MINIREVIEW Directed Mutation: Between Unicorns and Goats

Vol. 174, No. 6

JOURNAL OF BACTERIOLOGY, Mar. 1992, P. 1711-1716 0021-9193/92/061711-06$02.00/0

Copyright © 1992, American Society for Microbiology

MINIREVIEW

Directed Mutation: Between Unicorns and Goats PATRICIA L. FOSTER

Department of Environmental Health, Boston University School of Public Health, Boston University School of Medicine, 80 East Concord Street, Boston, Massachusetts 02118

cannot be readily explained by physiological factors. Thus, it seems specious at this point to argue that all late-arising mutants existed before selection was applied.

INTRODUCTION When populations of Escherichia coli are subjected to certain nonlethal selections, such as for utilization of a carbon source or reversion of an amino acid auxotrophy, spontaneous mutants accumulate with time, sometimes continuing to appear for weeks. This observation may not be startling to anyone who has forgotten a plate in the warm room, but it would be startling if the following were found to be true: (i) the mutants did not preexist but arose only after the cells were plated, (ii) the nonmutant cells were not growing, and (iii) the mutants would not have arisen had they not been selected for. These are the unexpected findings that led John Cairns and colleagues (10) to suggest that "bacteria, in stationary phase, have some way of producing (or selectively retaining) only the most appropriate mutations." The phenomenon has variously been called "directed," "Cairnsian," "adaptive," and "selection-induced" mutation. In this review, I will examine how the data have fared in the 3 years since reference 10 was published and will discuss some of the hypotheses to explain them. I have confined my review to E. coli, which has been the subject of most reports. My bias is that not all of the criteria for directed mutation need be met for there to be interesting and important phenomena to discover, so we need not have to choose between unicorns and goats (40, 41).

ARE THE CELLS GROWING?

The late appearance of mutants could be due to (i) growth of the population under selection because the selection is "leaky" or because the medium contains utilizable contaminants, (ii) growth of the population because of cross-feeding by early-appearing mutant colonies, or (iii) "cryptic" growth of cells that are cannibalizing their neighbors. All of these phenomena may occur, but the question is whether the amount of resulting cell growth can account for the numbers of mutants that arise. Replication-dependent mutations typically occur at about 10-' per cell per generation, give or take an order of magnitude. If postselection mutations are due to normal replication errors, 109 generation equivalents have to occur for every mutant that appears. In most cases, this amounts to the entire population under selection repeatedly replacing itself. In the 1950's and 1960's, Ryan and coworkers published a series of increasingly heroic experiments documenting that His' revertants were arising in cultures of His- cells that were not demonstrably dividing, turning over, or replicating their DNA (33, 34). More recently, Hall (20) has shown that within colonies of Trp- cells deprived of tryptophan, the numbers of viable cells were actually decreasing with time. Less heroically, we stabilized a population of revertible Lac- cells on lactose plates by adding scavenger cells and found that Lac' revertants continued to appear (8). By plating dilutions of the revertible Lac- cells with the scavengers, we also demonstrated that the number of Lac' revertants that arise per cell is independent of the number of preexisting Lac' colonies, thus eliminating cross-feeding as an issue (8). Although it is safe to conclude that gross population increases cannot explain the continued appearance of mutants after selection, cryptic growth is not so easy to dismiss. The turnover of some of the population at the expense of the majority would not necessarily be detectable as a change in cell numbers. Both Ryan (33) and Hall (20) did reconstruction experiments in the presence of penicillin (which kills only dividing cells), and they found no increase in death rates, implying there is no cell turnover. In addition, on the basis of a "true" death rate in the presence of chloramphenicol (which prevents protein synthesis), Hall (20) estimated the maximum possible amount of cell turnover and found it insufficient to account for the number of Trp+ mutants that appear. Nonetheless, it still appears possible that a subpopulation of cells continues to metabolize and give rise to postselection

