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Spontaneous mutations are thought to occur primarily in growing cells. However, spontaneous mutations also arise in nutritionally deprived cells, and in some cases this process appears to be adaptive. Here it is reported that when a Lac− strain of Escherichia coli is under selection for lactose use, the spectrum of Lac+ mutations that arises is different, and simpler, than that arising without selection. Mutations appearing during selection were mainly one-base deletions in runs of iterated bases. Similar mutations occurring in repetitive DNA elements are associated with a variety of human hereditary diseases and are increased in cells that cannot correct heteroduplex DNA.
The mechanisms by which spontaneous mutations arise in growing cells have been the subject of much research. Two causes of spontaneous mutation that are often cited are intrinsic polymerase errors and endogenous DNA lesions. Indeed, many of the mutations that arise spontaneously in bacteria and in their viruses are the same types of errors as those made by DNA polymerases replicating damaged and undamaged templates in vitro (1, 2). However, it is unclear if such mechanisms can account for the mutations that arise among cells that are apparently not growing or replicating their DNA (3). Many theories have been proposed to explain how mutations occur in nongrowing cells and why those mutations are adaptive (3–11). Comparisons between the types of mutations that arise during nonselected growth and those that arise under selective conditions may help to choose among these theories (12).
In previous publications (7, 8), we described the appearance of Lac+ revertants among populations of a strain of Escherichia coli, FC40, that cannot use lactose because of a frameshift mutation affecting the lacZ gene. Lac+ revertants occurring during exponential growth, which were detected 2 days after cells were plated with lactose as the only carbon source, arose at about 10−9 per cell per generation, a rate that is well within the normal range for mutations of this type. After day 2, Lac+ revertants continued to arise at a nearly constant rate of about 10−9 per cell per hour. The post-plating mutants quickly became the main class, and 90 to 95% of the Lac+ revertants that had appeared after a few days were apparently the result of mutations that occurred after plating. This class of mutants did not appear if the cells were starved in the absence of lactose or in the presence of lactose if there was another, unfulfilled growth requirement (7). In addition, mutation to Lac+ during lactose selection, but not during prior growth of the cultures, required some function or functions of the major recombination pathway, RecABC (7, 11). In the work reported here, we determined the sequence changes in Lac+ revertants that arose each day after FC40 was plated on minimal lactose plates.
The mutant lac allele, lacI33, carried by FC40 on an F′ episome, derives from an in-frame fusion of the lacI gene to the lacZ gene (13) but has a +1 frameshift mutation in lacI that is polar on lacZ (14). Because the lacI coding sequence is not essential, this allele can be reverted by any mutation that restores the reading frame but does not create a nonsense mutation. Such events include simple −1-bp deletions as well as more complex DNA rearrangements that restore the reading frame within the 130-bp target shown (Fig. 1).
Newly arising, independent Lac+ mutants of FC40 were collected on days 2 to 5 after the cells were plated on minimal lactose plates (15). The DNA from these mutants was analyzed by amplification and sequencing (16), and the results are summarized in Tables 1 and and22 and in Fig. 1. Nearly all of the Lac+ mutants that arose after day 2 carried −1-bp deletions, and most of these occurred in runs of three to five bases. In contrast, about 50% of the Lac+ revertants that were isolated on day 2 carried mutations that resulted in deletions, duplications, and rearrangements. Thus, complex mutations are frequent in the absence of selection but are rare during selection. Considering only these two classes of intragenic revertants, there were nine complex mutations and 11 −1-bp deletions among the mutants isolated on day 2 but only one complex mutation and 30 −1-bp deletions among the mutants isolated after day 2 (χ2 = 10.9, P = 0.001). Indeed, the one late-arising deletion mutant, isolated on day 3, was only weakly Lac+ (see Table 3), which suggests that this complex mutation may also have arisen during prior growth of the culture but took an extra day to appear as a Lac+ colony.
Because late-arising Lac+ colonies might have been the result of mutations that gave only a weak Lac+ phenotype, we measured the β-galactosidase activity of an assortment of mutants (Table 3). The mutant with the largest deletion had the greatest activity, 422 Miller units, which suggests that this deletion creates a strong promoter. Not surprisingly, the three extragenic mutants that were isolated had rather low β-galactosidase activity. Among the −1-bp deletion revertants, the levels of β-galactosidase activity were about the same—most ranged from about 200 to 300 Miller units regardless of the day on which they appeared.
These results limit the types of mechanisms that might be responsible for the Lac+ revertants of FC40 that arose during selection. Although we did not sequence regions outside the target region, we read approximately 200 bases of sequence for each mutant and found no sequence changes other than the ones that reverted the frameshift. In addition, the reverting mutations were found at seven different sites. The lack of silent mutations and the diversity of mutational sites argue against the theory that the late-arising mutations resulted from recombination with homeologous (similar but nonhomologous) sequences located elsewhere in the genome (10). A distinctive feature of the late-arising frameshift mutations is that 90% occurred in runs of three or more bases. This site specificity is typical of −1-bp frameshift mutations made by DNA polymerases in vitro (2) and can be explained by replication of a misaligned template (17). Thus, the late-arising revertants of FC40 might be the result of simple polymerase errors, which supports our previous conclusion, based on genetic evidence, that adaptive mutations require some form of DNA replication (8). In vivo, mutations at iterated sequences are greatly enhanced by the loss of mismatch repair functions (18), which raises the possibility, previously suggested (5), that in nutritionally deprived cells, error correction activity may be low, or that mispaired or misaligned DNA may be inaccessible to the error-correcting enzymes. Similar types of mutations, apparently due to slippage during replication, repair, or recombination of repetitive DNA elements, have recently been associated with a variety of human hereditary diseases (19).
The late-arising revertants of FC40 are a subset of the kinds of mutations that can revert lacI33, yet they are distinguished from the mutations that arise during nonselective growth by their requirement for RecABC (20). One straightforward explanation for this result is that − 1-bp deletions are produced by the same mechanism both before and during selection but that there is an extra requirement for some function or functions of RecABC in the latter case. For example, if the −1-bp deletions are the result of polymerase errors, RecABC may be required to initiate DNA synthesis, to preserve the products, or both. RecABC can initiate DNA synthesis by producing D-loops (21) and (perhaps by the same mechanism) by catalyzing unequal crossing-over between regions of homology (22). Both of these mechanisms would require that more than one copy of the lac region be present in a cell. Thus, the mutations might arise in a subpopulation that has duplicated the lac region (8, 23) or simply among cells (probably the majority) that have more than one copy of the episome. If DNA synthesis is initiated only infrequently or is restricted in extent, yet is error-prone, this would help to explain why the amount of DNA synthesis measured in non-dividing cells appears to be inadequate to account for the mutations that arise (3). If the products of this synthesis are transitory unless the cell achieves a useful mutation and begins to divide, this would explain why the only mutations that are recovered are adaptive (4, 7–9).
We thank J. Cairns, J. Drake, E. Eisenstadt, M. Marinus, and F. Stahl for discussions and comments on this manuscript. Supported by NSF grant MCB-9213137 to P.L.F.
Note added in proof: After submission of this manuscript, we learned that others (24) had obtained similar results.