Transient and Heritable Mutators in Adaptive Evolution in the Lab and in Nature

Susan M. Rosenberg,*,† Carl Thulin* and Reuben S. Harris*,† *Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada and †Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030

ABSTRACT Major advances in understanding the molecular mechanism of recombination-dependent stationary- phase mutation in Escherichia coli occurred this past year. These advances are reviewed here, and we also present new evidence that the mutagenic state responsible is transient. We ﬁnd that most stationary-phase mutants do not possess a heritable stationary-phase mutator phenotype, although a small proportion of heritable mutators was found previously. We outline similarities between this well-studied system and several recent examples of adaptive evolution associated with heritable mutator phenotype in a similarly small proportion of survivors of selection in nature and in the lab. We suggest the following: (1) Transient mutator states may also be a predominant source of adaptive mutations in these latter systems, the heritable mutators being a minority (Rosenberg 1997); (2) heritable mutators may sometimes be a product of, rather than the cause of, hypermutation that gives rise to adaptive mutations.

DAPTIVE mutations are those that allow organisms mental system, the stationary-phase mutation was dem- A to succeed in the face of natural or artificial selec- onstrated to exist as a process distinct from normal growth- tions. These mutations can arise by any of several routes. dependent mutation by virtue of using a different molecu- “Adaptive mutation” has also been used to denote a lar mechanism of mutation. [See Maenhaut-Michel and particular set of mutational routes, described in bacteria Shapiro (1994) and Maenhaut-Michel et al. (1997) for and yeast, that differ from canonical growth-dependent evidence that stationary-phase transposon-mediated de- mutations (e.g., Luria and Delbru¨ck 1943; Newcomb letion mutations are also mechanistically different from 1949; Lederberg and Lederberg 1952) in that the those occurring during growth.] mutations occur after cells are exposed to selection, in Recombination: Stationary-phase reversion of a lac ϩ 1 cells apparently not dividing, and only had been found frameshift mutation carried on an FЈ sex plasmid in in genes whose functions were selected (Cairns et al. Escherichia coli (Cairns and Foster 1991) requires func- 1988; reviewed by Drake 1991; Foster 1993; Hall tional proteins of the RecBCD double-strand break- 1993; Rosenberg 1994, 1997). This past year, the best- repair recombination system, whereas growth-depen- studied assay system for such mutations also has been dent reversion of the same allele does not (Harris et shown to produce unselected (nonadaptive) mutations al. 1994, 1996; Foster et al. 1996). This implies that (reviewed by Rosenberg 1997). Therefore, this special recombination is part of a unique mechanism by which mode of mutation is now called stationary-phase, or stress- the stationary-phase mutations form. Because RecBCD ful lifestyle-associated mutation (SLAM), of hypermuta- enzyme loads onto DNA only at double-strand ends, this tion. It may be a major route to formation of adaptive also implicates DNA double-strand breaks (DSBs) as a mutations as defined here. molecular intermediate in the stationary-phase mutation mechanism (Harris et al. 1994). [See Taddei et Stationary-Phase Mutation pre-1997 al. (1995, 1997a) for another stationary-phase mutation assay system with RecA- and RecB-dependence that may The mere existence of a mutagenic mode that ap- operate via a similar mechanism to that of lac system.] peared to produce adaptive mutations preferentially was One way that recombination might promote muta- controversial because of the possibility that mutations tion is illustrated in Figure 1. A recombinational strand- might be directed, in a Lamarckian manner, specifically exchange intermediate could prime DNA synthesis. Poly- Cairns to the selected gene ( et al. 1988). In one experi- merase errors made during this synthesis could become mutations. That recombinational strand-exchange intermediates are also intermediates in recombination- Corresponding author: Susan M. Rosenberg, Department of Molecular dependent mutation is implied by the requirement in and Human Genetics, Baylor College of Medicine, One Baylor Plaza, mutation for proteins that process such intermediates Houston, TX 77030. E-mail: [email protected] Foster Harris Although it is obvious that this issue of Genetics is dedicated to ( et al. 1996; et al. 1996) and by evidence Jan Drake, the authors wish to dedicate this article to Jan and Pam that a transient accumulation of these intermediates is Drake with heartfelt appreciation for their scrupulous and exhaustive mutagenic (Harris et al. 1996). service to the genetics community, and with particular appreciation for Jan’s support and encouragement of our efforts in entering the That DNA synthesis is part of recombination-depen- field of mutation. dent mutation was implied by the DNA sequences of

