy-number plasmid, even when themutational target is located on an F′

plasmid, where recombination is in-tense (52, 64, 103). There is no evi-dence that selective stress can generatean equivalently high level of DinB ac-tivity (as required by this model).

! Thus far, the mutagenic activity of DinBseems independent of RecA (103, 104),whereas reversion in the Cairns systemdepends on RecA.

! The Cairns experiment works whenDinB is deleted (although it producesfourfold-fewer revertants). A fourfoldcontribution of DinB to plasmid mu-tation rate is far less than the 100-foldDinB-dependent increase required toexplain revertants by the positional di-rection model.

The amplification model explains all of theavailable data and is supported by the evi-dence outlined above. Establishment of thismodel will require demonstrating (to the sat-isfaction of all) that revertant clones containmixtures of stable haploid Lac+ and unstableLac+ precursor cells (see below), that amplifi-cation is a prerequisite for reversion, and thatmutation rates do not change under selectiveconditions.

AREAS OF DISCUSSIONAn interesting feature of this controversy isthat there is general agreement on most of theexperimental evidence. The original system isrobust and the basic reversion experiment canbe repeated reliably. Supporters of the severalmodels agree on the following observations(but disagree on their interpretation).

Leakiness of the lac MutationStarvation in the absence of any carbonsource does not appear to be mutagenic


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(29, 51). Residual lac function is required inthe presence of lactose for reversion in theCairns system (3). In the amplification model,this provides the minimal function whose am-plification serves to improve growth.

In the hypermutation and the functionaland positional direction models, residual lacfunction does not support growth but isneeded to provide energy for plasmid replica-tion, recombination, and mutagenesis. Whenlactose is provided but growth is blocked bylack of an amino acid, no reversion is seen;thus protein synthesis is also required (11).These models propose that cells use availableenergy and protein synthesis to support plas-mid replication and mutagenesis in preferenceto chromosome replication and growth.

Role of RecombinationIn the several stress-induced mutation mod-els, recombination provides structures (re-combination intermediates) into which DinBcan load to initiate error-prone replication.Contrary to this, direct tests suggest thatDinB-mediated mutagenesis does not dependon recombination (64, 103, 104).

In the amplification model, recombinationfunctions catalyze the genetic exchanges thatamplify the growth-limiting lac gene and re-move the extra mutant copies following re-version. Thus the main role of recombina-tion is to change lac copy number. Thoughnot essential to the Cairns system, the mini-mal increase in mutation rate requires RecAfor SOS (DinB) induction and (with other re-combination functions) for coamplification ofdinB with lac in a few revertant clones.

The Question of Mixed Clones andProposal of Two Pathways ofAdaptive MutationThere is general agreement that amplificationof the leaky mutant lac allele can allow growth(3, 47, 49, 82). In the amplification model,growth and increased lac copy number pre-cede reversion and stimulate revertant yield

by providing more targets. This predicts thatrevertant colonies should include both stableLac+ cells (formed following reversion, seg-regation, and overgrowth) and unstable Lac+

cells (precursors that still harbor the ampli-fication). These mixed clones were observedin E. coli (at a slightly higher lactose con-centration than that used by others) and aremore easily observed in Salmonella. The ratioof stable to unstable Lac+ cells varies widelyfrom one clone to the next and increases withage of the colony as stable types overgrow(49).

Those who do not observe mixtures reportinstead two types of revertant colonies—a ma-jority type (90% at 5 days) composed entirelyof stable Lac+ cells and a minority type (10%)composed entirely of unstable Lac+ cells; thefraction of revertants with unstable Lac+ cellsincreased during the course of a reversion ex-periment (47). To explain this, it is proposedthat cells respond to selective stress in ei-ther of two mutually exclusive ways. In somecells, stress induces only point mutations, andin others, stress induces only amplification(47, 48).

NEW ISSUES IN ADAPTIVEMUTATIONSeveral new aspects of work on adaptive muta-tion promise to shed light on the controversy,but have not yet tipped the balance either way.

