Spencer Benson

Summary

A recent article by Galitski and Roth(’) characterizes adaptive reversion of chromosomal lac- mutations in Salmonella typhimurium LT2. Using a classical genetic approach they show that adaptive reversion, as characterized by the appearance of late revertant colonies, is an exception rather than a general phenomenon for reversion of nonsense, missense, frameshift and insertion mutations. For certain mutations, however, the number of late revertants exceeds the predicted number. These excess revertants suggest that adaptive mutability is applicable to chromosomal genes as well as to genetic changes involving F plasmids and lysogenic phages

Adaptive mutations The widely accepted neo-Darwinian view of genetic change postulates that mutation and selection are two distinct processes. Specifically, this view proposes that mutations occur without regard to their fitness, i.e. that they are ran- dom, and that the environment (selection) acts as a filter to retain those that are beneficial. Nearly half a century ago now classical experiments clearly demonstrated that muta- tions in bacteria can be r a n d ~ m ( ~ , ~ ) . These experiments, however, did not eliminate the possibility that certain selec- tion conditions induced adaptive mutations, i.e. preferential formation of beneficial mutations without a concurrent increase in nonselected mutations. During the last decade several groups have described and characterized systems in which ‘adaptive’ mutations are recovered following impo- sition of selections for a Lac+ p h e n ~ t y p e ( ~ - ~ ) . These systems have been offered as possible examples of adaptive muta- t i ~ n ( ~ - l l ) . The evolutionary implication of systems that could ‘direct’ mutational changes or programs in response to en- vironmental signals has sparked a lively debate among geneticists, molecular biologists and evolutionists(l

The two systems used most often to characterize adap- tive mutation are: (1) a +1 frameshift mutation in the plas- mid-encoded lacl33RlacZ hybrid gene(4’6,7,10,16-23); and (2) the Mu-Ara-Lac genetic construct that requires excision of the Mu prophage for formation of Lac+ araRlacZ gene f ~ s i o n s ( ~ 5 ~ ~ ) . As pointed out by Galitski and Roth, these sys- tems have a genetic complexity that ‘makes one suspect that they may be examples of limited generality(’). Several lines of evidence suggest that adaptive reversion of the plasmid-encoded lac/33QlacZ allele is linked to transfer of the p l a ~ m i d ( ~ , ~ , ~ ~ ) and requires recombination func-

t i o n ~ ( ~ ~ ~ ~ ~ ) , whereas others have suggested that transfer per se is not required, only transfer functions in the donor cell.^(^^^^^). When the lacl33QlacZallele is chromosomal the number of late-arising revertants is greatly reduced, and unlike reversion of the plasmid allele, reversion is no longer dependent on the recA gene p r o d ~ c t ( ~ $ ~ ~ ) .

The continued appearance of new mutants during extended selective conditions has been reported for many genetic systems, the majority of which involve chromosomal genes (for a review, see ref. 9). In all these systems the mechanism(s) that produce genetic change in the absence of cell division remain(s) one of the mysteries of biology. Several models have been proposed for how selective con- ditions could stimulate adaptive mutation. These include a starvation-induced hypermutable state(25), in which a small subpopulation of cells enters a state of extreme mutation formation. The net effect is that most cells die and a few indi- viduals acquire beneficial mutations that allow growth, resulting in escape from the hypermutable state and fixation of the genotype in the population. A second model suggests that multiple sequential mutations contribute to a general increase in fitness and eventually result in a revertant phenotype(26). A third model invokes amplification of gene segments with residual activity(27). For example, lac- genes that encode a low level of residual enzymatic activity might be amplified when cells are plated on selective lactose medium. This would increase the level of a needed enzy- matic activity and provide an increase in DNA that can ex- perience beneficial mutations. A fourth model invokes slowed repair of mismatched or damaged DNA during star- vation conditions. This allows preferential fixing of the muta- tion that relieves the nutrient block, which in turn allows DNA

replication and colony outgrowth(14). To address the general applicability of adaptive mutation for chromosomal genes, Galitski and Roth isolated and characterized 30 indepen- dent Lac- mutations, starting with a nontransposable Lac+ insertion in the S. typhimurium LT2 hisoperon. Salmonellae are naturally Lac-.

Do late ‘adaptive’ revertants fit predictions? If adaptive mutation is a general phenomenon then it stands to reason that it should apply to many mutational types and occur in a predictable fashion using known biochemical path- ways. Many mutations in lac and other operons do not yield late-arising revertant^(^,^^,^^). This finding was extended by Galitski and Roth, who showed that mutations that yield revertants during nonselective growth conditions do not nec- essarily yield late revertants(’). Late ‘adaptive’ revertants were defined as colonies that first appeared between days 3 and 7. Among the thirty Lac- alleles characterized by Galitski and Roth, opal (UGA) and frameshift mutations tended to yield late revertants while amber and insertion mutations (TnIWTc) tended not to. Among the various mutational types, however, there were no consistent patterns. For example, the allele that yielded the highest number of late revertants was an amber mutation, but the majority of amber mutations tested did not readily yield late revertants(’). One might expect that a good predictive parameter for late rever- sions would be the rate of reversion during exponential nons- elective growth. This appears not to be the case. There was no correlation of the ability to yield late revertants with the nonselective reversion rate(’). One reason for this might be that late reversion occurs by different mechanisms. For the /ac/33ihcZ plasmid allele the spectrum of late Lac+ rever- sions is known to be different from those that occur during nonselective growth(18,21). Mutations in genes with known biochemical functions in recombination effect adaptive reversion of the plasmid-encoded allele /ac/33l2/acZ allele(19,23). Where tested(l ,7.22), however, these mutations do not effect adaptive reversion of chromosomal alleles, including the chromosomal /ac/33id/acZallele.