Genetics 148: 1559–1566 (April, 1998) 1560 S. M. Rosenberg, C. Thulin and R. S. Harris

possibility that the mechanism might be specific to sex plasmids. First, FЈ-encoded transfer functions are required for efficient Lacϩ reversion on the FЈ (Foster and Trimarchi 1995; Galitski and Roth 1995). Sec- ond, one site on the E. coli chromosome (Foster and Figure 1.—Hypothesis: DNA synthesis is primed by a recom- Trimarchi 1995; Radicella et al. 1995; R. S. Harris binational strand-exchange intermediate (Harris et al. 1994, and S. M. Rosenberg, unpublished results), and one 1996; Foster et al. 1996). This idea incorporates three known on the Salmonella chromosome (Galitski and Roth features of recombination-dependent mutation: the require- 1995), do not support recombination-dependent Lacϩ mentfor homologous genetic recombination, the involvement of DNA double-strand breaks (DSBs), and the evidence that reversion. Although conjugative transfer is not required the mutations result from DNA polymerase errors. Each line for recombination-dependent mutation (Foster et al. represents a single DNA strand, close parallel lines represent 1995; Rosenberg et al. 1995), the suspicion lingered duplex DNA, arrowed ends represent 3Ј DNA ends, dashed that somehow bacterial sex must be responsible (e.g., line represents newly synthesized DNA, and “m” indicates a Galitski and Roth 1996; Benson 1997). DNA polymerase mistake that can become a mutation when not corrected by postsynthesis mismatch repair. As an example An alternative possibility is that the transfer (Tra) ofwhat DNAs could recombine, the homologous DNAs recom- proteins promote the DSBs required for recombination- bining are illustrated as sister molecules derived from replica- dependent mutation. Tra proteins cause single-strand tion which, although drawn as uncompleted replication prod- nicks at the FЈorigin of transfer, and these could become ucts, need not be. Aspects of this hypothesis that have not DSBs by any of several routes including endonuclease, been tested are discussed in the text. Figure reprinted with Kuzminov permission from Rosenberg (1997). or replication of the nicked template ( 1995; Rosenberg et al. 1995; Rosenberg et al. 1996). If DSBs were limiting in the chromosome, this could account the stationary-phase Lacϩreversions, which are nearly all for some sites being inactive and would not exclude the Ϫ1 deletions in small mononucleotide repeats (Foster possibility that DSBs formed in other ways would allow and Trimarchi 1994; Rosenberg et al. 1994). These are other sites to be active for recombination-dependent characteristic of DNA polymerase errors and are different mutation. from growth-dependent Lacϩ reversions, which are het- erogeneous (Foster and Trimarchi 1994; Rosenberg Stationary-Phase Mutation in 1997 et al. 1994). The major replicative polymerase of E. coli, PolIII, has been implicated in the stationary-phase muta- At least five important discoveries were made last year: genesis (Foster et al. 1995; Harris et al. 1997a). Not directed: Two groups provided evidence that the The idea in Figure 1 is similar to that hypothesized recombination-dependent mutation mechanism is not to explain origin-independent, inducible stable DNA directed in a Lamarckian manner specifically at genes replication in E. coli (iSDR; Kogoma 1997), which has whose functions are selected. Foster (1997) measured similar, but not identical, genetic requirements to recom- reversion of a frameshift mutation in a defective tetracy- bination-dependent stationary-phase mutation (Asai and cline-resistance gene in a transposon that was hopped Kogoma 1994; Foster et al. 1996; Harris et al. 1996). For onto the FЈ that carries lac. Foster found recombina- both, features that have not been tested experimentally tion-dependent accumulation