Origins of Duplications and Processof AmplificationPrior to selection, lac duplications are carriedby about 1% or 106 of the 108 cells plated(91, 92). The majority of the pre-existingduplications are large (135 kb) and flankedby direct copies of the transposable element,insertion sequence 3 (IS3) (E. Kugelberg,D. E. Andersson & J. R. Roth, unpublisheddata), the largest direct-order sequence re-peats flanking the lac region on the plasmidF′

128 (54). The number of plated duplication-bearing cells (106) is vastly greater than the

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number of revertant clones (102), suggestingthat the plated large duplications have a lowprobability of initiating a successful clone. Re-moval of IS3 from the plasmid reduces the re-vertant number two- to threefold, suggestingthat IS-mediated duplication initiates abouthalf of the revertants.

Amplifications found within revertantcolonies after selection include a few withthe common IS3 join points, but most (70%)include smaller regions (10 to 25 kb) andhave join points with short sequence repeats(3 to 12 bp) resembling the endpoints of dele-tions. Duplications of the SJ type are presentbut rare in the preselection population; theyform revertant colonies with high efficiency(E. Kugelberg, D. E. Andersson & J. R. Roth,unpublished data).

It is proposed that the common IS3 du-plications are highly unstable and inherentlycostly (perhaps owing to included genes thatare toxic when overexpressed). Therefore inthe Cairns experiment, the common IS3 du-plications rarely initiate a successful colonyunless they are first converted to the SJ typeby join point deletions, explaining their lowefficiency of revertant formation. The otherhalf of revertants may be initiated by the rarepre-existing SJ duplications that form rever-tants with high efficiency.

The frequency of duplications in a pop-ulation is constrained by their high segrega-tion frequency and is expected to approachsteady state as the product of duplication fre-quency and segregation rate approaches theproduct of haploid cell number and forma-tion rate. The time needed to reach this steadystate (about 100 generations) is dictated bythe reciprocal of the fastest rate (segrega-tion), about 1% per generation. Nearly halfof the steady-state level is achieved duringgrowth of a cell to a full-density culture (30generations). Thus the failure to see fluctua-tion in revertant yield (11) is consistent withrevertants initiated by pre-existing duplica-tion cells. Duplications are thus resistant toLuria-Delbruck fluctuation because of theirreversibility.

Mu a bacteriophagethat forms aprophage byinserting itssequence at randominto the bacterialchromosome

Role of RpoS and PpkRecently mutations eliminating thestationary-phase sigma factor (RpoS) (57, 62)or polyphosphate kinase (Ppk) (97) have beenseen to reduce the yield of revertants arisingin the Cairns system. RpoS contributes to awide variety of phenomena, many involvingcontrol of gene expression when growth isstrongly limited; polyphosphate is involvedin control of RpoS synthesis (50). The effectsof these mutants have been interpreted asevidence that RpoS controls DinB expressionin stationary-phase cells, thereby providinga connection between growth limitationand increased mutation rate that is key tothe stress-induced mutability models (57,62). However, direct tests revealed smalleffects of these mutations on dinB expression,suggesting that RpoS may act primarily atsome other stage in the reversion process,e.g., recombination, mismatch repair, tra ex-pression, or (consistent with the amplificationmodel) residual growth on lactose.

OTHER SYSTEMS THAT SEEMTO SHOW STRESS-INDUCEDMUTAGENESISThe first two cases below are among thefounding works on adaptive mutation. Weoutline these and several other systems thatsuggest mutagenesis during selection.

Deletions that Remove a MuProphage and Fuse the ArabinosePromoter to a lacZ GeneThese deletions are more frequent after ex-tended periods of selection on lactose (88). Itwas ultimately shown that the detected mu-tants depend on events occurring prior to se-lection (30, 90, 92a). However, it is not clearwhy these deletions are detected more eas-ily following prolonged selection or why se-lection appears to affect the position of dele-tion endpoints. We suggest that several eventsare necessary to complete these revertants; a


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rate-limiting event prior to selection may ini-tiate a process of phenotype improvementduring growth of the colony.