DO THE MUTANTS PREEXIST? As part of the evidence for directed mutation, Cairns et al. (10) presented the results of fluctuation tests showing deviations from the Luria-Delbruck distribution (expected of preselection mutations) toward the Poisson distribution (expected of postselection mutations). This emphasis was, perhaps, unfortunate because the Luria-Delbruck distribution can be skewed toward the Poisson distribution by various factors, such as differential fitness between mutants and nonmutants, phenotypic lag, and poor plating efficiency (25, 35, 42). Thus, the occurrence of a Poisson distribution, of itself, cannot demonstrate that mutants arise after selection. However, the case for postselection mutation does not rely solely upon the Poisson distribution. In some cases, the mutants that appear after selection are due to mutational events that simply are never detected during exponential growth (5, 10, 19, 37). More typically, mutants arise both before and after selection, but most of the former make their appearance by 24 to 72 h after selection (4, 8, 20, 32). Late-arising mutants are not distinguished by exceptional growth defects (8, 10, 20). Common sense tells us that at some point all preexisting slowly growing mutants will surely have appeared, but mutants continue to accumulate for days or weeks (8, 10, 20, 37). The distribution of these mutants has a larger Poisson component with time (8, 10), which 1711

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mutations. Global metabolic changes occur as cells enter into stationary phase, and the resulting population is heterogeneous (reviewed in reference 39). The penicillin experiments would not detect a metabolizing but nondividing or only slowly dividing population of cells. However, the amount of de novo DNA synthesis that occurs in starving cells is small. Some net DNA synthesis, amounting to a 30 to 70% increase, can occur after cells cease to divide, but this synthesis rapidly falls to zero within a few hours (28, 34). Ryan (34) attempted to detect DNA turnover during starvation with a density shift experiment but found less than 5% hybrid DNA after 400-h incubation of His- cells in the absence of histidine. Boe (5) also reported 5% as the maximum amount of [3H]thymidine incorporated into starved cells during a 24-h period. Of course, neither experiment precludes the possibility that DNA is being synthesized mainly from endogenous precursors. Even with the limitations of these experiments, the magnitude of turnover, either of cells or of DNA, cannot realistically be more than 10% of that required to account for postselection mutations by normal replication errors. Thus, if cryptic growth gives rise to postselection mutations, the cells engaged in this process must have an increased mutation rate. DOES STARVATION PER SE INDUCE MUTATIONS? Cairns et al. (10) introduced the delayed-overlay experiment to show that starvation in the absence of a specific selection is not mutagenic. In these experiments, Lac- cells were plated on medium with no sugar and then overlaid at various times with top agar containing lactose. No increase in Lac+ mutants occurred during the period without lactose. Using this method, we also demonstrated that depriving a Lac- Trp- strain of tryptophan did not induce Lac+ revertants even when lactose was present (8). Hall (20) used a filter lift technique to show that starving a Trp- Cys- strain for tryptophan did not induce Cys+ revertants and vice versa. Thus, for these point mutations, starvation per se does not appear to be mutagenic. However, for movement of mobile genetic elements, the evidence is contradictory. One controversial example involves a strain in which deletions of a Mu element can fuse lacZ in frame to araB, giving a Lac' phenotype on lactose medium with arabinose as the inducer (37). Both Shapiro (37) and Cairns et al. (10) found that the fusion did not occur during exponential growth or during prolonged starvation unless lactose was present. In stark contrast, Mittler and Lenski (26) reported that araB::lacZ fusions could be stimulated by incubation of this strain with aeration in liquid glucose minimal medium for 9 days. Several laboratories, including mine, have tried to reconcile this discrepancy, but it remains as yet unresolved (9). Shapiro and Leach (38) have provided a model that could explain both results. The initial steps of the excision process, which require Mu transposition functions, could be stimulated by starvation, but resolution, which requires the coordinated activity of various host functions, may be differently regulated. Of particular relevance, after the initial strand transfer reaction a hybrid template could form, allowing transcription of an araB::lacZ fusion from the ara promoter before the rearrangements are completed. In-frame hybrids would result in ,B-galactosidase production, which in the presence of lactose could stimulate completion of the process. Thus, lactose, although not always required for fusion formation, would greatly accelerate its rate (38).

