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Rates and Mechanisms of Bacterial Mutagenesis from Maximum-Depth Sequencing

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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.


Substitution spectra(a) Frequency of base substitutions recovered in our sequencing protocol at t=20 generations and t=120 generations in rpoB CDS. Values are not normalized by number of generations and thus are true frequencies, not mutation rates. Experiments are biological quadruplicates. Error bars are 95% CI upper bound. (b) The high frequency of C->A substitutions is consistent even as R increases. If these substitutions were polymerase errors due to damaged nucleotides, they should decline with increasing R faster than the line representing a model in which the polymerase makes C->A errors with 50% frequency for a subpopulation of DNA molecules (see Supplemental Information: Model of Damaged Base Pairs). (c) C->A substitutions in vivo cluster in nucleotides with at least 2 neighboring Cs within a 2bp radius, unlike polymerase errors (*=p<0.01 by t-test).
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Figure 3: Substitution spectra(a) Frequency of base substitutions recovered in our sequencing protocol at t=20 generations and t=120 generations in rpoB CDS. Values are not normalized by number of generations and thus are true frequencies, not mutation rates. Experiments are biological quadruplicates. Error bars are 95% CI upper bound. (b) The high frequency of C->A substitutions is consistent even as R increases. If these substitutions were polymerase errors due to damaged nucleotides, they should decline with increasing R faster than the line representing a model in which the polymerase makes C->A errors with 50% frequency for a subpopulation of DNA molecules (see Supplemental Information: Model of Damaged Base Pairs). (c) C->A substitutions in vivo cluster in nucleotides with at least 2 neighboring Cs within a 2bp radius, unlike polymerase errors (*=p<0.01 by t-test).

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
Substitution spectra(a) Frequency of base substitutions recovered in our sequencing protocol at t=20 generations and t=120 generations in rpoB CDS. Values are not normalized by number of generations and thus are true frequencies, not mutation rates. Experiments are biological quadruplicates. Error bars are 95% CI upper bound. (b) The high frequency of C->A substitutions is consistent even as R increases. If these substitutions were polymerase errors due to damaged nucleotides, they should decline with increasing R faster than the line representing a model in which the polymerase makes C->A errors with 50% frequency for a subpopulation of DNA molecules (see Supplemental Information: Model of Damaged Base Pairs). (c) C->A substitutions in vivo cluster in nucleotides with at least 2 neighboring Cs within a 2bp radius, unlike polymerase errors (*=p<0.01 by t-test).
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Related In: Results  -  Collection

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Figure 3: Substitution spectra(a) Frequency of base substitutions recovered in our sequencing protocol at t=20 generations and t=120 generations in rpoB CDS. Values are not normalized by number of generations and thus are true frequencies, not mutation rates. Experiments are biological quadruplicates. Error bars are 95% CI upper bound. (b) The high frequency of C->A substitutions is consistent even as R increases. If these substitutions were polymerase errors due to damaged nucleotides, they should decline with increasing R faster than the line representing a model in which the polymerase makes C->A errors with 50% frequency for a subpopulation of DNA molecules (see Supplemental Information: Model of Damaged Base Pairs). (c) C->A substitutions in vivo cluster in nucleotides with at least 2 neighboring Cs within a 2bp radius, unlike polymerase errors (*=p<0.01 by t-test).
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 Delbr&uuml;ck 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 &ldquo;hot&rdquo; or &ldquo;cold&rdquo; 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.