Evolved β-GalactosidaseIn this system, Lac+ cells arise in a strainof E. coli whose lac operon has been deleted(43, 44). This requires two mutations affect-ing an operon (ebg) that is normally unable tosupport use of lactose. One mutation elimi-nates the repressor (allowing operon expres-sion) and the second alters an enzyme, increas-ing its ability to hydrolyze lactose. The firstmutation is expected to be common (on theorder of 10−6) and the other rare (on the or-der of 10−9). Strains with both required muta-tions are expected to be present at a frequencynear 10−15 and are not found in unselectedcultures. However, they do appear within aparent colony (less than 109 cells) growing onrich medium containing lactose, which allowsgrowth but still selects for improved ability touse lactose. After many days incubation, thesecolonies show Lac+ papillae, which containcells with both ebg mutations.

The number of total cells in each colony isfar too few to assure coincidental occurrenceof the two required mutations with a standardmutation rate. The result has been interpretedas evidence for directed mutation in responseto stress, but the possibility has not been elim-inated that the observed double mutants arisesequentially during serial expansion of nestedclones resulting from stepwise improvementof growth ability. We suggest that the morecommon repressor mutant arises first and ini-tiates a slowly growing clone within which thesecond rarer mutation type arises.

Accumulation of Mutantsin Aging ColoniesWild-type E. coli cells are plated on richmedium and allowed to grow for about 1 day.Some colonies are tested at this point for theirfrequency of Rif R mutant cells, while othersare incubated for an additional week, during

which the cell population increases only about10-fold. After one week, many of these “aged”colonies contain a greatly increased frequencyof Rif R mutants (on the order of 50-fold)(6). This phenomenon has been referred toas ROSE (resting organisms in a structuredenvironment) (98, 99) or MAC (mutagenesisin aging colonies) (6). It is attributed to stress-induced mutagenesis of cells and depends onCrp/cAMP, RpoS, and RecA. While the in-duced mutagenesis is inferred to be general,the frequency of mutants resistant to otherantibiotics increases marginally or not at allcompared with that of Rif R mutants.

Recently, M. Wrande & D. Hughes (un-published data) have shown that the Rif R fre-quency increases owing to clonal growth ofmutants that pre-exist the aging period. Forreasons that are not yet clear, some typesof Rif R mutants grow more than the parentstrain under the aging conditions and giverise to about 50-fold-more descendants overone week. Results would be explained if someRif R mutations changed RNA polymerase soas to increase expression of genes that enhancegrowth in old colonies. We suggest that mu-tants resistant to other drugs sometimes ariseduring growth of Rif R clones and hitchhiketo a slightly increased frequency. Thus thephenomenon can be explained by growth un-der selection without any increase in mutationrate.

Loss of Fitness in Stationary-PhaseCulturesLiquid bacterial cultures are grown to fulldensity and then incubated for extendedperiods, during which there is little netgrowth (61). Samples are removed at varioustimes and used to initiate new cultures, whosefitness is assessed by measuring growth ratein fresh medium. Fitness appears to drop withtime and the results are analyzed to infer anincreased mutation rate in the growth-limitedculture.

It has been suggested that this exper-iment involves unappreciated growth (17).

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Stationary-phase cultures are known to se-lect for extensive growth by rare individu-als in the population, even when the popula-tion as a whole remains constant or decreasesslightly—the GASP (growth advantage in sta-tionary phase) phenomenon, in which a fewcells grow at the expense of some dead cells(24, 106). This growth provides opportunitiesfor mutations to arise. Selected mutations thatenhance survival or growth during stationaryphase may be deleterious in fresh medium. Nomutagenesis is necessary to explain the resultsand no mutagenesis has been demonstrated bythe experiments.

Selection for Loss of KanamycinResistance in Stationary-Phase CellsMutation accumulation was examined instationary-phase colonies held under condi-tions that would kill any minority popula-tion that initiated growth. The lethal selectionwas then removed to allow detection of anymutants that arose during the period of sus-pended growth (5, 78). These are interesting,cleverly designed experiments, but they leaveopen the possibility that growth occurredin a subpopulation within the stationary-phase colony after relaxation of the lethalcounterselection.

