Adaptive Mutation in Escherichia coli

Abstract

In 1988 John Cairns, Julie Overbaugh, and Stephan Miller published a paper entitled "The origin of mutants," in which they suggested that bacteria could choose which mutations to make (11). Shortly thereafter, Cairns and I began collaborating on a National Science Foundation-funded project to investigate the genetic basis of what was popularly called "directed mutation" (although at the time we were calling it "selectiondependent mutation," which now seems as good a name as any). Our plan was to mutagenize an appropriate strain of Escherichia coli and identify and characterize variants defective in selection-dependent mutation. Cairns and coworkers had been investigating a strain called SM195, described in their original paper (11). However, the mechanisms of mutation in this strain had proved to be complicated, and we wanted a new experimental subject. We were setting up to investigate selection-induced activation of the cryptic bgl operon (36), when Jeffrey Miller gave us a Lac Ϫ strain, called ␣45, that he said had a high rate of reversion to Lac ϩ after plating on minimum lactose medium. The lac allele in this strain is a fusion of lacI to lacZ that eliminates the lac regulatory region as well as several residues of lacI and lacZ; transcription of the fusion is constitutive, initiating at the lacI promoter (7). ␣45 has a ϩ1 frameshift mutation, lacI33, in the lacI coding region (12). Miller and coworkers have used several similar strains to study various mutational mechanisms (52, 67). As in many of the strains that originated with Jacob and Monod, proAB and lac are deleted from the chromosome and carried on an episome, FЈ128. This arrangement greatly facilitates genetic manipulations and turned out to be important to adaptive mutation. We mated the episome from ␣45 into a ⌬(lac-pro) recipient that we had made rifampin resistant and named the new strain FC40 (Foster and Cairns #40).In our first paper (10), we showed that Lac ϩ revertants of FC40 accumulate at a constant rate for about a week after the cells are plated on minimal lactose plates. Two days after plating (the first day that Lac ϩ colonies appear), the numbers of mutants among cultures has a Luria-Delbrück distribution (meaning that the mutations occurred prior to plating), whereas on subsequent days the distribution becomes Poisson (meaning that the mutations occurred after plating). After 5 days on lactose plates, there are about 100 Lac ϩ colonies per 10 8 cells plated. We calculated that the normal preplating mutation rate was about 10 Ϫ9 Lac ϩ revertants per cell per generation, whereas the postplating mutation rate was about 10 Ϫ9 per cell per h; considering that the doubling time in minimal medium is about an hour, this means that per unit time, the two mutation rates are the same. We have never figured out if this similarity is informative or merely a coincidence.The high postplating reversion rate of FC40 allowed us to eliminate a number of artifactual explanations for the phenomenon. In our first paper (10) we ruled out the possibility ...

Stress responses and genetic variation in bacteria

“…The mechanism of adaptive Lac + mutation in FC40 has been recently reviewed [46]. The following is a summary of the relevant characteristics:…”

“…The rate of adaptive mutation is reduced two-to four-fold if Pol IV is eliminated [57,58]. E. coli's other inducible error-prone polymerase, Pol V, is not involved in adaptive mutation [36,59].…”

“…Our current model for adaptive mutation to Lac + is as follows [46]. When FC40 is incubating on lactose, the cells are not proliferating but replication is occasionally initiated at one of the episome's vegetative origins.…”

Determinants of spontaneous mutation in the bacteriumEscherichia colias revealed by whole-genome sequencing

“…In contrast to Pol V, Pol IV is abundant even in normally growing cells (∼250 molecules) and can be induced 10-to 30-fold by DNA damage and other stresses (25,26). Pol IV is required for stationary-phase or adaptive mutation (27,28) and has a substantial role in producing mutations on the F´episome (29,30). However, in normally growing cells Pol IV contributes only 10%, at most, to spontaneous mutations on the chromosome (20,29,31).…”

Proficient and Accurate Bypass of Persistent DNA Lesions by DinB DNA Polymerases

“…The molecular details of the mechanism responsible for this mutagenesis are quite controversial, but the involvement of dinB is well-established. [41][42][43][44][45][46][47][48][49] The fact that deletion of the chromosomal dinB gene does not profoundly alter spontaneous mutagenesis suggests that DinB function may not always be mutagenic. Indeed, it has been suggested that the -1 frameshift phenotype observed in adaptive mutagenesis arises as a consequence of its substrate specificity 50 and the increased levels of DinB produced from the episomal copy.…”