Adaptive mutation: General mutagenesis is not a programmed response to stress but results from rare coamplification of dinB with lac

Abstract

In a particular genetic system, selection stimulates reversion of a lac mutation and causes genome-wide mutagenesis (adaptive mutation). Selection allows rare plated cells with a duplication of the leaky lac allele to initiate clones within which further lac amplification improves growth rate. Growth and amplification add mutational targets to each clone and thereby increase the likelihood of reversion. We suggest that general mutagenesis occurs only in clones whose lac amplification includes the nearby dinB ؉ gene (for error-prone DNA polymerase IV). Thus mutagenesis is not a programmed response to stress but a side effect of amplification in a few clones; it is not central to the effect of selection on reversion.W hen a particular lac mutant of Escherichia coli is plated on selective medium, revertant colonies accumulate over several days (1, 2). Two models assume that mutations arise in the nongrowing population (3). Directed mutation proposes that stress preferentially induces beneficial (i.e., Lac ϩ ) mutations (1, 2). The hypermutable state proposes that stress induces general (genome-wide, undirected) mutagenesis in a subset of cells (Ϸ0.1%), and this mutagenesis produces the Lac ϩ revertants (4-6).An alternative model, amplification mutagenesis, proposes that reversion occurs in cells growing under selection and requires no change in mutability. On selective medium, rare preexisting cells with a lac duplication initiate slow-growing clones within which the growth rate increases progressively as amplification increases the copy number of the partially functional mutant lac allele (7,8). The probability of reversion within each clone increases with the number of target lac copies. After reversion, selection holds the revertant lac ϩ allele and favors cells that stabilize this allele by loss of mutant copies. This model is a specific form of a more general hypothesis proposed by Lenski et al. (9).Genomewide mutagenesis (an Ϸ100-fold increase in rate) is experienced by some revertants. This mutagenesis depends on the error-prone DNA polymerase, DinB (10-12), which may be induced when the SOS regulon is activated by DNA fragments released during segregation of the amplified lac region (8). Three problems complicate understanding how selection might cause general mutagenesis:(i) Induction of SOS does not mutagenize strains with a single wild-type dinB ϩ gene (13-15).(ii) Only 10% of Lac ϩ revertants arising under selection experience general mutagenesis (16,17).(iii) Selection causes mutagenesis only when lac is near the dinB gene (16,(18)(19)(20)(21).Evidence is presented that general mutagenesis occurs only in those developing clones whose amplified lac region includes the nearby dinB ϩ gene. Thus general mutagenesis is not a programmed response to stress in stationary phase but rather a side effect in a subset of developing clones growing under strong selection. Materials and MethodsSupporting Information. More detailed descriptions of methods and complete genotypes of all strains are published as supportin...

Multiple pathways of selected gene amplification during adaptive mutation

“…The behavior of the system does not require a stress-induced increase in the formation rate of either revertant alleles (lac ϩ ) or the deletions that form SJ duplications. Increases in general mutation rate that have been reported are due to occasional coamplification of dinB with lac, as described previously (8) and in supporting information.…”

“…This suggested a lower level of LacZ activity, indicating that the large IS3-flanked lac duplication (and the smaller REP duplications) found in these cells amplify poorly under selection and allow only slow growth on lactose. Previously published evidence shows that these faint blue-sectored clones had 10-15 lac copies, whereas the dark-blue sectored clones found in unstable-rich colonies (below) had 50-100 lac copies (8).…”

“…The IS3A and IS3C elements were replaced by the Kan R and Rif R genes by linear transformation (8). Three similarly constructed lac duplications had lac in the middle of the repeat and a Cm R determinant at the junction between copies.…”

“…The difficulty of amplifying larger duplications may reflect in part their expected higher rate of loss by segregation. To test the effect of repeat size on segregational loss, three lac duplications were constructed by linear transformation, as described (8). The stability of these duplications was estimated by following the loss of a Cm R element present at the duplication junction during nonselective growth.…”

“…The selective gene amplification model explains the effect of selection without stress-induced mutagenesis (8)(9)(10). Rare cells with a duplication of the leaky lac allele initiate slow-growing clones on selective medium.…”