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Rates and Mechanisms of Bacterial Mutagenesis from Maximum-Depth Sequencing

View Article: PubMed Central - PubMed

ABSTRACT

In 1943, Luria and Delbrück used a phage resistance assay to establish spontaneous mutation as a driving force of microbial diversity1. Mutation rates are still studied using such assays, but these can only examine the small minority of mutations conferring survival in a particular condition. Newer approaches, such as long-term evolution followed by whole-genome sequencing 2, 3, may be skewed by mutational “hot” or “cold” spots 3, 4. Both approaches are affected by numerous caveats 5, 6, 7 (see Supplemental Information). We devise a method, Maximum-Depth Sequencing (MDS), to detect extremely rare variants in a population of cells through error-corrected, high-throughput sequencing. We directly measure locus-specific mutation rates in E. coli and show that they vary across the genome by at least an order of magnitude. Our data suggest that certain types of nucleotide misincorporation occur 104-fold more frequently than the basal rate of mutations, but are repaired in vivo. Our data also suggest specific mechanisms of antibiotic-induced mutagenesis, including downregulation of mismatch repair via oxidative stress; transcription-replication conflicts; and in the case of fluoroquinolones, direct damage to DNA.

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Mutational spectra and contextsa) Substitution frequencies of all ROIs after ~120 generations of growth. Note that values are not normalized for the number of generations and are thus true frequencies, rather than mutation rates. b) Mutation frequencies are shown in context of their 5’ (A, C, G, or T on the x axis) and 3’ (A, C, G, or T on the y axis) neighbors. c) The relative relationship between in vivo substitution frequencies and expected errors due to sequencing and PCR (from in vitro DNA assays) is poorly described by a linear approximation (R2 = 0.27). Furthermore, the recovered frequency from in vivo substitutions (R=3) is higher than the rate of error (equivalent frequencies would be represented by the dotted line), even with the relatively relaxed read-cutoff threshold of R=2 (The sequencing + PCR error with an R=3 cutoff is approximately an order of magnitude lower). Templates are rpoB CDS and mrcA ROIs.
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Figure 9: Mutational spectra and contextsa) Substitution frequencies of all ROIs after ~120 generations of growth. Note that values are not normalized for the number of generations and are thus true frequencies, rather than mutation rates. b) Mutation frequencies are shown in context of their 5’ (A, C, G, or T on the x axis) and 3’ (A, C, G, or T on the y axis) neighbors. c) The relative relationship between in vivo substitution frequencies and expected errors due to sequencing and PCR (from in vitro DNA assays) is poorly described by a linear approximation (R2 = 0.27). Furthermore, the recovered frequency from in vivo substitutions (R=3) is higher than the rate of error (equivalent frequencies would be represented by the dotted line), even with the relatively relaxed read-cutoff threshold of R=2 (The sequencing + PCR error with an R=3 cutoff is approximately an order of magnitude lower). Templates are rpoB CDS and mrcA ROIs.

Mentions: The mutational spectrum from MDS matches that found in long-term sequencing experiments, with transition mutations favored over transversions (Fig. 3A, Extended Data Fig. 4, 5a). We also note an unexpected high frequency of C->A substitutions. These appear not to be lasting mutations, as complementary G->T substitutions emerged with less than 0.1-fold frequency. A similar effect was found to a lesser extent for G->A/C->T substitutions. Increasing R did not significantly reduce these high substitution frequencies (Fig. 3B, SI: Model of Damaged Base Pairs), suggesting that the majority of in vivo C->A substitutions are not due to damaged nucleotides. We found that in vitro templates synthesized with 8-oxoG resulted in low C->A substitution rates (Extended Data Fig. 3C), and treatment of in vivo DNA with fpg did not change the observed substitution frequency (Extended Data Fig. 3C), further confirming that these C->A substitutions are unlikely due to 8-oxoG. It is possible that As, or rAs, are misincorporated into the genome at C sites in vivo. We found that neighboring Cs are predictive of a higher frequency of C->A substitutions, suggesting that these transient substitutions cluster spatially along the genome, unlike polymerase or sequencing errors (Fig. 3C, Extended Data Fig. 3b, 4, 5b).