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In another disputed case, Hall (19) reported that excision of an insertion element from the bglF gene, which codes for the 3-glucoside transport protein, only occurred in the presence of salicin, the substrate of the bgl operon. This example is particularly dramatic, since two events, the excision and a point mutation in bglR, the regulatory region, are required before the cell becomes Sal'. However, Mittler and Lenski (27) have reported that a low frequency of excision events occurs in nondividing cells even in the absence of salicin. These experiments will be discussed in more detail below. Although these reports are somewhat conflicting, they all support the conclusion that starvation is not generally mutagenic, but different environmental factors can stimulate the movement of mobile genetic elements in nondividing cells. Indeed, increased movement of insertion elements during stationary phase has previously been observed (3, 30, 45) and could be related to changes in DNA topology that occur in starving cells (39). What factor actually triggers excision might depend on how the cells entered into stationary phase. IS THE PROCESS SPECIFIC? Perhaps the most astounding aspect of directed mutation is, of course, that it is directed, i.e., only the mutants that are selected for arise in the population. Cells under selection are not, apparently, accumulating useless mutations. Unfortunately, this is the aspect that is the least supported by experimental evidence. It is not easy to find a second, neutral phenotype which can be readily assayed while the population is under selection for some other factor. Since the cells are not growing, this second phenotype, like the first, has to be dominant, making drug and phage resistances useless. In addition, the second event should also be subject to postselection mutation. Valine resistance, which meets these criteria, has been used repeatedly to show that neutral mutations do not occur during selection (4, 10, 19, 20). Although valine resistance can result from a number of mutational events and thus provides a fairly broad target, it has been argued that it might be a special case (14, 41). Hall (20) found no increase in LacI- mutants within colonies starved for tryptophan. As mentioned above, he also demonstrated that colonies of a Trp- Cys- strain, grown on medium limiting for only one amino acid, produced no new revertants (scored as papillae) 40 h after transfer to medium missing only the other (20). One reservation about this experiment is whether a cell, starved for one amino acid but supplied with another, could respond to the switch by synthesizing the required biosynthetic enzymes in time to produce visible papilla. Despite these reservations, the several examples of point mutations that are not induced by starvation per se and the few experiments showing that selection for these same events does not induce neutral mutations are consistent with the basic idea that a process of trial and error takes place before mutations are immortalized (see below).

ARE MULTIPLE MUTATIONAL EVENTS DIRECTED? Bacteria apparently have an array of "cryptic" genes which can be mutationally activated to allow novel carbon sources to be utilized. In some cases, two mutational events are required. Cairns et al. (10) cited these as possible cases of directed mutation, since the probability of both events occurring by chance is unreasonably low. However, underlying this argument is the assumption that neither mutation

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alone confers a selective advantage. Any low-frequency event can be modeled as a sequence of higher-frequency events, and Lenski et al. (25) developed such a model to fit the distribution of Lac' mutants reported by Cairns et al. (10). In this model, a mutant with an intermediate phenotype that could grow slowly on lactose and then mutate again to the full Lac' phenotype was hypothesized. However, to fit the data and allow the growth of the intermediate to be undetectable, the second mutation rate had to be set unreasonably high (10-5 per cell per generation). If we constrain mutation rates to normal replication-dependent levels and assume that the experimenter would notice massive growth of an intermediate, sequential models do not satisfactorily describe postselection mutants arising at frequencies of about 10-8 to 10-9 per cell. For very rare events, however, they may. Hall has presented two dramatic examples of double mutants appearing at frequencies orders of magnitude higher than expected. The first, activation of the cryptic bgl operon, which requires two mutations (19), was mentioned above. The probable first event, excision of an insertion element from the bglF gene, was estimated to occur in up to 10% of the cells in an aging colony on MacConkey plates containing salicin. From within this population of about 106 cells per colony, the second mutation, activating bglR, could then appear and give rise to a Sal' papilla. Since Hall (19) found that the excision event alone did not allow cells to grow on salicin, the results suggested some form of "anticipatory mutagenesis" (43). However, if the excision event were to confer even a slight growth advantage, a small number of bglF+ mutants could easily grow to the population size required to produce the second mutation (27, 43). In contrast to Hall's results (19), Mittler and Lenski (27) have found that intermediates, presumably bglF+ mutants, can overgrow the parental strain on MacConkey plates containing salicin. The second double event reported by Hall (21) was reversion of a trpA trpB mutant strain to Trp+. Both alleles carry missense mutations, and neither single revertant can grow on its own without tryptophan. When this strain was plated on medium with limiting amounts of tryptophan, Trp+ papillae appeared on the aging colonies at frequencies orders of magnitude greater than expected. However, here too, a small amount of growth of an intermediate, achieving an average of about 105 cells per colony, could account for the results (7). The TrpA protein converts indoleglycerol phosphate to indole, and the TrpB protein converts indole to tryptophan. trpA trpB+ mutants can readily grow on indole excreted by neighboring trpA + trpB cells (16). A more abundant source of indole might be available within aging colonies from spontaneous cleavage of the indoleglycerol phosphate produced by the trpA trpB majority (23). A subpopulation of trpA trpB+ cells, growing on indole, could then, by a single mutation, give rise to the Trp+ papillae at the frequencies observed. I have dealt at length with these experiments because they have been widely quoted as spectacular examples of directed mutation. Hall found no sequence changes in the Trp+ revertants other than the reverted bases themselves, a result which none of the current models for directed mutation can explain (21) (see below). However, this result is readily explained by the sequence of events described above. Experiments in several laboratories are in progress to clarify these ambiguities.

