John R. Roth

The Escherichia coli system of Cairns and Foster employs a lac frameshift mutation that reverts rarely (10⁻⁹/cell/division) during unrestricted growth. However, when 10⁸ cells are plated on lactose medium, the nongrowing lawn produces ∼50 Lac⁺ revertant colonies that accumulate linearly with time over 5 days. Revertants carry very few associated mutations. This behavior has been attributed to an evolved mechanism (“adaptive mutation” or “stress-induced mutagenesis”) that responds to starvation by preferentially creating mutations that improve growth. We describe an alternative model, “selective inbreeding,” in which natural selection acts during intercellular transfer of the plasmid that carries the mutant lac allele and the dinB gene for an error-prone polymerase. Revertant genome sequences show that the plasmid is more intensely mutagenized than the chromosome. Revertants vary widely in their number of plasmid and chromosomal mutations. Plasmid mutations are distributed evenly, but chromosomal mutations are focused near the replication origin. Rare, heavily mutagenized, revertants have acquired a plasmid tra mutation that eliminates conjugation ability. These findings support the new model, in which revertants are initiated by rare pre-existing cells (10⁵) with many copies of the F’lac plasmid. These cells divide under selection, producing daughters that mate. Recombination between donor and recipient plasmids initiates rolling-circle plasmid over-replication, causing a mutagenic elevation of DinB level. A lac⁺ reversion event starts chromosome replication and mutagenesis by accumulated DinB. After reversion, plasmid transfer moves the revertant lac⁺ allele into an unmutagenized cell, and away from associated mutations. Thus, natural selection explains why mutagenesis appears stress-induced and directed.

In the Cairns–Foster adaptive mutation system, a +1 lac frameshift mutant of Escherichia coli is plated on lactose medium, where the nondividing population gives rise to Lac⁺ revertant colonies during a week under selection. Reversion requires the mutant lac allele to be located on a conjugative F′lac plasmid that also encodes the error-prone DNA polymerase, DinB. Rare plated cells with multiple copies of the mutant F′lac plasmid initiate the clones that develop into revertants under selection. These initiator cells arise before plating, and their extra lac copies allow them to divide on lactose and produce identical F′lac-bearing daughter cells that can mate with each other. DNA breaks can form during plasmid transfer and their recombinational repair can initiate rolling-circle replication of the recipient plasmid. This replication is mutagenic because the amplified plasmid encodes the error-prone DinB polymerase. A new model proposes that Lac⁺ revertants arise during mutagenic over-replication of the F′lac plasmid under selection. This mutagenesis is focused on the plasmid because the cell chromosome replicates very little. The outer membrane protein OmpA is essential for reversion under selection. OmpA helps cells conserve energy and may stabilize the long-term mating pairs that produce revertants.

To test whether growth limitation induces mutations, Cairns and Foster constructed an Escherichia coli strain whose mutant lac allele provides 1-2% of normal ability to use lactose. This strain cannot grow on lactose, but produces similar to 50 Lac(+) revertant colonies per 108 plated cells over 5 days. About 80% of revertants carry a stable lac(+) mutation made by the error-prone DinB polymerase, which may be induced during growth limitation; 10% of Lac(+) revertants are stable but form without DinB; and the remaining 10% grow by amplifying their mutant lac allele and are unstably Lac(+). Induced DinB mutagenesis has been explained in two ways: (1) upregulation of dinB expression in nongrowing cells ("stress-induced mutagenesis") or (2) selected local overreplication of the lac and dinB(+) genes on lactose medium (selected amplification) in cells that are not dividing. Transcription of dinB is necessary but not sufficient for mutagenesis. Evidence is presented that DinB enhances reversion only when encoded somewhere on the F'lac plasmid that carries the mutant lac gene. A new model will propose that rare preexisting cells (1 in a 1000) have similar to 10 copies of the F'lac plasmid, providing them with enough energy to divide, mate, and overreplicate their F'lac plasmid under selective conditions. In these clones, repeated replication of F'lac in nondividing cells directs opportunities for lac reversion and increases the copy number of the dinB(+) gene. Amplification of dinB(+) increases the error rate of replication and increases the number of lac(+) revertants. Thus, reversion is enhanced in nondividing cells not by stress-induced mutagenesis, but by selected coamplification of the dinB and lac genes, both of which happen to lie on the F'lac plasmid.

