The Origin of Mutants Under Selection: How Natural Selection Mimics Mutagenesis (Adaptive Mutation)

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.     

Donor Strains of the Soft-Rot Bacterium Erwinia chrysanthemi and Conjugational Transfer of the Pectolytic Capacity

Donor strains of Erwinia chrysanthemi ICPB EC16, a member of the soft-rot (pectolytic) section of the enterobacterial genus Erwinia, were obtained by chromosomal integration of an F′lac⁺ plasmid originating from Escherichia coli. These stable donor strains, selected from an unstable F′lac⁺ heterogenote by repeated platings of single Lac⁺ colonies on lactose minimal agar, do not segregate (as does the parent F′lac⁺ heterogenote) into Lac⁻ or F⁻ clones, in either the presence or absence of acridine orange. One representative donor strain (from the 12 that have been selected) has been examined in more detail; it can transfer ade⁺, gal⁺, gtu⁺ (utilization of galacturonate), his⁺, lac⁺, leu⁺, lys⁺, mcu⁺ (multiple carbohydrate utilization), pat⁺ (production of polygalacturonic acid trans-eliminase), thr⁺, and trp⁺ in a polarized manner to appropriate recipient strains of E. chrysanthemi; the frequencies of ade⁺, leu⁺, and thr⁺ transfer were higher than those of the other markers tested to date. This donor strain transfers lac⁺ genes during a 6-h mating on membranes; most of the Lac⁺ recombinants are donors of chromosomal markers. The kinetics of entry as well as the frequencies of transfer of chromosomal markers indicate that thr⁺ and leu⁺ enter the recipient as proximal markers and that lac⁺ enters as a distal marker. Analysis of the recombinants demonstrates close linkage between thr and leu, ade and thr, his and pat, and his and trp loci. The results suggest that the integration of F′lac⁺ into the chromosome of E. chrysanthemi has occurred at a region adjacent to the leu-thr loci, and that the chromosome is transferred in the following sequence: origin----leu--thr--ade--lys--mcu--pat--his--trp--gal--gtu--lac--F. Plant-tissue maceration occurs in Pat⁺ recombinants and not in Pat⁻ recombinants, even though both form another pectolytic enzyme, hydrolytic polygalacturonase. This genetic evidence supports the idea that the E. chrysanthemi polygalacturonic acid trans-eliminase plays an essential role in bringing about plant-tissue maceration.     

Adaptive amplification and point mutation are independent mechanisms: evidence for various stress-inducible mutation mechanisms

“Adaptive mutation” denotes a collection of processes in which cells respond to growth-limiting environments by producing compensatory mutants that grow well, apparently violating fundamental principles of evolution. In a well-studied model, starvation of stationary-phase lac⁻ Escherichia coli cells on lactose medium induces Lac⁺ revertants at higher frequencies than predicted by usual mutation models. These revertants carry either a compensatory frameshift mutation or a greater than 20-fold amplification of the leaky lac allele. A crucial distinction between alternative hypotheses for the mechanisms of adaptive mutation hinges on whether these amplification and frameshift mutation events are distinct, or whether amplification is a molecular intermediate, producing an intermediate cell type, in colonies on a pathway to frameshift mutation. The latter model allows the evolutionarily conservative idea of increased mutations (per cell) without increased mutation rate (by virtue of extra gene copies per cell), whereas the former requires an increase in mutation rate, potentially accelerating evolution. To resolve these models, we probed early events leading to rare adaptive mutations and report several results that show that amplification is not the precursor to frameshift mutation but rather is an independent adaptive outcome. (i) Using new high-resolution selection methods and stringent analysis of all cells in very young (micro)colonies (500–10,000 cells), we find that most mutant colonies contain no detectable lac-amplified cells, in contrast with previous reports. (ii) Analysis of nascent colonies, as young as the two-cell stage, revealed mutant Lac⁺ cells with no lac-amplified cells present. (iii) Stringent colony-fate experiments show that microcolonies of lac-amplified cells grow to form visible colonies of lac-amplified, not mutant, cells. (iv) Mutant cells do not overgrow lac-amplified cells in microcolonies fast enough to mask the lac-amplified cells. (v) lac-amplified cells are not SOS-induced, as was proposed to explain elevated mutation in a sequential model. (vi) Amplification, and not frameshift mutation, requires DNA polymerase I, demonstrating that mutation is separable from amplification, and also illuminating the amplification mechanism. We conclude that amplification and mutation are independent outcomes of adaptive genetic change. We suggest that the availability of alternative pathways for genetic/evolutionary adaptation and clonal expansion under stress may be exploited during processes ranging from the evolution of drug resistance to cancer progression.     