What effects the formation of late revertants? Revertant colonies reflect the product of two sequential processes, reversal or suppression of the genetic defect by a heritable genetic change, and selective outgrowth of the revertant cell to a visible colony. An underlying assumption in studies on adaptive mutation is that the time required for a revertant colony to form in a lawn of nongrowing siblings is the same, irrespective of when the reversion event occurs (generally 2-3 days). When this assumption has been tested by reconstruction or cell-seeding experiments this assump- tion is ~ p h e l d ( l ~ ’ ~ ~ ~ ~ ) , which suggests that late-appearing revertants are not preexisting revertants that experience an extended phenotypic lag.

A second important consideration in formation of late revertants is allele leakiness. Even a small amount of P- galactosidase activity might provide energy necessary for biochemical reactions involved in the reversion process. This idea is supported by work with the plasmid /ac/33Q/acZ allele, where adding a small amount of a metabolizable car- bon source stimulates the number of late revertants(17). When Galitski and Roth(’) looked at allele leakiness they found that cells lacking detectable P-galactosidase did not yield late revertants(’). They conclude that some residual metabolic activity is necessary for late reversions. This suggests that the amount of residual P-galactosidase activity might correlate with the propensity to form late revertants, i.e. increased leakiness would result in increased numbers of late revertants. Although they observed a positive correlation between these two para- meters, it was low, and a high residual activity did not guar- antee a high number of late revertants(’). Thus, factors beside metabolic capacity must be involved in formation of late-arising revertant colonies. One common measure of metabolic capability is the ability of the cells to grow and divide. It follows that high residual activity might result in growth on the selective medium, which in turn allows rever- sion via replication errors. To test this Galitski and Roth monitored population growth on the selective medium. As expected, residual P-galactosidase activity showed a high correlation with growth on the selective lactose medium. There was not a ‘strict’ proportionality between growth on the selective medium and the number of late revertants, however. For example, mutations that allowed the greatest number of cell doublings on the selection media did not nec- essarily yield the greatest number of late revertants. This finding is consistent with previous findings obtained with the plasmid-encoded /ac/33il/acZmutation in E. cob, where late revertants occur in the absence of population growth. In this system population growth on the selective medium is mini- mized by the addition of a tenfold excess of nonrevertable Lac- scavenger cells to metabolize contaminants and pre- vent cross-feeding from Lac+ revertantd4). The scavenger population reduces the number of late revertants but adds complexity to the system, since lac+ plasmids often are recovered in this p o p ~ l a t i o n ( ~ ~ * ~ ) . Galitski and Roth show that the addition of scavenger cells reduces the number of late revertants by approximately 50% without significantly reducing the doubling rate of the revertible cells after the initial lag period(’). This suggests that increased competition on fresh selective media influences the ability of the popula- tion to yield late revertant colonies. Whether this effect stems from a reduction in the frequency of genetic change or an inability to form a visible colony, or both, is unknown. The findings underscore the complexity of the selection environment, the many interactions that can occur among cells during selection(20), and our limited knowledge of the environmental parameters that influence colony formation in lawns of nongrowing or slow-growing cells.

Is there solid evidence for adaptive mutations of chromosomal origin? There is no question that extended nonlethal selections can yield late mutants when the experimental system allows some limited g r o ~ t h ( ~ O > ~ l ) , or involves parachromosomal elements such as plasmids or p r o p h a g e ~ ( ~ - ~ l ~ ~ , ~ ~ ) . To assess if adaptive mutation occurs in cells that have limited growth on the selective medium, Galitski and Roth com- pared the actual number of late Lac+ revertants to the expected number (the product of the nonselective reversion rate and the number of cell divisions on the selective media). For many of the alleles the number of late revertants exceeded the expected number, in some cases by more than fivefold(l). When scavenger cells were included, the difference in observed versus expected increased. Whether reversion of these alleles is truly adaptive remains an open question. An estimate of the frequency of mutation at other loci was not taken('), but in other systems where the rate of mutation at nonselected loci has been monitored there is no evidence that other unselected mutations occur at an increased rate(g,30). It is likely that this will be true in the S. typhimurium system.

Conclusion The elegant and robust study by Galitski and Roth supports the idea that adaptive mutation can occur for some, but not all, revertible chromosomal mutations. They show that trivial explanations such as residual enzymatic activity and limited growth are not sufficient to account for formation of the late revertants. They also demonstrate the residual P-galactosi- dase activity is a necessary but not a sufficient requirement and that growth ability on the selective medium is related to late reversion. What remains unknown is whether the late revertants will be fundamentally different from revertants that occur during nonselective growth. This has been demonstrated for the plasmid-encoded lacl33QlacZ allele(18,21) and for revertants of an ochre mutation in the S. typhimurium his o p e r ~ n ( ~ ~ ) . One striking feature of this, and previous studies, is that each mutation and selection system appears to have a unique adaptive mutational signature. Determining whether these signatures are part of a coher- ent mutational program of widespread applicability, or sim- ply a collection of individual characteristics unique to each mutational system, is the real challenge that lies before us.

Acknowledgements Work on adaptive mutation in the author's laboratory is sup- ported by NSF grant MCB-9218625.

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Spencer Benson is at the Dept of Microbiology, University of Maryland, College Park, Maryland 20742, USA