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Evidence for Retromutagenesis as a Mechanism for Adaptive Mutation in Escherichia coli.

Morreall J, Kim A, Liu Y, Degtyareva N, Weiss B, Doetsch PW - PLoS Genet. (2015)

Bottom Line: When residual lacZ transcription was blocked beforehand by catabolite repression, both strands were mutated about equally, but if revertants were selected immediately after nitrous acid exposure, transcribed-strand mutations predominated among the revertants, implicating retromutagenesis as the mechanism.This result was not affected by gene orientation.Retromutagenesis is apt to be a universal method of evolutionary adaptation, which enables the emergence of new mutants from mutations acquired during counterselection rather than beforehand, and it may have roles in processes as diverse as the development of antibiotic resistance and neoplasia.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, United States of America; Graduate Program in Genetics and Molecular Biology, Emory University School of Medicine, Atlanta, Georgia, United States of America.

ABSTRACT
Adaptive mutation refers to the continuous outgrowth of new mutants from a non-dividing cell population during selection, in apparent violation of the neo-Darwinian principle that mutation precedes selection. One explanation is that of retromutagenesis, in which a DNA lesion causes a transcriptional mutation that yields a mutant protein, allowing escape from selection. This enables a round of DNA replication that establishes heritability. Because the model requires that gene expression precedes DNA replication, it predicts that during selection, new mutants will arise from damage only to the transcribed DNA strand. As a test, we used a lacZ amber mutant of Escherichia coli that can revert by nitrous acid-induced deamination of adenine residues on either strand of the TAG stop codon, each causing different DNA mutations. When stationary-phase, mutagenized cells were grown in rich broth before being plated on lactose-selective media, only non-transcribed strand mutations appeared in the revertants. This result was consistent with the known high sensitivity to deamination of the single-stranded DNA in a transcription bubble, and it provided an important control because it demonstrated that the genetic system we would use to detect transcribed-strand mutations could also detect a bias toward the non-transcribed strand. When residual lacZ transcription was blocked beforehand by catabolite repression, both strands were mutated about equally, but if revertants were selected immediately after nitrous acid exposure, transcribed-strand mutations predominated among the revertants, implicating retromutagenesis as the mechanism. This result was not affected by gene orientation. Retromutagenesis is apt to be a universal method of evolutionary adaptation, which enables the emergence of new mutants from mutations acquired during counterselection rather than beforehand, and it may have roles in processes as diverse as the development of antibiotic resistance and neoplasia.

No MeSH data available.


Related in: MedlinePlus

Experimental scheme.A stationary phase, saturated culture of lac mutants in LB broth + glucose was divided into many samples, washed by centrifigation, starved by incubation in 10 mM MgSO4, resuspended in acidic buffer, and incubated with or without added NaNO2. Each sample was divided in half. One part was grown in LB broth before being plated on lactose-minimal agar. The other was plated directly on the lactose-minimal agar. After 48 h, Lac+ revertant colonies were picked for DNA sequencing.
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pgen.1005477.g003: Experimental scheme.A stationary phase, saturated culture of lac mutants in LB broth + glucose was divided into many samples, washed by centrifigation, starved by incubation in 10 mM MgSO4, resuspended in acidic buffer, and incubated with or without added NaNO2. Each sample was divided in half. One part was grown in LB broth before being plated on lactose-minimal agar. The other was plated directly on the lactose-minimal agar. After 48 h, Lac+ revertant colonies were picked for DNA sequencing.

Mentions: The strand specificity for mutagenesis was determined as detailed under Methods and outlined in Fig 3. Briefly, the amber mutant tester strains were grown to saturation with glucose, washed, and starved. Multiple samples were treated with NaNO2 in an acidic buffer, with a survival of 29 to 33%. After the treatment was stopped with a neutral buffer, each sample was divided in two. One portion was spread immediately on the selective medium; the other was grown in LB broth before selection (Fig 3). If reversion occurred entirely by retromutagenesis, direct plating would reveal that only TS mutations were selected, whereas after intermediate growth, both TS and NTS mutations would appear among the revertants.