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WHAT ARE THE GENETIC CONTROLS? The class of genetic defects which are potentially the most interesting are those that affect postselection mutation but have no effect on mutations occurring during normal growth. Such defects might alter the pathway(s) by which DNA errors are generated or immortalized or might change the physiology of cells in stationary phase. To date, three mutant strains with this specificity have been described, as well as one mutant that has increased rates of both pre- and postselection mutation. Cairns et al. (10) showed that a defect in the uvrB-bio region (but not uvrB or bio) specifically increases postselection mutation. Interestingly, although the A(uvrB-bio) strain produces more Lac' revertants than the wild type when plated on lactose minimal medium, it fails to papillate on MacConkey plates containing lactose even though Lac' revertants are being produced (7). This suggests that the defect is interfering with the orderly entry and exit from stationary phase; thus, the mutant may have an increased postselection mutation rate, because it enters into stationary phase early, but then it has difficulty resuming growth in rich medium (7). Strains that are defective in both the ada and ogt genes also have enhanced spontaneous mutation rates after selection, but not during normal growth (31). Both ada and ogt encode DNA alkyltransferases, which remove alkyl groups from guanines and thymines in double-stranded DNA. Thus, some endogenous substrate of these enzymes accumulates in nondividing cells, giving rise to mutations (31). We have shown that GC-to-AT transitions are particularly enhanced, implicating alkyl guanines as the mutagenic lesions (17). recA430, which codes for a partially defective RecA protein, reduces the postselection reversion of a lacZ frameshift allele (8). Although recA430 is defective for SOS functions, SOS mutagenesis is not involved in this phenotype (8). Since the lacZ frameshift can be reverted by deletions occurring between small regions of partial homology (1), RecA430 may be unable to perform this type of recombination in nondividing cells, implicating recombination in at least some cases of postselection mutation. Alternatively, RecA430 may be deficient in "stable replication", a recA+-dependent form of replication that is induced under certain physiological conditions (24). Boe has demonstrated that defects in methyl-directed mismatch repair (MMR) enhance postselection mutations (5). MMR is a postreplication correction system that reduces replication errors by about 100-fold; thus, MMR- mutants are also powerful mutators during normal growth. Interestingly, Boe discovered that MMR-defective strains survive poorly during starvation, which suggests that the MMR pathway is usually active in nondividing cells (5). However, it is also possible that lethal errors occur as the cells attempt to resume growth when plated for viability (5), and there is evidence that MMR may be involved in repairing some DNA lesions (15). Thus, it is not clear that the poor viability of MMR- cells is related to postselection mutation. Although not a genetic defect, the addition of caffeine dramatically and reversibly inhibits the appearance of His' revertants during histidine starvation; since caffeine is a mild mutagen during exponential growth, the inhibition is specific for postselection mutations (18). Caffeine intercalates into DNA, interfering with the ability of DNA repair enzymes to properly recognize and/or bind to their substrates (36). Thus, this result supports Stahl's (40) proposal that DNA repair synthesis, which serves to prevent or correct DNA errors