Selection for Loss of RepressorFunction in Nongrowing PopulationsIn this interesting system (105, 105a), a nor-mal lac operon is fused to the promoter ofa purine biosynthetic gene ( purD) and itsexpression is limited by an unlinked super-repressor mutation ( purR S). When this testeris plated on lactose as sole carbon source, Lac+

mutants accumulate linearly over 6 days andthe tester lawn shows no residual growth. Therevertants carry either purR Null mutations orpurD operator mutations, both of which areexpected to allow expression of the lac operonfusion. Surprisingly, in the absence of selec-tion the frequency of operator mutations (ad-jacent to the purD::lac fusion) approaches that

GASP: growthadvantage instationary phase

of null mutations in the distant purR gene de-spite the huge difference in target sizes. Underselection, the frequency of operator types isonly 4% but still higher than expected. Thissystem does not involve the F′ plasmid, andselection is made for loss-of-function muta-tions (in the case of purR) that are expectedto be recessive. To our knowledge, no testshave been made for either general mutagen-esis or lac amplification in the course of thisexperiment. Amplification of the purD::lac fu-sion (with its operator region) seems a possiblecontributing factor.

Reversion of Auxotrophs in MultiplyMarked Strains of Bacteria and YeastSeveral laboratories have reported experi-ments involving strains of bacteria and yeastwith multiple auxotrophic requirements (72,96). Revertant frequency is assessed dur-ing starvation for individual nutrients. Forany particular mutation, more revertants arefound during selection for its restored func-tion than during starvation for a differentnutrient. While these results, like those forthe Cairns system, suggest a stress-inducedchange in mutation rates, no tests have ruledout growth of a subpopulation or stepwiseimprovement of common partially revertantcells during development of revertant clonesunder selection.

THEORETICAL COSTS ANDBENEFITS OF MUTATIONThe intuitive appeal of stress-induced muta-genesis may wane upon closer examination.

What Selective Forces DictateEvolution of Mutation Rates inNatural Populations?Organism persistence depends on mainte-nance of genetic information. This is achievedby reducing mutation rates and by selec-tively removing deleterious mutations fromthe population. However, adaptation to new


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situations requires some level of geneticvariation to provide rare beneficial mutations.These forces must be balanced. The numberof mutations generated in a population is de-termined by the population size and the muta-tion rate of the organism. Therefore, for anygiven population size, an organism is expectedto develop a mutation rate that optimizes thebalance between common deleterious muta-tions and much rarer mutations that increaselong-term fitness (18, 76, 93). The optimal ra-tio of cost to benefit is expected to change withcircumstances and lifestyle. A high mutationrate might be more costly for a well-adaptedorganism in a constant environment than fora poorly adapted organism in a highly variableenvironment.

The cost of genetic variation also will beinfluenced by genome size and the frequencyof sexual recombination. Genome size dic-tates the target size for deleterious mutations(19, 20) and recombination increases the effi-cacy of purifying selection (23, 69). The tol-erated mutation rate should increase in pro-portion to population size and recombinationfrequency and decrease in proportion to thenumber of genes in the genome (the target fordeleterious mutations). Organisms can satisfythis relationship with various combinations ofparameter values. For example, an organismsuch as HIV, which has a large populationsize, small genome, and sexual recombination,can tolerate a high mutation rate without riskof extinction. Conversely, an organism with asmall population, large genome, and no sexualrecombination needs to keep mutation rateslow (2, 14, 23, 65, 69). However, once somesuitable combination is established, these val-ues are expected to be difficult and costly tochange.

Evidence that Bacteria MinimizeMutation RatesMost organisms invest heavily in minimiz-ing mutation rates (e.g., exporting or destroy-ing mutagenic compounds, proofreading byDNA polymerases, expressing DNA repair

functions). These mechanisms may be as effi-cient as the cell can afford to make them, givenpressure to replicate rapidly and use resourcesefficiently. In most DNA-based microbes, thesubstitution rate per base pair is low: 10−11 to10−9 cells per generation. Cells of E. coli canaccomplish 100 to 1000 replications withoutany error (19, 20). It would appear that theselow rates provide sufficient beneficial muta-tions for long-term survival.

Mutators Are DemonstrablyAdvantageous in Some SituationsDespite the evidence that mutation rates havebeen minimized by selection, theoretical ar-guments and experiments demonstrate thatcells with a high mutation rate (mutators) canbe positively selected during growth in cer-tain environments—when selection requiresrepeated rare mutations and the available vari-ability is limiting (67, 100, 101). This occurswhen the population is small and rare mutantsprovide a selective advantage (e.g., antibioticresistance) that is larger than the fitness cost ofmany of the deleterious mutations (34). Heremutators win.