Van Hofwegen et al. demonstrated that Escherichia coli rapidly evolves the ability to use citrate when long selective periods are provided (D. J. Van Hofwegen, C. J. Hovde, and S. A. Minnich, J Bacteriol 198:1022–1034, 2016, http://dx.doi.org/10.1128/JB.00831-15 ). This contrasts with the extreme delay (15 years of daily transfers) seen in the long-term evolution experiments of Lenski and coworkers. Their idea of “historical contingency” may require reinterpretation. Rapid evolution seems to involve selection for duplications of the whole cit locus that are too unstable to contribute when selection is provided in short pulses.

Selection detects mutants but does not cause mutations. Contrary to this dictum, Cairns and Foster plated a leaky lac mutant of Escherichia coli on lactose medium and saw revertant (Lac⁺) colonies accumulate with time above a nongrowing lawn. This result suggested that bacteria might mutagenize their own genome when growth is blocked. However, this conclusion is suspect in the light of recent evidence that revertant colonies are initiated by preexisting cells with multiple copies the conjugative F′lac plasmid, which carries the lac mutation. Some plated cells have multiple copies of the simple F′lac plasmid. This provides sufficient LacZ activity to support plasmid replication but not cell division. In nongrowing cells, repeated plasmid replication increases the likelihood of a reversion event. Reversion to lac⁺ triggers exponential cell growth leading to a stable Lac⁺ revertant colony. In 10% of these plated cells, the high-copy plasmid includes an internal tandem lac duplication, which provides even more LacZ activity—sufficient to support slow growth and formation of an unstable Lac⁺ colony. Cells with multiple copies of the F′lac plasmid have an increased mutation rate, because the plasmid encodes the error-prone (mutagenic) DNA polymerase, DinB. Without DinB, unstable and stable Lac⁺ revertant types form in equal numbers and both types arise with no mutagenesis. Amplification and selection are central to behavior of the Cairns–Foster system, whereas mutagenesis is a system-specific side effect or artifact caused by coamplification of dinB with lac. Study of this system has revealed several broadly applicable principles. In all populations, gene duplications are frequent stable genetic polymorphisms, common near-neutral mutant alleles can gain a positive phenotype when amplified under selection, and natural selection can operate without cell division when variability is generated by overreplication of local genome subregions.

Changes in gene copy number are among the most frequent mutational events in all genomes and were among the mutations for which a physical basis was first known. Yet mechanisms of gene duplication remain uncertain because formation rates are difficult to measure and mechanisms may vary with position in a genome. Duplications are compared here to deletions, which seem formally similar but can arise at very different rates by distinct mechanisms. Methods of assessing duplication rates and dependencies are described with several proposed formation mechanisms. Emphasis is placed on duplications formed in extensively studied experimental situations. Duplications studied in microbes are compared with those observed in metazoan cells, specifically those in genomes of cancer cells. Duplications, and especially their derived amplifications, are suggested to form by multistep processes often under positive selection for increased copy number.

The origin of mutations under selection has been intensively studied using the Cairns-Foster system, in which cells of an Escherichia colilac mutant are plated on lactose and give rise to 100 Lac⁺ revertants over several days. These revertants have been attributed variously to stress-induced mutagenesis of nongrowing cells or to selective improvement of preexisting weakly Lac⁺ cells with no mutagenesis. Most revertant colonies (90%) contain stably Lac⁺ cells, while others (10%) contain cells with an unstable amplification of the leaky mutant lac allele. Evidence is presented that both stable and unstable Lac⁺ revertant colonies are initiated by preexisting cells with multiple copies of the F′lac plasmid, which carries the mutant lac allele. The tetracycline analog anhydrotetracycline (AnTc) inhibits growth of cells with multiple copies of the tetA gene. Populations with tetA on their F′lac plasmid include rare cells with an elevated plasmid copy number and multiple copies of both the tetA and lac genes. Pregrowth of such populations with AnTc reduces the number of cells with multiple F′lac copies and consequently the number of Lac⁺ colonies appearing under selection. Revertant yield is restored rapidly by a few generations of growth without AnTc. We suggest that preexisting cells with multiple F′lac copies divide very little under selection but have enough energy to replicate their F′lac plasmids repeatedly until reversion initiates a stable Lac⁺ colony. Preexisting cells whose high-copy plasmid includes an internal lac duplication grow under selection and produce an unstable Lac⁺ colony. In this model, all revertant colonies are initiated by preexisting cells and cannot be stress induced.