Stabilization of Lactose Metabolism in Streptococcus lactis C2

The integration of the lactose plasmid from lactic streptococci into the host chromosome could stabilize this trait for dairy fermentations. Sixty lactose-positive (Lac⁺) transductants of lactose- and proteinase-negative (Lac⁻ Prt⁻) LM0220 were induced for temperature phage by UV irradiation or mitomycin C. Four of the transductants, designated KB18, KB21, KB54, and KB58, yielded lysates demonstrating less than one Lac⁺ transductant per 0.2 ml of phage lysate. Successive transferring in the presence of acriflavine did not yield Lac⁻ segregants from KB18, KB21, KB54, or KB58, whereas Streptococcus lactis C2 (parent culture) and three other Lac⁺ transductants showed 12 to 88% conversion from Lac⁺ to Lac⁻ within 6 to 10 repetitive transfers. When grown in continuous culture, KB21 did not show any Lac⁻ variants in 168 h, while S. lactis C2 had 96% conversion from Lac⁺ to Lac⁻ in 144 h. Agarose gel electrophoresis of plasmid DNA isolated from KB18, KB21, KB54, and KB58 revealed that the lactose plasmid, pLM2103, normally present in Lac⁺ transductants, was missing. This suggested integration of the transferred lactose plasmid into the chromosome. In contrast to phage lysates induced from S. lactis C2, which exhibited an exponential decrease in the number of Lac⁺ transductants after exposure to small doses of UV irradiation, the transduction frequency for lactose metabolism was stimulated by UV irradiation of lysates from KB58. The latter indicated chromosomal linkage for lac and that integration of the lactose genes plasmid into the chromosome had occurred.     

Carbohydrate Accumulation and Metabolism in Escherichia coli: Characteristics of the Reversions of ctr Mutations

The reversion behavior of pleiotropic carbohydrate mutants, previously designated as ctr, was studied. The mutants revert to complete restoration of the wild-type phenotype, as well as to a spectrum of partial wild-type phenotypes. Lac⁺ reversions were found in the lac region (11 min) and some Mal⁺ reversions occurred at malB (79 min), at a distance from the site of the ctr mutations (46 to 47 min). About one-third of Lac⁺ and Mal⁺ revertants were constitutive for uptake of their respective substrates, and one-third modified for inducibility. The remaining third were not distinguishable from wild type. Induction of a ctr mutation in a lac constitutive strain, either operator or repressor mutant, did not affect lactose metabolism. A polar-like ctr mutant, deficient in both enzyme I and heat-stable protein of the phosphoenolpyruvate-dependent phosphotransferase strain was also described. Partial revertants of ctr were still found to lack enzyme I.     

High rotational mobility of DNA in animal cells and its modulation by histone acetylation

Summary Several spontaneous Lac⁻ deletion derivatives of the β-galactosidase gene ofLactobacillus bulgaricus were analyzed for their phenotypic stability. We found that one of these mutants,lac139, carrying a deletion of 30 by within the gene, was able to revert to a Lac⁺ phenotype. Genetical analysis of revertants indicated that an internal region of 72 by was duplicated immediately next to the deletion site. The region involved in the duplication event is flanked by direct repeated sequences of 13 by in length. Both events, the deletion and the duplication, were mediated by the presence of such short direct repeats. Enzymatic studies of the purified proteins indicated identical kinetic parameters, but showed considerable instability of the revertant protein.     