The difficulty with using this as evidencefor beneficial effects of a high mutation rateis that the advantage of mutators persists onlyas long as these conditions prevail. As soon asmore typical conditions resume (i.e., the drugis removed), previously accumulated delete-rious mutations (34) become costly and themutator strain is eliminated. The short-termbenefit of mutagenesis is obtained at a cost oflong-term extinction.

The beneficial effect of a high mutationrate was demonstrated for E. coli mutatorsduring colonization of a mouse gut (36, 37).The mutator advantage was seen when theinitial inoculum was a poorly adapted labo-ratory strain placed under strong selection toadapt to a different situation. In strains thatwere well-adapted to life in a mouse, muta-tor derivatives were strongly counterselected.Thus the mutator had a short-term advantage,but was a poor risk for long-term survival,

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because it accumulated deleterious mutationsand had little prospect of restoring its muta-tion avoidance system (60). In summary, “evo-lution of a disease is not the equivalent of long-term evolution of a pathogen.”

Mutagenesis Is Often UnnecessaryBacterial populations are large enough to in-clude most beneficial mutations even withstandard low basal mutation rates. Change inany particular base pair is expected to occurwith a frequency of 10−10 cells per genera-tion, which is within reach for most bacte-rial populations. If multiple mutations are re-quired, we expect they will arise sequentiallywith intermediate growth of the initial mu-tant. Adaptation seems likely to be initiated bycommon small-effect mutations. In the Cairnssystem, these are duplications. Typically oneSalmonella cell in a 1000 carries a duplicationof any specified chromosomal gene prior toany selective enrichment (1); such mutationsare likely to exist even in small populations.A fitter mutant that arises without a muta-tor has the advantage of competing long-termwithout an increased burden of deleteriousmutations.

Mutagenesis Is Often FutileBacterial populations are estimated to spendmost of their time under strong growth limi-tation (53, 55), and many of these stresses can-not be relieved by mutation (e.g., lack of anycarbon source, exposure to extreme physicalconditions). If mutagenesis were induced bysuch stresses, it would create deleterious mu-tations without hope of benefit. A mechanismfor stress-induced mutagenesis might be ad-vantageous if cells could determine (a) that theproblem is solvable by mutation and (b) thatdeath is assured if no mutation is forthcoming.Without such prescience, cells with a mech-anism for stress-induced mutagenesis wouldbe eliminated by repeated rounds of futilemutagenesis.

Temporary Increases in MutationRates Are Still CostlyWhether mutations are introduced slowlyover time or during short periods of intensestress-induced mutagenesis, the costly dele-terious mutations produced remain part ofthe genome and continue to reduce fitnessof the cell that produced them. If a popula-tion were maintained long term under con-ditions that favor a high mutation rate, thenit would have to readjust its whole lifestyleand find a new set of values that satisfy therelationship described above—increase re-combination, increase population size, and/orreduce genome size. Even temporary dis-turbance of the relationship is likely to becostly.

Mutators Are Common AmongNatural IsolatesMany natural isolates carry defects in theirmutation avoidance systems and show highmutation rates (4, 7, 58, 71, 94). Some havebeen isolated from environments that arestressful (see above). However, even in the ab-sence of strong selection, mutators are likelyto be present at a high steady-state frequencybecause their counterselection is delayed. Thesteady-state level of a mutator is dictated bythe combined effects of its rate of formationand its rate of removal by purifying selec-tion. Counterselection depends on secondaryaccumulation of deleterious mutations. Thatis, a new mutator cell may show unimpairedgrowth for many generations, but will ulti-mately accumulate mutations that allow itselimination from the population. Its persis-tence in the population may be extended bysexual exchanges that separate it from its dele-terious products. Delayed counterselectionallows mutators to persist in the populationfor a longer time and therefore to exist at ahigher steady-state level under any growthconditions. Thus mutators may be commonin populations even if they never provide anybeneficial mutations.