of tetracycline-resistant include the following: (1) Whether there is a direct associa- (TetR) mutants over time after exposure to lactose as tion of recombination with the DNA synthesis that leads the sole carbon source, coincident with the accumula- to mutation. Alternatively, synthesis might occur in one tion of Lacϩ mutants. Independently, Torkelson et al. genomic region with recombination required elsewhere, (1997) found high frequencies of unselected frameshift- for example, for cell survival, or mutation fixation; (2) reversion, loss-of-function, and temperature-sensitive mu- whether 3Ј end invasions initiate the DNA synthesis (lead- tations at sites on a cloning vector (pBR322)-style plasmid, ing strand) as drawn. Alternatively, 5Ј end invasions, evi- in the bacterial chromosome, and on the FЈ during recom- dence for which also exists (Dutreix et al. 1991; Rosen- bination-dependent stationary-phase Lacϩ reversion. The berg and Hastings 1991; Miesel and Roth 1996; frameshift-reversion targets used displayed a preference Razavy et al. 1996; Roca and Cox 1997), could allow for Ϫ1 deletions in small mononucleotide repeats, the assembly of whole replication forks; and (3) whether characteristic mutation spectrum observed previously the homologous DNAs that recombine are sister mole- in lac. These results demonstrate that the mutational cules as drawn. Torkelson et al. (1997) provide evidence process is not directed at the selected lac gene. that the homologies used are not regions of amplified Not sex plasmid-specific: Models in which recombina- DNA, and discuss other possible homology sources. tion-dependent stationary phase mutation was held to Are themutations confined to sex plasmids? Although be specific to bacterial sex plasmids predicted that the a novel mutational mechanism was obvious in recombi- mutagenic process would not operate on other repli- nation-dependent stationary-phase mutation, doubt was cons (e.g., Foster and Trimarchi 1995; Galitski and cast on the generality of this mechanism because of the Roth 1995; Radicella et al. 1995; Galitski and Roth Transient and Heritable Mutators 1561

1996; Benson 1997). This contrasts with the detection phase mutation. This was done by replacing the Lacϩ- by Torkelson et al. (1997) of high-level mutation in conferring sequences with the original lacϪ frameshift mu- three replicons, including the bacterial chromosome. tation and then by measuring rates of stationary-phase To compare the ability of chromosomal and FЈ-located Lacϩreversion in these strains (Table 1). None of the 12 sites to undergo mutation, Torkelson et al. (1997) recycled stationary-phase mutants possesses a mutator screened for mutations that confer resistance to the phenotype for stationary-phase mutation (Table 1). This nucleotide analog, 5-fluorocytosine (FC). These can oc- indicates that most cells that have undergone recombi- cur by loss of function of the FЈ-located codAB locus, or nation-dependent stationary-phase Lacϩ reversion are by loss of function of the chromosomal upp gene. The descended from a transiently hypermutable subpopula- FC-resistant mutations fell about equally into these two tion of all of the stressed cells. Two of the 12 are deficient classes, as demonstrated by mapping and by a separate in stationary-phase mutation (antimutators) (Table 1). phenotype specific to upp mutants. This argues for a Although the origin of the two antimutator mutations mechanism of chromosomal mutation similar to that is not known, we found that the antimutator phenotype on the FЈ. However, the recombination-dependence of is not linked with the lac locus: 20 out of 20 transductants the chromosomal mutations still needs to be tested. derived from each antimutator did not cotransduce the Hypermutation in a subpopulation of stressed cells: antimutator phenotype with a selectable transposon The way that Torkelson et al. (1997) performed the marker linked to lac (marker described in Table 1). It screen for unselected mutations allowed us to observe is possible that the antimutator phenotype results from that genome-wide hypermutation underlies stationary- unselected mutation(s) that occurred during the first phase Lacϩ reversion and occurs in a subpopulation of round of stationary-phase mutation. the cells exposed to selection on lactose medium. They The hypermutable subpopulation: The finding of screened for the unselected mutations by replica plat- Torkelson et al. (1997) that