Transduction of Lactose Metabolism by Streptococcus cremoris C3 Temperate Phage

Temperate phage was induced from Streptococcus cremoris C3 and morphologically characterized by high-resolution electron micrographic techniques. Interspecies genetic transfer of lactose-fermenting ability by the temperate phage was demonstrated, using two lactose-negative (Lac⁻) S. lactis strains as recipients. Plasmid transfer was confirmed by agarose gel electrophoresis. Transductant plasmid profiles were of three types—those containing no visible plasmid deoxyribonucleic acid, those possessing a 23-megadalton (Mdal) plasmid, and those containing a 23-Mdal plasmid and a 30-Mdal plasmid. A Lac⁺ transductant could serve as a donor of the lac determinants during solid-surface matings. These results add to previously published reports of inter- and intraspecies genetic transfer in dairy starter cultures.     

Recombination between an Flac and a mini-FKm plasmid deleted for an origin of replication

The mini-F plasmid specifying resistance to kanamycin (Km), pML31, contains an origin of replication at kilobase coordinate 42.6 in the F DNA sequences. In previous research we found that this origin could be deleted by recombinant DNA techniques without the loss of plasmid maintenance functions. In this report we show that the deleted plasmid, designated pMF21, has normal incompatibility properties and a recA⁺-dependent ability to form cointegrates with an Flac plasmid. By comparison, pML31 does not form cointegrates with the Flac plasmid at a detectable frequency. The frequency for spontaneous loss of the Lac⁺ phenotype in strains containing pMF21:Flac cointegrates resembles that of the Flac plasmid; however, in some Lac⁻ variants the Km^r phenotype is retained. Examination of the plasmid DNA in four of these Lac⁻Km^r clones revealed two with normal pMF21 plasmids and two with plasmids intermediate in size between pMF21 and the Flac.     

Adaptive mutation inEscherichia coli strain FC40

Mutations can arise in static populations of cells that are subjected to nonlethal selective pressure, a phenomenon that has been called ‘adaptive mutation’. This phenomenon has been extensively studied in FC40, a strain ofEscherichia coli that cannot metabolize lactose (Lac⁻) but that reverts to lactose utilization (Lac⁺) when lactose is its sole energy and carbon source. The adaptive Lac⁺ mutations arise by two mutational processes: a recombination-dependent process that is highly active on the episome carrying the Lac⁻ allele, and an unknown process that affects the whole genome. Most of the Lac⁺ mutations are due to the first process, which also produces nonselected mutations on the F′ episome. However, about 10% of the Lac⁺ mutations arise in a subpopulation of cells that experience a period of transient hypermutation. Although minor contributors to any one type of mutation, the hypermutators account for nearly all cases of multiple mutations. The evolutionary implications of these results are: (i) DNA synthesis associated with recombination may be an important source of spontaneous mutation, particularly in cells that are not actively growing; (ii) the efficient mutational mechanism that occurs on the episome could result in the horizontal transfer of new alleles among species that carry and exchange conjugal plasmids; and (iii) a subpopulation of transient hypermutators could be a source of multiple mutations that would allow for rapid adaptive evolution under adverse conditions.     

Multiple Pathways of Duplication Formation with and Without Recombination (RecA) in Salmonella enterica

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.     

Genetic and physical studies of restriction-deficient mutants of the Inc FIV plasmids R124 and R124/3

Summary R124 and R124/3 are R plasmids that carry the genes for two different restriction and modification systems. The phenotype of strains carrying either of these plasmids along with the F'lac ⁺ plasmid, is restriction-deficient (Res^-). The Res^- phenotype is not due to selection of preexisting mutants but rather to a complex mutational event caused by the F plasmid. Restriction-deficient mutants carry extensive deletions and other DNA rearrangements. Tn7 insertion is used to locate the restriction gene. Many of the Res^- mutants are genetically unstable and revert at exceptionally high frequencies. Reversion is accompanied by DNA rearrangements which result in a net gain of 9 kb of DNA. F derivates of F⁺ which do not cause restriction-deficiency but do cause deletion were used to distinguish between the DNA rearrangements associated with restriction-deficiency and those associated with deletion. From Res⁺ revertants of strains carrying F'lac ⁺ and R124 or R124/3 we have isolated F plasmids that now carry the genes for the R124 or R124/3 restriction and modification systems. It is suggested that interaction between part of the F plasmid and that segment of the R plasmid which controls the switch in Res-Mod specificity which has been observed (Glover et al. 1983) is responsible for the production of restriction-deficiency.     