Microcompartments are loose protein cages that encapsulate enzymes for particular bacterial metabolic pathways. These structures are thought to retain and perhaps concentrate pools of small, uncharged intermediates that would otherwise diffuse from the cell. In Salmonella enterica , a microcompartment encloses enzymes for ethanolamine catabolism. The cage has been thought to retain the volatile intermediate acetaldehyde but allow diffusion of the much larger cofactors NAD and coenzyme A (CoA). Genetic tests support an alternative idea that the microcompartment contains and recycles private pools of the large cofactors NAD and CoA. Two central enzymes convert ethanolamine to acetaldehyde (EutBC) and then to acetyl-CoA (EutE). Two seemingly peripheral redundant enzymes encoded by the eut operon proved to be essential for ethanolamine utilization, when subjected to sufficiently stringent tests. These are EutD (acetyl-CoA to acetyl phosphate) and EutG (acetaldehyde to ethanol). Obligatory recycling of cofactors couples the three reactions and drives acetaldehyde consumption. Loss and toxic effects of acetaldehyde are minimized by accelerating its consumption. In a eutD mutant, acetyl-CoA cannot escape the compartment but is released by mutations that disrupt the structure. The model predicts that EutBC (ethanolamine-ammonia lyase) lies outside the compartment, using external coenzyme B ₁₂ and injecting its product, acetaldehyde, into the lumen, where it is degraded by the EutE, EutD, and EutG enzymes using private pools of CoA and NAD. The compartment appears to allow free diffusion of the intermediates ethanol and acetyl-PO ₄ but (to our great surprise) restricts diffusion of acetaldehyde.

In several bacterial systems, mutant cell populations plated on growth-restricting medium give rise to revertant colonies that accumulate over several days. One model suggests that nongrowing parent cells mutagenize their own genome and thereby create beneficial mutations (stress-induced mutagenesis). By this model, the first-order induction of new mutations in a nongrowing parent cell population leads to the delayed accumulation of visible colonies. In an alternative model (selection only), selective conditions allow preexisting small-effect mutants to initiate clones that grow and give rise to faster-growing mutants. By the selection-only model, the delay in appearance of revertant colonies reflects (1) the time required for initial clones to reach a size sufficient to allow the second mutation plus (2) the time required for growth of the improved subclone. We previously characterized a system in which revertant colonies accumulate slowly and contain cells with two mutations, one formed before plating and one after. This left open the question of whether mutation rates increase under selection. Here we measure the unselected formation rate and the growth contribution of each mutant type. When these parameters are used in a graphic model of revertant colony development, they demonstrate that no increase in mutation rate is required to explain the number and delayed appearance of two of the revertant types.

Duplications are often attributed to “unequal recombination” between separated, directly repeated sequence elements (>100 bp), events that leave a recombinant element at the duplication junction. However, in the bacterial chromosome, duplications form at high rates (10−3–10−5/cell/division) even without recombination (RecA). Here we describe 1800 spontaneous lac duplications trapped nonselectively on the low-copy F′128 plasmid, where lac is flanked by direct repeats of the transposable element IS3 (1258 bp) and by numerous quasipalindromic REP elements (30 bp). Duplications form at a high rate (10−4/cell/division) that is reduced only about 11-fold in the absence of RecA. With and without RecA, most duplications arise by recombination between IS3 elements (97%). Formation of these duplications is stimulated by IS3 transposase (Tnp) and plasmid transfer functions (TraI). Three duplication pathways are proposed. First, plasmid dimers form at a high rate stimulated by RecA and are then modified by deletions between IS3 elements (resolution) that leave a monomeric plasmid with an IS3-flanked lac duplication. Second, without RecA, duplications occur by single-strand annealing of DNA ends generated in different sister chromosomes after transposase nicks DNA near participating IS3 elements. The absence of RecA may stimulate annealing by allowing chromosome breaks to persist. Third, a minority of lac duplications (3%) have short (0–36 bp) junction sequences (SJ), some of which are located within REP elements. These duplication types form without RecA, Tnp, or Tra by a pathway in which the palindromic junctions of a tandem inversion duplication (TID) may stimulate deletions that leave the final duplication.