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Mutators Sometimes Win inLong-Term Growth ExperimentsIn the classic experiments of Lenski andcoworkers (22), cultures maintained underlong-term cultivation are often taken over bymutators. While this could be attributed tostrong selective pressures for rare mutationsthat improve growth in the short term (byproducing beneficial mutations, as outlinedabove), the late appearance of mutators (aftercells have already been substantially adaptedto the culture conditions) argues againstthis.

We propose that mutators arise frequently(because many genes are involved in mutationavoidance) and are removed slowly (because ofdelayed counterselection). Early in the growthexperiment, the frequency of mutators is keptlow by periodic selection for favorable muta-tions that arise in the predominant nonmuta-tor cell type. Beneficial mutations are unlikelyin the small mutator population. (If a muta-tor has a 100-fold-higher mutation rate andis present at a frequency of 10−6, then only1 mutation in 10,000 will arise in the mutatorsubpopulation.) However, a common mutatorwill likely arise in an expanding subclone witha pre-existing beneficial mutation. (If muta-tors arise at 10−5/cell/division, 10% of newfitter mutations should acquire a mutator bythe time their subclone reaches 104 cells.) Thefrequency of this mutator will increase to 10−4

in the whole culture if the subclone sweeps thepopulation. Thus the new mutator can hitch-hike on a beneficial mutation that was not of itsmaking. The proposed process should applyto any frequent mutation that is not strongly(or promptly) counterselected. By this means,a mutator could increase in frequency with-out generating any advantageous mutations.Once a mutator is present at 10−4, 1% of newmutations will occur in the mutator subpop-ulation and there is a greater chance that anew advantageous mutation will allow hitch-hiking to higher frequency. Counterselectionof the mutator is assumed to be weak on thetimescale of these experiments.

Directing MutagenesisIn view of the cost of genome-wide mutage-nesis, bacteria would be well served if theycould focus their mutability on sites that arelikely to contribute a fitness increase. Thiswas proposed by the initial functional direc-tion model for adaptive mutation. Bacteriahave achieved this goal by making partic-ular genes highly mutable. These “contin-gency loci” encode proteins that affect theprogress of a host infection—often by dic-tating cell surface features (68). These genespossess base runs in which frequent frameshiftmutations cause inactivation and reactivationof the gene. These base runs affect only thegene in which they occur and thus do notput the entire genome at risk. In essence, asmall number of genes (about 30) are individ-ually and stochastically turned on and off—rather like a phase-variation mechanism—generating in the population a vast array ofexpression patterns (230), some of which pro-duce a cell surface that solves the particularselective problem (68). Because each inactiva-tion is reversible, there is no long-term loss ofinformation. This mechanism would not beuseful for most genes.

Unlike contingency loci, the positional di-rection model for adaptive mutation does notprovide low-cost mutagenesis. Positional di-rection proposes that all recombination be-comes mutagenic under stress. This would af-fect the whole genome. The posited directionto the F′ plasmid occurs because this target issubject to heavy recombination for other rea-sons (plasmid transfer functions), not becausevariation here is particularly valuable to thecell.

BROADER IMPLICATIONS OFADAPTIVE MUTATIONThe special features of the Cairns systemsuggest that its behavior cannot be general-ized easily. On the contrary, we submit thatthe fundamental events are general but aremore easily observed because of idiosyncrasies

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of the system. In the case of the amplifica-tion model, duplication, selective amplifica-tion, mutation, and segregation are expectedto occur in any organism.

The principle of adapting by amplifyinga rate-limiting gene and the principle of in-creasing mutant yield by adding target copiesare general. The unique feature of the sys-tem is that it can produce detectable resultswithin a week because recombination rates arehigh in the neighborhood of lac, and muta-tion rate increases slightly because the nearbydinB gene is occasionally coamplified with lac.In effect, the system does a “fast-forward” ongenetic adaptation and allows a complex se-ries of events to be completed within 20 to 30generations (73). The system promises to re-veal more principles that can be generalizedbroadly (e.g., selective remodeling and stabi-lizing of rearrangements).