pHG276: A multiple cloning site pBR322 copy number vector expressing a functional lacα peptide from the bacteriophage lambda PR promoter

The bacteriophage lambda early promoter P_R has been used to direct the synthesis of lacα in a plasmid containing the multiple cloning site of pUC8. The copy number of the plasmid is controlled by the rop(rom) gene and the plasmid incorporates the cI⁸⁵⁷ gene for temperature regulation of lacα expression. Isolation of recombinant derivatives with DNA inserts in the multiple cloning region can be identified by insertional inactivation of lacα and consequently, a Lac⁻ phenotype in a host carrying the M15 deletion of lac. The potential of pHG276 as a fully regulated expression vector is examined.     

Involvement of a Plasmid in Production of Ropiness (Mucoidness) in Milk Cultures by Streptococcus cremoris MS

Curing and genetic transfer experiments showed that lactose-fermenting ability (Lac⁺) and the ability to produce mucoidness in milk cultures (Muc⁺) in Streptococcus cremoris MS were coded on plasmids. The Lac⁺ phenotype was associated with a 75.8-megadalton plasmid, pSRQ2201. The Muc⁺ phenotype was associated with a 18.5-megadalton plasmid, pSRQ2202. The Lac plasmid, pSRQ2201, was first conjugatively transferred from S. cremoris MS to Lac⁻S. lactis ML-3/2.2. Later, the Muc plasmid, pSRQ2202, was conjugatively transferred from Lac⁻ Muc⁺S. cremoris MS04 to Lac⁺ nonmucoid S. lactis transconjugant ML-3/2.201. Subsequently, pSRQ2201 and pSRQ2202 were cotransferred from Lac⁺ Muc⁺S. lactis transconjugant ML-3/2.202 to Lac⁻, nonmucoid, malty S. lactis 4/4.2 and S. lactis subsp. diacetylactis SLA3.25. Transconjugants showing pSRQ2201 were Lac⁺; those containing pSRQ2202 were Muc⁺. With the transfer of pSRQ2202, the transconjugants S. lactis ML-3/2.202 and S. lactis subsp. diacetylactis SLA3.2501 not only acquired the Muc⁺ phenotype but also resistance to bacteriophages, which were lytic to the respective parent strains S. lactis ML-3/2.201 and S. lactis subsp. diacetylactis SLA3.25.     

LAC4 Is the Structural Gene for β-Galactosidase in KLUYVEROMYCES LACTIS

Using genetic and biochemical techniques, we have determined that β-galactosidase in the yeast Kluyveromyces lactis is coded by the LAC4 locus. The following data support this conclusion: (1) mutations in this locus result in levels of β-galactosidase activity 100-fold lower than levels in uninduced wild type and all other lac^- mutants; (2) three of five lac4 mutations are suppressible by an unlinked suppressor whose phenotype suggests that it codes for a nonsense suppressor tRNA; (3) a Lac⁺ revertant, bearing lac4–14 and this unlinked suppressor, has subnormal levels of β-galactosidase activity, and the K_m for hydrolysis of o-nitrophenyl-β, D-galactoside and the thermal stability of the enzyme are altered; (4) the level of β-galactosidase activity per cell is directly proportional to the number of copies of LAC4; (5) analysis of cell-free extracts of strains bearing mutations in LAC4 by two-dimensional acryl-amide gel electrophoresis shows that strains bearing lac4–23 and lac4–30 contain an inactive β-galactosidase whose subunit co-electrophoreses with the wild-type subunit, while no subunit or fragment of the subunit is observable in lac4–8, lac4–14 or lac4–29 mutants; (6) of all lac4 mutants, only those bearing lac4–23 or lac4–30 contain a protein that cross-reacts with anti-β-galactosidase antibody, a finding consistent with the previous result; and (7) β-galactosidase activity in several Lac⁺ revertants of strains carrying lac4–23 or lac4–30 has greatly decreased thermostability.     