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during normal growth, may serve to create DNA errors in nondividing cells (see below). WHAT IS THE MECHANISM OF DIRECTED MUTATION? Because Cairns et al. (10) suggested that the phenomenon they were describing was, in some sense, Lamarckian, the scientific community responded with a storm of alternative hypotheses to explain the observations by non-Lamarckian mechanisms. In the rush to defend orthodoxy, it was largely overlooked (but not by science historians and philosophers [22, 35]) that none of the mechanisms postulated by Cairns et al. (10) violated any principles of molecular biology. Some of the alternative hypotheses involving experimental artifacts have been discussed above. Other hypotheses, however, incorporate interesting new ideas, which are discussed below. With the exception of the Davis model, all include the notion that cells under selection have some mechanism for trial and error, i.e., genetic variants are continuously produced but are repaired or discarded unless the cell achieves success. The most unorthodox hypothesis suggested by Cairns et al. (10) was that the selective conditions "instruct" the cell which mutations to create or retain. They proposed that variants could arise at the level of transcription and, if a successful protein resulted, be immortalized as mutations in the DNA by reverse transcriptase. If a transcript and its protein were associated and the relevant transcript were targeted by success for reverse transcription, this mechanism could result in a direct information flow from the environment to the DNA. Although elegant, this hypothesis is likely to be wrong. First, reverse transcriptase, although subsequently found in some strains of E. coli, has not been found in K-12 strains (reviewed in reference 46), which are the subjects of most of the experiments on postselection mutation. Second, the instructional model predicts that the only sequence changes that occur are those in the gene which codes for the successful protein. Using reversion of a lacZ amber allele, we have found that tRNA suppressors arise after selection and have all the characteristics of directed mutations (17). In the case of these suppressors, the information that restores activity to the mutant protein is not present in the DNA or the RNA that encodes it, eliminating the possibility of a direct link of the kind proposed. Most examples of directed mutation involve inducible genes. Davis (12) proposed that the selective agent induces transcription, resulting in regions of single-stranded DNA in the transcribed gene. Since single-stranded DNA is more vulnerable to damage, a biased mutation rate would occur in the relevant gene. While Davis' case for the mutagenic consequences of transcription is convincing, it cannot explain the postselection appearance of tRNA suppressors (12), which we have observed (see above). In addition, the presence of isopropyl-,-D-thiogalactopyranoside (IPTG), a gratuitous inducer of the lac operon, increases the speed at which Lac' mutants appear after lactose is added but does not change their numbers even if a second, metabolizable carbon source is available (17). Finally, strains carrying a constitutively expressed lacZ allele do not accumulate Lac' mutants in the absence of lactose (8). Thus, although transcription may very well be mutagenic and transcription is obviously required to recover any mutant, it is not sufficient to explain the role of the selective agent in directed mutation. Stahl (40) and, subsequently, Boe (5) proposed the slow-

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repair model. While in stationary phase, cells may be sporadically replicating or repairing their DNA. Because of their nutritionally deprived state, the normal error-correction enzymes are slow to act, and errors persist in the newly synthesized strand. If this DNA is transcribed and produces a useful protein, the cell replicates its DNA, immortalizing the error. If success is not achieved, the error is eventually corrected. The true beauty of this model is in the choice of the repair pathway. Since methylation lags behind replication, the newly synthesized DNA would be unmethylated; thus, the MMR system, which corrects in favor of the methylated strand, would always recreate the original sequence. However, this model can also work for other repair enzymes, such as the alkyltransferases, which restore DNA bases to their original state (31). The only problem with the slow-repair model is the absence of direct data to support it. As mentioned above, defects in either the MMR or alkylation repair pathway increase postselection mutations (5, 31), but the critical prediction of the model is that the loss of the repair enzymes should increase mutations in nondividing cells in the absence of selection. With both MMR- and ada ogt mutant strains, we have found no accumulation of Lac' revertants in starving cells in the absence of lactose (17). The ultimate trial-and-error model was proposed by Hall (20). He hypothesized that under selection, some fraction of cells enter at random into an "hypermutable state" during which DNA errors are produced at a high rate. If the cell achieves success, it exits this state and resumes growth. If the cell does not succeed, it eventually dies. This model differs from the slow-repair model mainly in the consequence of failure. All of the random-trial-and-error models predict that unselected mutations should not be enhanced in the population at large, but they should be enhanced in cells that achieve success. As the successful cell begins to grow, the genetic variation that allowed it to do so would, of necessity, be preserved, but other variants extant at that time would also have some probability of escaping repair. The frequency at which nonselected mutations should appear depends on the model of choice. Hall (20) found that 2 of 110 Trp+ revertants, versus 0 of 4,530 nonreverted cells, carried auxotrophies. Boe (5) also reported that 20 of 2,000 Asn+ revertants had additional growth defects but gave too few experimental details to allow this result to be evaluated. Although these very small numbers can provide only tentative support for the trial-and-error models, clustered mutations may not have been detected in these screens. Clustering would be predicted by the Davis and MMR-deficient models (5, 21) or by any mutational mechanism that involved only small regions of the chromosome (10, 21). As mentioned above, Hall (21) sequenced 700 bases surrounding the trpA and trpB loci from 11 Trp+ double revertants and found no sequence changes other than the reverted bases themselves. Since this mutation frequency was far lower than expected, he dismissed all the models discussed above (21). However, if the observed frequency of double revertants is artifactual (see above), this result is not relevant. A few other speculative ideas that may be applicable to postselection mutation should be briefly mentioned. First, carbon starvation and iron deficiency both impair tRNA modification; a mutation in miaA that prevents modification of some tRNAs is a spontaneous mutator (11). Although the reason for this mutator phenotype is unclear, global changes in the levels of repair enzymes and nucleotide pools could result from the decreased translational efficiency of undermodified tRNA (11). Second, Ninio (29) has proposed that

