Different characteristics distinguish early versus late arising adaptive mutations in Escherichia coli FC40

Powell, Sandra; Wartell, Roger M.

doi:10.1016/s0027-5107(00)00149-4

Cited by 31 publications

(24 citation statements)

References 27 publications

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“…About 2% of the FC40 Lac ϩ clones isolated on days 5 to 7 are composed mostly of cells that have amplified the Lac Ϫ allele (20,25). If incubation is continued for 10 days, up to 60% of the newly arising Lac ϩ colonies consist of amplifiers (41,56). The late appearance of these colonies reflects the fact that they are slower to develop than colonies composed of true Lac ϩ revertants (41).…”

Section: Amplificationmentioning

confidence: 99%

Adaptive Mutation in Escherichia coli

Foster¹

2000

Cold Spring Harbor Symposia on Quantitative Biology

102

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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 ...

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Section: Amplificationmentioning

confidence: 99%

Adaptive Mutation in Escherichia coli

Foster¹

2000

Cold Spring Harbor Symposia on Quantitative Biology

102

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“…Amplification is involved in the reversion of the episomal lac allele, although its true role is debated (4,10,11,17). Since adaptive reversion of the chromosomal lac allele in FC691 is not dependent on RecA and therefore does not involve amplification (6), it appears that Red may induce an entirely new pathway for adaptive change on the chromosome.…”

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confidence: 99%

Phage λ Red-Mediated Adaptive Mutation

2002

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Replacement of the recBCD genes of Escherichia coli with the red recombination genes of bacteriophage lambda results in a strain in which adaptive mutation occurs at an elevated frequency. Like RecBCDdependent adaptive mutation, Red-mediated adaptive mutation is dependent upon recA and ruvABC functions.Adaptive mutation is a process by which an organism under nonlethal selective pressure produces mutations that relieve the selective pressure (see reference 5 for a review). It is illustrated particularly well by Escherichia coli strain FC40, a strain bearing an FЈ episome carrying a lac allele with a frameshift mutation that makes it phenotypically Lac Ϫ . When FC40 is plated on lactose-minimal medium, Lac ϩ revertants appear at a high rate over several days, largely in the absence of cell growth, death, or turnover (2, 4). The formation of these adaptive mutations, unlike mutations that occur during growth under nonselective conditions, depends on the RecA-RecBCD recombination pathway (2,8).E. coli strains in which the RecBCD function is replaced by the Red recombination system of bacteriophage exhibit a hyperrecombination phenotype (13,14). The possibility that the activity of the cell's recombination system might be a ratelimiting factor in the production of adaptive revertants led us to test whether Red ϩ bacteria might produce such revertants at an elevated rate. We report here that they do.Bacterial strains. P1 transduction was used to replace the recC-ptr-recB-recD gene cluster in FC40 and FC691 with a sequence bearing both P tac -gam-bet-exo and the cat gene. (The P tac -gam-bet-exo sequence is designated red.) Recombinants were selected for chloramphenicol resistance, producing strains TP694 and TP730. Strains TP705 and TP732 were made by transforming TP694 and TP730 with linear DNA resulting from the digestion of plasmid pTP822 (16). Recombinants, in which ⌬recBCD::red-cat was replaced by ⌬recBCD::red-paecI822, were selected for immunity to phage and screened for sensitivity to chloramphenicol and kanamycin. These and other strains employed in this study are described in Table 1.Media. The lysogeny broth (LB) used contained, per liter, 10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, and 1 ml of 1 M NaOH. It was solidified with 15 g of agar per liter and supplemented as necessary with 25 g of tetracycline/ml, 20 g of chloramphenicol/ml, 0.1 mM isopropylthiogalactopyranoside (IPTG), or 80 g of 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside (X-Gal)/ml.M9 salt solution contained, per liter, 6.3 g of Na 2 HPO 4 , 3.0 g of KH 2 PO 4 , 0.5 g of NaCl, and 1.0 g of NH 4 Cl. M9 glycerolminimal medium consisted of M9 salt solution plus 1 mM MgSO 4 , 5 g of thiamine/ml, 0.1 mM CaCl 2 , 10 g of gelatin/ ml, and 1 mg of glycerol/ml. M9 lactose-minimal agar consisted of M9 salt solution plus 1 mM MgSO 4 , 5 g of thiamine/ml, 1 mg of lactose/ml, and 15 mg of agar/ml. Measurement of stationary-phase reversion to Lac؉ . The strains to be tested were scraped from frozen cultures in storage vials and streaked on LB ...