unselected mutations were ing. They compared the frequency of unselected muta- elevated only among Lacϩ revertants does not conflict tions among recombination-dependent Lacϩ mutant with Foster’s (1997) finding of unselected mutants that colonies with two important control populations: colo- were LacϪ, because Foster’s unselected mutants oc- nies from lacϪ cells that were never exposed to selection curred orders of magnitude less frequently than could (LacϪ unstressed cells) and colonies derived from LacϪ have been detected in the replica plating screen of Tor- cells exposed to prolonged lactose selection, rescued kelson et al. (1997). However, Foster’s data do bear from the same plates that generated Lacϩ revertants, on the nature of the hypermutable subpopulation dis- but which did not become Lacϩ (LacϪ stressed cells). covered by Torkelson et al. (1997). Hall (1990) sug- The Lacϩ revertants displayed frequencies of unselected gested that stressed cells might enter a hypermutable mutations 10–100 or more times greater than in either state that they could leave only by generating the se- control population, indicating that they experienced lected (adaptive) mutation, or by death if they failed to hypermutation. Moreover, starvation on lactose is not do so. This would produce survivors that all carry an sufficient to cause hypermutation, because only some of adaptive mutation, in contrast with Foster’s (1997) the starved cells (the Lacϩ mutants) were hypermutated data. Thus, current data suggest that the hypermutable (LacϪ stressed cells were not). state is not lethal, or at least not always lethal, in the Hypermutation in the subpopulation is transient: Two absence of an adaptive mutation (Rosenberg 1997; possibilities for the basis of subpopulation hypermuta- Torkelson et al. 1997). bility exist: The cells in the subpopulation could be Hot and cold sites: The multiple chromosomal muta- either (1) transiently or (2) heritably hypermutable. tion targets assayed by Torkelson et al. (1997) varied Previously, Longerich et al. (1995), and more recently dramatically in their mutability, showing no relationship Torkelson et al. (1997), found that most recombination- between target size and frequency of mutation. Two dependentLacϩrevertants do not possess heritable muta- fermentation regulons displayed fewer loss-of-function tor phenotypes [although a small fraction of those ex- mutations than one single gene target. This implies the amined by Torkelson et al. (1997) did, as discussed existence of hot and cold regions for the recombination- below]. However, the assays they used measured growth- dependent stationary-phase mutation mechanism. This dependent mutation. At least two mutator mutations finding could explain the previous failure to detect re- that are specific to recombination-dependent mutation combination-dependent mutation to Lacϩ at the lac have been described previously: recD (Harris et al. 1994) chromosomal locus (Foster and Trimarchi 1995; Rad- and recG (Foster et al. 1996; Harris et al. 1996) null icella et al. 1995; R. S. Harris and S. M. Rosenberg, alleles. Such mutators could have been missed in tests unpublished results) and at one locus on the Salmonella for growth-dependent mutators. chromosome (Galitski and Roth 1995), and the previ- To ask whether Lacϩ revertants are heritable station- ous lack of accumulation of a specific substitution muta- ary-phase mutators, we “recycled” 12 independent re- tion coincident with recombination-dependent Lacϩre- combination-dependent stationary-phase Lacϩ revertants version (Foster 1994). Also, stationary-phase mutation to LacϪ and tested them in another round of stationary- systems that operate independently of recombination 1562 S. M. Rosenberg, C. Thulin and R. S. Harris

TABLE 1 Most stationary-phase Lac؉ revertants do not display heritable stationary-phase mutator phenotypes

