Adaptive mutation in Escherichia coli.

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 strain, called 45, that he said had a high rate of reversion to Lac 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 1 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, F 128. 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 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 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 colonies per 10 cells plated. We calculated that the normal preplating mutation rate was about 10 9 Lac revertants per cell per generation, whereas the postplating mutation rate was about 10 9 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 that the appearance of Lac mutants was due simply to proliferation of the Lac population. We demonstrated that the mutations only appeared when lactose was present, not if the cells were merely starving, and thus were adaptive. Importantly, we showed that reversion to Lac during lactose selection, but not during nonselective growth, was dependent on some function or functions of RecA. Thus, our results demonstrated that the mechanism by which adaptive mutations occur is different from the mechanism by which growth-dependent mutations occur. It was this feature of FC40—a high rate of adaptive mutation that occurs by a distinct mechanism—that made it attractive for further study.