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“…The relative viability was monitored as described previously (23). Lac ϩ colonies originate either by a Ϫ1-bp frameshift point mutation (15,48) or by the amplification of the leaky lac allele to multiple copies (24,43). Because amplification accounts for a small portion of the Lac ϩ colonies arising on day 5 and earlier (24), we have not corrected for levels of amplification.…”

Section: Methodsmentioning

confidence: 99%

“…Starvation in stationary phase without lactose produces a similar effect (42). The Lac reversions occur either by compensatory frameshift (point) mutations (15,48) or by the amplification of the weakly functional lac gene to 20 to 50 copies (24,43). Stress-induced point mutagenesis in lac (33,34), ampD (41), and tet (42), as well as stress-induced lac amplification (34), require the RpoS-controlled general stress response.…”

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Separate DNA Pol II- and Pol IV-Dependent Pathways of Stress-Induced Mutation during Double-Strand-Break Repair in Escherichia coli Are Controlled by RpoS

Frisch¹,

Su²,

Thornton³

et al. 2010

J Bacteriol

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Previous work showed that about 85% of stress-induced mutations associated with DNA double-strand break repair in carbon-starved Escherichia coli result from error-prone DNA polymerase IV (Pol IV) (DinB) and that the mutagenesis is controlled by the RpoS stress response, which upregulates dinB. We report that the remaining mutagenesis requires high-fidelity Pol II, and that this component also requires RpoS. The results identify a second DNA polymerase contributing to stress-induced mutagenesis and show that RpoS promotes mutagenesis by more than the simple upregulation of dinB.Stress-induced mutagenesis is a collection of mechanisms observed in bacterial, yeast, and human cells in which mutation pathways are activated in response to adverse conditions, such as starvation or antibiotic stresses, under the control of stress responses (18). The coupling of mutagenesis to stress responses generates genetic diversity upon which natural selection can act specifically when cells are maladapted to their environment, i.e., are stressed. These mechanisms are potentially important models for mutagenesis that drives pathogenhost adaptation (5, 44), antibiotic resistance (8,9,18,31,35), cancer progression, and resistance (3).Perhaps the most well-characterized mechanism of stressinduced mutagenesis is mutagenesis associated with DNA double-strand break (DSB) repair in carbon-starved Escherichia coli cells, as characterized in the E. coli Lac assay (7) and related assays. In the Lac assay, cells with an FЈ-borne lac ϩ1-bp frameshift allele are starved on lactose medium on which they accumulate Lac ϩ reversion mutations (7), chromosomal tet frameshift (6), or ampD base substitution and frameshift mutations (41). Starvation in stationary phase without lactose produces a similar effect (42). The Lac reversions occur either by compensatory frameshift (point) mutations (15,48) or by the amplification of the weakly functional lac gene to 20 to 50 copies (24, 43). Stress-induced point mutagenesis in lac (33, 34), ampD (41), and tet (42), as well as stress-induced lac amplification (34), require the RpoS-controlled general stress response. The point mutagenesis mechanism is addressed here.The point mutagenesis mechanism appears to be a switch from high-fidelity to error-prone DSB repair under stress, and it is controlled by the SOS DNA damage and RpoS stress responses. Point mutagenesis requires the induction of the SOS (36), RpoS (33,34), and E -controlled extracytoplasmic unfolded protein (20) stress responses, the proteins of DSB repair by homologous recombination (16,22,23,28), either the F-encoded TraI single-strand endonuclease (42) or a DSB delivered near lac by I-SceI endonuclease expressed in vivo (42), and the DinB error-prone DNA polymerase IV (Pol IV) (13, 37), which SOS (10, 30) and RpoS (33) upregulate transcriptionally. The sole role of the SOS response in point mutagenesis is the upregulation of Pol IV (17). The mutations occur in acts of DSB repair that become mutagenic under the influence of the SOS and RpoS respon...

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Different characteristics distinguish early versus late arising adaptive mutations in Escherichia coli FC40

Cited by 31 publications

References 27 publications

Adaptive Mutation in Escherichia coli

Adaptive Mutation in Escherichia coli

Phage λ Red-Mediated Adaptive Mutation

Separate DNA Pol II- and Pol IV-Dependent Pathways of Stress-Induced Mutation during Double-Strand-Break Repair in Escherichia coli Are Controlled by RpoS

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