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translational or transcriptional mistakes leading to an errorprone polymerase or to nonfunctional error-correction enzymes could produce transient mutators. Although the effect on the frequencies of single mutations would be small, transient mutators could account for rare double events (29). Finally, gene duplications occur at most chromosomal locations at frequencies of 10-3 to 10-4 per cell (2). These events are inherently unstable, being both created and resolved by recombination, and are also substrates for amplification (44). Thus, bacteria are continually producing extra gene copies upon which mutagenic mechanisms could work and which could then be retained or discarded. This mechanism has the advantage of providing a role for RecA, which would be required both for the creation and immortalization of the genetic variation. Indeed, duplications are stimulated by a recA allele that is constitutive for SOS functions (13), which correlates well with our finding that recA430, which is SOS defective, is deficient in some postselection mutational process (8). ARE THERE ANY UNICORNS HERE? Since reference 10 was published, it has become widely appreciated that mutations can arise in nondividing cells, vindicating the work of Ryan (28, 32-34) which had been largely ignored for 30 years. While this fact may not achieve unicorn status (41), it is certainly not uninteresting. In the case of insertion elements, a number of environmental factors can apparently trigger excision events. These could be mediated through changes in the physical structure of the DNA, in which case the cell might be considered passive, or by a dynamic feedback from the environment, as suggested by Shapiro and Leach (38). In contrast, the mechanism by which point mutations arise in nondividing cells is not at all clear. Some form of DNA replication would appear to be required, but there is no convincing evidence that it occurs. To account for the mutations, the small amount of DNA synthesis that presumably takes place must be error prone, either because the replication enzymes have reduced fidelity or because repair enzymes are slow. An increase in error rate of only 10-fold, easily imaginable by either mechanism, may be sufficient. It remains to be seen whether this increase is due to an inducible process, by analogy to the SOS response, or is simply an unavoidable consequence of a nutritionally deprived state. The evidence suggests that the role of selection in the mutational process is not to "direct" the process, but to define success. While this may appear to be just a version of "you get what you select for," there is an important distinction. If the idea of transient genetic variants is correct, it is not the organism, but its informational molecules that are under selection (10). Thus, a nondividing cell is, potentially, multiphenotypic, a characteristic previously thought to be true of the population, not the individual. If a bacterium can, either by design or accident, increase its genetic variability under stress while maintaining its genome more or less intact, this is clearly of evolutionary significance. Finally, it is probably a mistake to expect all the diverse phenomena labeled "directed mutation" to be due to a single mechanism. Not surprisingly, some unexpected results simply have other, unrelated, causes (4, 6). We can already distinguish mechanisms that result in the movement of mobile genetic elements from mechanisms that may induce point mutations. On the basis of the specificity of the genetic defects mentioned above, it is likely that mutations occurring by recombinational events are under different controls

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from those occurring by base substitutions (16). We know little about how mutational processes may be influenced by the physiology of cells in different nutritionally deprived states. Thus, the research stimulated by the directed mutation hypothesis may well lead to a variety of insights into how cells manage their genetic affairs.

ACKNOWLEDGMENTS I thank J. Miller, E. Eisenstadt, M. Fox, J. Shapiro, J. Drake, R. Kolter, F. Stahl, S. Sarkar, and, of course, J. Cairns for ideas and discussions. I also thank J. Mittler and R. Lenski for communicating results before publication, and the participants in the Population Biology and Evolution of Microorganisms Gordon Conference, 1991, who cordially met me halfway. This work was supported by National Science Foundation grant DMB-8905004.

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