Evidence for Retromutagenesis as a Mechanism for Adaptive Mutation in Escherichia coli.

Morreall J, Kim A, Liu Y, Degtyareva N, Weiss B, Doetsch PW - PLoS Genet. (2015)

Experimental scheme.A stationary phase, saturated culture of lac mutants in LB broth + glucose was divided into many samples, washed by centrifigation, starved by incubation in 10 mM MgSO4, resuspended in acidic buffer, and incubated with or without added NaNO2. Each sample was divided in half. One part was grown in LB broth before being plated on lactose-minimal agar. The other was plated directly on the lactose-minimal agar. After 48 h, Lac+ revertant colonies were picked for DNA sequencing.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4548950&req=5

pgen.1005477.g003: Experimental scheme.A stationary phase, saturated culture of lac mutants in LB broth + glucose was divided into many samples, washed by centrifigation, starved by incubation in 10 mM MgSO4, resuspended in acidic buffer, and incubated with or without added NaNO2. Each sample was divided in half. One part was grown in LB broth before being plated on lactose-minimal agar. The other was plated directly on the lactose-minimal agar. After 48 h, Lac+ revertant colonies were picked for DNA sequencing.
Mentions: The strand specificity for mutagenesis was determined as detailed under Methods and outlined in Fig 3. Briefly, the amber mutant tester strains were grown to saturation with glucose, washed, and starved. Multiple samples were treated with NaNO2 in an acidic buffer, with a survival of 29 to 33%. After the treatment was stopped with a neutral buffer, each sample was divided in two. One portion was spread immediately on the selective medium; the other was grown in LB broth before selection (Fig 3). If reversion occurred entirely by retromutagenesis, direct plating would reveal that only TS mutations were selected, whereas after intermediate growth, both TS and NTS mutations would appear among the revertants.

Bottom Line: When residual lacZ transcription was blocked beforehand by catabolite repression, both strands were mutated about equally, but if revertants were selected immediately after nitrous acid exposure, transcribed-strand mutations predominated among the revertants, implicating retromutagenesis as the mechanism.This result was not affected by gene orientation.Retromutagenesis is apt to be a universal method of evolutionary adaptation, which enables the emergence of new mutants from mutations acquired during counterselection rather than beforehand, and it may have roles in processes as diverse as the development of antibiotic resistance and neoplasia.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, United States of America; Graduate Program in Genetics and Molecular Biology, Emory University School of Medicine, Atlanta, Georgia, United States of America.

ABSTRACT
Adaptive mutation refers to the continuous outgrowth of new mutants from a non-dividing cell population during selection, in apparent violation of the neo-Darwinian principle that mutation precedes selection. One explanation is that of retromutagenesis, in which a DNA lesion causes a transcriptional mutation that yields a mutant protein, allowing escape from selection. This enables a round of DNA replication that establishes heritability. Because the model requires that gene expression precedes DNA replication, it predicts that during selection, new mutants will arise from damage only to the transcribed DNA strand. As a test, we used a lacZ amber mutant of Escherichia coli that can revert by nitrous acid-induced deamination of adenine residues on either strand of the TAG stop codon, each causing different DNA mutations. When stationary-phase, mutagenized cells were grown in rich broth before being plated on lactose-selective media, only non-transcribed strand mutations appeared in the revertants. This result was consistent with the known high sensitivity to deamination of the single-stranded DNA in a transcription bubble, and it provided an important control because it demonstrated that the genetic system we would use to detect transcribed-strand mutations could also detect a bias toward the non-transcribed strand. When residual lacZ transcription was blocked beforehand by catabolite repression, both strands were mutated about equally, but if revertants were selected immediately after nitrous acid exposure, transcribed-strand mutations predominated among the revertants, implicating retromutagenesis as the mechanism. This result was not affected by gene orientation. Retromutagenesis is apt to be a universal method of evolutionary adaptation, which enables the emergence of new mutants from mutations acquired during counterselection rather than beforehand, and it may have roles in processes as diverse as the development of antibiotic resistance and neoplasia.

No MeSH data available.


Related in: MedlinePlus