THE LEGACY OF THECAIRNS SYSTEMWe dedicate this review to John Cairns, whodeveloped the system described here andraised fundamental issues regarding the ori-gins of mutations under selection. Cairns hascontributed heavily to thinking about the con-tribution of mutation to the origins of cancer(9, 10). In both the bacterial system and inthe origins of malignancies, a population ofcells is maintained with a strongly restrictedability to grow (shortage of lactose in bac-teria, multilevel growth control mechanismsin metazoans). Each cell is under selection toescape this restriction and divide—a normalselfish response for a bacterium or a singlemetazoan cell viewed apart from the organ-ism as a whole. In both cases, growing cells

arise after extended time periods and are morecommon than can be explained given the ap-parent growth and ambient mutation rates.In the case of the E. coli lac mutation, rever-tant clones accumulate over 6 days of selec-tion and exceed their predicted frequency byabout 100-fold. In the case of cancer, a big-ger problem is posed. If five mutations arerequired, each arising at 10−5 cells per gen-eration, the expected likelihood of a cancercell is about (105)5 or 10−25. Because the hu-man body has only about 1014 cells, cancer ispredicted to be extremely rare, but in fact it iscommon.

Both paradoxes might be resolved if cellsunder strong selection increased or directedtheir mutation rate, and initially this seemedto be true for the bacterial system. However,the amplification model proposes that selec-tion does not change the rate or target speci-ficity of mutation but rather detects small im-provements in growth and initiates a seriesof clonal expansions—a powerful process thatis already favored for explaining cancer. Thisprocess requires no dedicated mechanism andexplains how rare, multiply mutant cells canarise in small populations even at low muta-tion rates.

The parallels between the Cairns systemand current views of cancer are even closer inthat amplifications are found in many tumorsand may amplify functions that help cells es-cape growth limitation. The remodeling of re-arrangements has been seen in both systemsand may reduce the fitness cost of selectedgene amplifications (70). Thus a bacterial sys-tem developed to give insight into origins ofcancer may prove to be a good model for thatdisease even if the system does not work in themanner initially suggested by its designer.

SUMMARY POINTS

1. Both mutation rate (formation) and selection (relative reproductive ability of themutant and parent) influence mutant frequency in a population, and their effects aredifficult to separate.


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2. Claims that stress increases mutation rate rely on the assumption that mutants arisein a nongrowing population (and must therefore be independent of selection). Thisassumption is unwarranted without data to exclude growth of subpopulations.

3. Models that propose an evolved mechanism for stress-induced mutagenesis disregardthe long-term fitness costs associated with accumulation of deleterious mutations.

4. The behavior of the Cairns system can be explained with no change in mutation rate ifone assumes that the rate-limiting gene amplifies during growth of a revertant clone.Reversion is then made more probable by an increase in number of mutation targetswithin the clone.

5. Coamplification of dinB with lac can explain the associated general mutagenesis seenin 10% to 15% of revertant colonies.

6. Each species has a characteristic mutation rate presumed to be optimized for congru-ence with long-term life-history parameters, including population size, genome size,and recombination rate.

7. Temporary increases in mutation rate do not circumvent the long-term cost of dele-terious mutations.

8. The Cairns system shows many analogies to cancer development in that cell popula-tions are under strong selection to escape growth restrictions.

ACKNOWLEDGMENTSWe thank people in our labs and collaborators who have contributed their thoughts and ex-periments to the effort to understand the “adaptive mutation” phenomena, including OttoBerg, Ulfar Bergthorsson, Kim Bunny, Tim Galitski, Shawn Gerum, Heather Hendrickson,Jeffrey Lawrence, Sophie Maisnier-Patin, Mats Pettersson, Emiko Sano, Semarhy QuinonesSoto, Fritz Roth, Jon Seger, Sue Slechta, and Cristina Tun-Garrido. We also thank colleaguesand friends who have provided helpful suggestions and encouragement, including SherwoodCasjens, Mick Chandler, Francois Cornet, Maurice Fox, Glenn Herrick, Pat Higgins, DiarmaidHughes, Jeffrey Lawrence, Frank Stahl, Beth Traxler, and Marie Wrande. We thank PhilHastings for sharing unpublished results. Our work has been supported by NIH grantGM27068 (to JRR), grants from the Swedish Research Council (to DIA), and a fellowshipfrom the Wenner-Gren Foundations (to EK).

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origin of mutations under selection: the adaptive mutation

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