Reversion Cumulative Lacϩ Reversion relative to Frameshift- colonies on day 5 relative to Tn-carrying bearing strain Expt. per 108 viable cellsa Tn-less parent control strain Phenotype Tn-less parent FC40 1 97.9 Ϯ 11 1 2 36.1 Ϯ 8.2 1 3 21.5 Ϯ 10 1 Tn10-containing control strain SMR3739 1 158 Ϯ 13 1.6 2 110 Ϯ 14 3 3 62.1 Ϯ 12 2.9 Recycled LacϪ strains SMR3768 1 9.1 Ϯ 1.3 0.06 antimutator SMR3769 1 132 Ϯ 10 0.8 nonmutator SMR3770 1 279 Ϯ 35 1.8 338Ϯ3 0.6 nonmutator SMR3771 1 238 Ϯ 31 1.5 nonmutator SMR3772 1 14.3 Ϯ 2.7 0.09 3 5.2 Ϯ 1.5 0.08 antimutator SMR3773 2 200 Ϯ 64 1.8 3 39.3 Ϯ 4.1 0.6 nonmutator SMR3774 2 78.7 Ϯ 11 0.7 nonmutator SMR3775 2 61.5 Ϯ 6.6 0.6 nonmutator SMR3776 2 62.3 Ϯ 6.3 0.6 nonmutator SMR3777 2 43.6 Ϯ 3.9 0.4 nonmutator SMR3778 2 54.7 Ϯ 7.4 0.5 nonmutator SMR3779 2 73.4 Ϯ 4.8 0.7 nonmutator Nonmutator mean ϮSE 0.83 Ϯ 0.1 Totals 10 nonmutator 2 antimutator 0 mutator Twelve independent recombination-dependent stationary-phase Lacϩ revertants were “recycled” back to carrying their original lac ϩ 1 frameshift mutation by transduction with P1 grown on zah-281::Tn10 lacI33- carrying strain SMR3739 (this work). SMR3739 was made by transduction of zah-281::Tn10, a transposon linked with lac, from strain CAG12080 (Singer et al. 1989) into the lac ϩ 1 frameshift-bearing strain FC40 (Cairns and Foster 1991). By this means, each recycled LacϪ strain (SMR3768-3779; this work) carries the original lac ϩ 1 frameshift allele and now also carries the transposon zah-281::Tn10 linked with lac. (The Tn was used as a selectable marker in these transductions.) These recycled LacϪ strains also carry any unselected mutations that may have occurred in association with recombination-dependent hypermutation (Torkelson et al. 1997) and are tested here for whether or not they possess a heritable mutator phenotype for stationary-phase Lacϩ reversion as follows: Recycled LacϪ strains were subjected to prolonged incubation on minimal medium with lactose as the sole carbon source, and Lacϩ stationary-phase mutants were scored over five days’ incubation as described previously (e.g., Harris et al. 1994, 1996). For each strain in each experiment (Expt.), 8–12 independent cultures were used and the mean frequency of Lacϩ colonies Ϯ1 SE is given. The presence of zah-281::Tn10 on the FЈ appears to cause a roughly twofold increase in stationary-phase Lacϩ reversion relative to the Tn-less parent. This might be caused by the transposon insertion itself or by transposon hopping or excision in stationary phase that could provide an additional source of DNA double-strand breaks, which are necessary for recombination- dependent stationary-phase mutation (Harris et al. 1994; reviewed by Rosenberg 1997). The recycled strains that have been through one round of recombination-dependent stationary-phase mutation, in addition to carrying zah-281::Tn10, do not display obvious increases in stationary-phase mutability relative to the Tn-carrying control strain. In addition, two recycled strains seem to have accrued mutations that inhibit their ability to engage in recombination-dependent Lac reversion and are designated antimutators. These two strains are not merely slow colony formers (data not shown) but appear to be genuine stationary-phase mutation-impaired strains. a Values are mean Ϯ SEM. Transient and Heritable Mutators 1563 proteins (e.g., Bridges 1993; Hall 1995; Galitski and reasoning that if one were limiting, its production would Roth 1996) may show recombination-independence restore MMR function and decrease stationary-phase mu- because their mutation targets reside in cold regions. tation. MutS gave no such effect, suggesting that its station- [It is also possible that those with no known genetic ary-phasedeclineistoalevelappropriateforthedecreased requirements may simply assay growth-dependent mu- replication. MutL overproduction decreased mutation tations under circumstances in which growth is diffi- (Harris et al. 1997b), but the levels of measurable MutL cult to measure. However, the RecA-independent muta- protein remained constant during the stationary phase tions of McKenzie et al. (1998) and Galitski and Roth (Feng et al. 1996; Harris et al. 1997b). We suggest (1996) do not occur during normal growth or reflect several mechanisms by which MutL might be visible on normal growth-dependent mutation rates, respectively.] Western blots but not active for mismatch repair during Because recombination-dependent mutation requires stationary-phase mutation (Harris et al. 1997b). What- DSBs, region-to-region variability in DSB-formation ever mechanism ultimately proves to be responsible, might be responsible for mutational hot and cold sites these results demonstrate a transient limitation of mis- (Rosenberg et al. 1995; Rosenberg 1997). DSBs can match repair capacity, caused at the level of MutL pro- be caused by arrest of replication forks (Michel et al. tein, that is specific to stationary-phase mutation. (We 1997), by oxidative damage (Bridges 1997), and by show that MutL is not limiting