Rates and Mechanisms of Bacterial Mutagenesis from Maximum-Depth Sequencing
Mutational spectra and contextsa) Substitution frequencies of all ROIs after ~120 generations of growth. Note that values are not normalized for the number of generations and are thus true frequencies, rather than mutation rates. b) Mutation frequencies are shown in context of their 5’ (A, C, G, or T on the x axis) and 3’ (A, C, G, or T on the y axis) neighbors. c) The relative relationship between in vivo substitution frequencies and expected errors due to sequencing and PCR (from in vitro DNA assays) is poorly described by a linear approximation (R2 = 0.27). Furthermore, the recovered frequency from in vivo substitutions (R=3) is higher than the rate of error (equivalent frequencies would be represented by the dotted line), even with the relatively relaxed read-cutoff threshold of R=2 (The sequencing + PCR error with an R=3 cutoff is approximately an order of magnitude lower). Templates are rpoB CDS and mrcA ROIs.
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Related In: Results  -  Collection

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Figure 9: Mutational spectra and contextsa) Substitution frequencies of all ROIs after ~120 generations of growth. Note that values are not normalized for the number of generations and are thus true frequencies, rather than mutation rates. b) Mutation frequencies are shown in context of their 5’ (A, C, G, or T on the x axis) and 3’ (A, C, G, or T on the y axis) neighbors. c) The relative relationship between in vivo substitution frequencies and expected errors due to sequencing and PCR (from in vitro DNA assays) is poorly described by a linear approximation (R2 = 0.27). Furthermore, the recovered frequency from in vivo substitutions (R=3) is higher than the rate of error (equivalent frequencies would be represented by the dotted line), even with the relatively relaxed read-cutoff threshold of R=2 (The sequencing + PCR error with an R=3 cutoff is approximately an order of magnitude lower). Templates are rpoB CDS and mrcA ROIs.
Mentions: The mutational spectrum from MDS matches that found in long-term sequencing experiments, with transition mutations favored over transversions (Fig. 3A, Extended Data Fig. 4, 5a). We also note an unexpected high frequency of C->A substitutions. These appear not to be lasting mutations, as complementary G->T substitutions emerged with less than 0.1-fold frequency. A similar effect was found to a lesser extent for G->A/C->T substitutions. Increasing R did not significantly reduce these high substitution frequencies (Fig. 3B, SI: Model of Damaged Base Pairs), suggesting that the majority of in vivo C->A substitutions are not due to damaged nucleotides. We found that in vitro templates synthesized with 8-oxoG resulted in low C->A substitution rates (Extended Data Fig. 3C), and treatment of in vivo DNA with fpg did not change the observed substitution frequency (Extended Data Fig. 3C), further confirming that these C->A substitutions are unlikely due to 8-oxoG. It is possible that As, or rAs, are misincorporated into the genome at C sites in vivo. We found that neighboring Cs are predictive of a higher frequency of C->A substitutions, suggesting that these transient substitutions cluster spatially along the genome, unlike polymerase or sequencing errors (Fig. 3C, Extended Data Fig. 3b, 4, 5b).

View Article: PubMed Central - PubMed

ABSTRACT

In 1943, Luria and Delbrück used a phage resistance assay to establish spontaneous mutation as a driving force of microbial diversity1. Mutation rates are still studied using such assays, but these can only examine the small minority of mutations conferring survival in a particular condition. Newer approaches, such as long-term evolution followed by whole-genome sequencing 2, 3, may be skewed by mutational “hot” or “cold” spots 3, 4. Both approaches are affected by numerous caveats 5, 6, 7 (see Supplemental Information). We devise a method, Maximum-Depth Sequencing (MDS), to detect extremely rare variants in a population of cells through error-corrected, high-throughput sequencing. We directly measure locus-specific mutation rates in E. coli and show that they vary across the genome by at least an order of magnitude. Our data suggest that certain types of nucleotide misincorporation occur 104-fold more frequently than the basal rate of mutations, but are repaired in vivo. Our data also suggest specific mechanisms of antibiotic-induced mutagenesis, including downregulation of mismatch repair via oxidative stress; transcription-replication conflicts; and in the case of fluoroquinolones, direct damage to DNA.

No MeSH data available.


Related in: MedlinePlus