The DNA gyrase of Escherichia coli participates in the formation of a spontaneous deletion by recA-independent recombination in vivo

Summary A system for detecting a spontaneous deletion in Escherichia coli was developed and the role of DNA gyrase in deletion formation was studied. A derivative of plac5, AM36, was isolated in which whole pBR322 DNA was inserted in the lacZ gene and 227 by of the lac gene duplicated at both sides of the pBR322 DNA. E. coli lac ^–strains lysogenized by AM36 had a Lac^– phenotype and segregated Lac^– revertants. Sequence analyses showed that the revertant was formed by a deletion that eliminated the inserted pBR322 DNA and one copy of the duplicated segments. The frequency of lac ^– revertant formation was independent of recA function, was increased by oxolinic acid, an inhibitor of DNA gyrase, but was not increased in a lysogen of a nalidixic acid-resistant derivative. The reversion frequencies of temperature sensitive mutants of gyrA gene are 10 to 100 times lower than that of the wild-type strain. These results indicate that the DNA gyrase of E. coli participated in the in vivo deletion formation resulting from the direct repeats.     

Construction of Streptococcus lactis subsp. lactis Strains with a Single Plasmid Associated with Mucoid Phenotype

Lactose-fermenting mucoid (Lac⁺ Muc⁺) variants of plasmid-free Streptococcus lactis subsp. lactis MG1614 were obtained by protoplast transformation with total plasmid DNA from Muc⁺S. lactis subsp. cremoris ARH87. By using plasmid DNA from these variants for further transformations followed by novobiocininduced plasmid curing, Lac⁻ Muc⁺ MG1614 strains containing only a single 30-megadalton plasmid could be constructed. This plasmid, designated pVS5, appeared to be associated with the Muc⁺ phenotype.     

Pathways of Genetic Adaptation: Multistep Origin of Mutants Under Selection Without Induced Mutagenesis in Salmonella enterica

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.     

Selection and timing of replication of plasmids R1drd-19 and F'lac in Escherichia coli.

The selection and timing of plasmid replication was studied in exponentially growing cultures of Escherichia coli K-12 carrying the plasmid R1drd-19 and E. coli strains B/r A and B/r F carrying the plasmid F′lac. In all cases plasmid replication was studied by analysis of covalently closed circular (CCC) DNA. The turnover time of replicating plasmid DNA into CCC-DNA was found to be less than 4 min. Density shift experiments (from ¹⁵NH₄⁺, D₂O to ¹⁴NH₄⁺, H₂O) showed that plasmids R1drd-19 and F′lac are selected randomly for replication. This means that one of the plasmid copies in a cell is selected and replicated. There is no further plasmid replication in the cell until all plasmid copies, including the newly formed ones, have the same probability of being selected for replication. The early kinetics of the appearance of light plasmid DNA after the density shift showed that the time interval between successive replications of plasmids R1drd-19 and F′lac is $\text{τ}\text{n}$, where τ is the generation time and n is the average number of plasmid replications per cell and cell cycle. In a second type of experiment, exponentially growing cells were separated into a series of size classes by low-speed centrifugation in sucrose step gradients. Replication of plasmids R1drd-19 and F′lac was equally frequent in all size classes. This result is in accordance with the results of the density shift experiment. It can therefore be concluded that replication of plasmids R1drd-19 and F′lac is evenly spread over the whole cell cycle, which means that one plasmid replication occurs every time the cell volume has increased by one initiation mass.     

Genetic Characterization of a Stable F' lac Plasmid

A mutant F′ plasmid has been isolated in a strain of Salmonella typhimurium harboring F_ts114lac. This mutant, designated FlacS, exhibits unique genetic stability in strains of S. typhimurium and Escherichia coli. It shows no thermolability and is lost at frequencies of 20 to 100 times less than the wild-type F′lac (F42) in the same genetic backgrounds. The FlacS is also insensitive to conventional plasmid curing agents, whereas both F_ts114lac and F42 are readily cured. The nature of the mutation(s) conferring stability to the FlacS is unclear, but plasmid linkage has been established. The high frequency of conjugal transfer of the FlacS and its behavior in recombination-deficient strains of S. typhimurium and E. coli argue against its stability being due to stable chromosomal integration. The FlacS is also capable of transferring chromosomal markers in S. typhimurium and E. coli mating systems. No major differences in chromosomal mobilization have been observed among F42, F_ts114lac, and FlacS donors of either genus.