during growth.) It remains other enzymatic and chemical means (Rosenberg et al. to be tested whether the whole population or the sub- 1996), any of which might occur heterogeneously in population experiences down-modulated MMR. We have the bacterial chromosome. Understanding the cause of suggested previously that the subpopulation could be hot and cold regions will provide important insights differentiated, not by MMR capacity, but rather by experi- into bacterial hypermutation. encing DSBs, as these are the sole loading sites for Mismatch repair modulation: The post-replicative RecBCD and so represent a key control point for the mismatch repair (MMR) system of E. coli (reviewed by ability to perform RecBCD-mediated recombination Modrich and Lahue 1996) is the single largest contrib- (Harris et al. 1994; Rosenberg et al. 1995; Rosenberg utor to mutation avoidance and influences multiple as- et al. 1996; Rosenberg 1997; Torkelson et al. 1997). pects of genetic stability, including genome rearrange- Stationary phase in bacteria is both a response to envi- ment (Petit et al. 1991), other forms of DNA repair ronmental conditions and a differentiated state (Seigele (e.g., Lieb and Shehnaz 1995; Mellon and Champe and Kolter 1992). After regulation of MMR in response 1996), and interspecies gene transfer (Rayssiguier et to environmental conditions or cell differentiation al. 1989; Matic et al. 1995, 1996; Zahrt and Maloy would have profound consequences for evolution, de- 1997). Homologs of the E. coli MMR proteins appear velopment, and cancer formation. See Richard et al. to play similar roles in other bacteria and in simple and (1997) for a similar modulation in stationary-phase hu- complex eukaryotes (e.g., Modrich 1995; Radman et al. man cells. 1995; Kolodner 1996). A 1997 study of recombination- A model: A model for the mechanism of recombina- dependent stationary-phase mutation in E. coli has pro- tion-dependent stationary-phase mutation is shown in duced the first evidence from any organism that MMR Figure 2. Some of the features of this model that have can be modulated and is not constitutively active (Har- not been tested are discussed above. ris et al. 1997b). Further unknowns include how a cell enters the hyper- Transient inhibition of MMR during stationary-phase mutable subpopulation. DSBs may be part of the differ- mutation was suggested by the sequences of the station- entiation (Rosenberg et al. 1994, 1996) but may not ary-phase Lacϩ reversions. These are nearly all Ϫ1 dele- account for all of it, and moreover, how DSBs form is tions in small mononucleotide repeats, unlike growth- unknown. The DSBs may trigger an SOS response, dependent Lac reversions (Foster and Trimarchi which can be induced by starvation (Taddei et al. 1995) 1994; Rosenberg et al. 1994), but identical to growth- and which is required for efficient recombination- dependent reversions in MMR-defective cells (Longer- dependent hypermutation (Harris 1997), and so may ich et al. 1995). Because most stationary-phase re- be part of a signal transduction process leading to a vertants are not heritably MMR-defective (Longerich hypermutable state. [The first links to SOS were pro- et al. 1995; Torkelson et al. 1997, and Table 1 above), vided by Cairns and Foster (1991). Some of these data these results implied a transient insufficiency of MMR were retracted by Foster et al. (1996). Data providing activity during stationary-phase mutation. Cellular levels evidence for an SOS involvement are shown by Harris of MutS and MutH MMR proteins were shown to de- (1997).] SOS may not be the whole signal transduction crease during the stationary phase (Feng et al. 1996), pathway because there are aspects of recombinational but did these proteins become limiting for MMR, or did hypermutation (e.g., diminished mismatch repair) not they simply fall to levels appropriate for the decreased seen in a normal SOS response. Note that standard SOS replication in the stationary phase? mutagenesis caused by UmuDC is not part of recombi- Harris et al. (1997b) overproduced MMR proteins dur- national hypermutation (Cairns and Foster 1991). ing recombination-dependent stationary-phase mutation, How hypermutation is regulated will have profound im- 1564 S. M. Rosenberg, C. Thulin and R. S. Harris

Figure 2.—A model for the mechanism of recombination-dependent stationary-phase mutation (Rosen- berg 1997). Untested aspects of this model are described in the text.

plications in biology and is a subject whose surface has mutating populations) cells was not measured for barely been scratched. comparison. In the other systems showing a similarly small proportion of heritable mutators, it is also not Transient and Heritable Mutators in Adaptive Evolution known whether these were the cause or consequence of mutability that generated adaptive mutations. Successful pathogenic (LeClerc et al. 1996) and com- 2. In all of these systems, the ability to alter genomes mensal (Matic et al. 1997) enteric bacteria in nature, radically under stressful conditions via transient, in- survivors of long-term laboratory culture (Sniegowski creased mutability mechanisms may be the predomi- et al. 1997) and of selection in laboratory conditions nant source of adaptive mutations, heritable muta- (Mao et al. 1997), as well as the winners in computer- tors being the minority (Rosenberg 1997). That simulated selection wars (Taddei et al. 1997b), carry a regulation, rather than heritable loss, of MMR would few to several percent heritable mutator mutants in the be more subtle and less risky has been noted surviving populations. Those examined so far are defec- (LeClerc et al. 1996). [See also Ninio (1991) for a tive in MMR (LeClerc et al. 1996; Matic et al. 1997; specific model predicting the fraction of spontane- Sniegowski et al. 1997). The heritable mutators are a ous mutations that should arise from transient and minority. In these systems the mechanism(s) of accumu- heritable mutators.] lation of the adaptive mutations in the majority of win- 3. With such normal circumstances as a commensal life- ners has not yet been examined. style being associated with apparent increased muta- These systems seem similar to recombination-depen- bility, one wonders whether the laboratory may be dent hypermutation in their small percentage of herita- the only environment in which selection for mutabil- ble mutators. Torkelson et al. (1997) found that 6 out ity is normally avoided. of 55 Lacϩ stationary-phase mutants with one or more 4. Regarding the possible generality of strategies like associated unselected mutations were heritable mutator recombination-dependent hypermutation in E. coli, mutants. At least five of these have lost the function of events have been described in multicellular eukary- an essential component of the MMR system. Four carry otes (e.g., McClintock 1978) that looked similar to mutations in mutL, and one carries a mutation in mutS the radical genomic upheavals implied by the DSBs, (Torkelson 1997). We present evidence demonstrating recombination, and loss of mismatch repair. Also, that the mutability experienced by most cells undergo- association of mutations with recombination, sex, or ing recombination-dependent stationary-phase muta- both has been reported in bacteria (Demerec 1962, tion is transient (Table 1), and that occurs an entire 1963), in yeast (Magni and von Borstel 1962; Es- novel mechanism for transient mutability in this system posito and Bruschi 1993; Strathern et al. 1995), (reviewed above). and in filamentous fungi (Paszewski and Surzycki Several points follow from comparison of recombina- 1964). Finally, stationary-phase adaptive mutability tional hypermutation with these other natural and labo- has been reported in other bacteria (Kasak et al. ratory systems in which mutators have been found: 1997) and in yeast (Hall 1992; Steele and Jinks- Robertson 1992; Baranowska et al. 1995; Heiden- 1. Because of the high frequency of unselected muta- reich and Wintersberger 1997), although whether tions among Lacϩ stationary-phase mutants, it is pos- these share mechanistic similarities with the system sible that the few MMR-defective mutations observed reviewed here remains to be seen. are also unselected (neutral), rather than selected (beneficial) mutations. In this context it is notewor- Conclusions: Recombination-dependent stationary- thy that antimutators are also relatively frequent (2 phase mutation was shown this past year to be neither out of 12) among the recycled adaptive mutants in directed in a Lamarckian way to selected genes, nor Table 1, although the frequency of antimutators specific to sex plasmids. Transient, genome-wide hyper- among LacϪ stressed and unstressed (i.e., nonhyper- mutation occurs in only a subpopulation of the stressed Transient and Heritable Mutators 1565 cells. Hot and cold sites may explain previous failures Foster, P. L., 1994 Population dynamics of a LacϪ strain of Esche- richia coli during selection for lactose utilization. Genetics 138: to find an unselected mutation and to find adaptive 253–261. mutations in some chromosomal regions. The MMR Foster, P. L., 1997 Nonadaptive mutations occur in the FЈ episome system’s activity is limited during recombination-depen- during adaptive mutation conditions in Escherichia coli. J. Bacte- riol. 179: 1550–1554. dent stationary-phase mutation by a lack of functional Foster, P. L., and J. M. Trimarchi, 1994 Adaptive reversion of a MutL, providing the first evidence that this important frameshift mutation in Escherichia coli by simple base deletions in system for maintenance of genetic stability is not consti- homopolymeric runs. Science 265: 407–409. Foster, P. L., and J. M. Trimarchi, 1995 Adaptive reversion of an tutive. 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