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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.

No MeSH data available.


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(a) Empirically, average substitution frequency (with high frequency substitutions such as C>A excluded) stabilizes as R increases. Note substitution frequencies are not normalized by number of generations. (b) Empirical sequencing C>A error rate at C>A mutational hotspots with neighboring Cs (same as those in Fig. 3c) vs. all other positions. (c) C>A substitution frequencies when 10% 8-oxoG is synthetically added to in vitro DNA and in fpg-treated samples. Frequencies are reported from ROI positions with potential 8-oxoG incorporations as described in template “rpoB_reverse_complement_8-oxo-dG.” Frequencies are reported at R=2 level. For R>2, no C>A substitutions were found in 72,646 in vitro template sites. Data represent biological triplicates. Error bars are standard deviation.
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Figure 7: (a) Empirically, average substitution frequency (with high frequency substitutions such as C>A excluded) stabilizes as R increases. Note substitution frequencies are not normalized by number of generations. (b) Empirical sequencing C>A error rate at C>A mutational hotspots with neighboring Cs (same as those in Fig. 3c) vs. all other positions. (c) C>A substitution frequencies when 10% 8-oxoG is synthetically added to in vitro DNA and in fpg-treated samples. Frequencies are reported from ROI positions with potential 8-oxoG incorporations as described in template “rpoB_reverse_complement_8-oxo-dG.” Frequencies are reported at R=2 level. For R>2, no C>A substitutions were found in 72,646 in vitro template sites. Data represent biological triplicates. Error bars are standard deviation.

Mentions: Mutation rates in E. coli have been reported from 0.2×10−10 to 5×10−10 nucleotides/generation3, 16, 17. Our calculated rate of mutation in rpoB CDS using synonymous substitutions is 4.1×10−10 nucleotides/generation, comparable to the rate obtained in 17 and at least one long-term evolution experiment using MG16552. Yet it is also higher than rates calculated by fluctuation assay and long-term evolution on other strains (Fig. 2A, Extended Data Fig. 3). We performed fluctuation assays and recovered a similar spectrum and low rate of mutation to others using such approaches16. It is likely that the higher rate of mutation in rpoB obtained with MDS indicates a rate uninfluenced by negative selection, phenotypic lag, or imperfect plating efficiency5.


Rates and Mechanisms of Bacterial Mutagenesis from Maximum-Depth Sequencing
(a) Empirically, average substitution frequency (with high frequency substitutions such as C>A excluded) stabilizes as R increases. Note substitution frequencies are not normalized by number of generations. (b) Empirical sequencing C>A error rate at C>A mutational hotspots with neighboring Cs (same as those in Fig. 3c) vs. all other positions. (c) C>A substitution frequencies when 10% 8-oxoG is synthetically added to in vitro DNA and in fpg-treated samples. Frequencies are reported from ROI positions with potential 8-oxoG incorporations as described in template “rpoB_reverse_complement_8-oxo-dG.” Frequencies are reported at R=2 level. For R>2, no C>A substitutions were found in 72,646 in vitro template sites. Data represent biological triplicates. Error bars are standard deviation.
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Related In: Results  -  Collection

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Figure 7: (a) Empirically, average substitution frequency (with high frequency substitutions such as C>A excluded) stabilizes as R increases. Note substitution frequencies are not normalized by number of generations. (b) Empirical sequencing C>A error rate at C>A mutational hotspots with neighboring Cs (same as those in Fig. 3c) vs. all other positions. (c) C>A substitution frequencies when 10% 8-oxoG is synthetically added to in vitro DNA and in fpg-treated samples. Frequencies are reported from ROI positions with potential 8-oxoG incorporations as described in template “rpoB_reverse_complement_8-oxo-dG.” Frequencies are reported at R=2 level. For R>2, no C>A substitutions were found in 72,646 in vitro template sites. Data represent biological triplicates. Error bars are standard deviation.
Mentions: Mutation rates in E. coli have been reported from 0.2×10−10 to 5×10−10 nucleotides/generation3, 16, 17. Our calculated rate of mutation in rpoB CDS using synonymous substitutions is 4.1×10−10 nucleotides/generation, comparable to the rate obtained in 17 and at least one long-term evolution experiment using MG16552. Yet it is also higher than rates calculated by fluctuation assay and long-term evolution on other strains (Fig. 2A, Extended Data Fig. 3). We performed fluctuation assays and recovered a similar spectrum and low rate of mutation to others using such approaches16. It is likely that the higher rate of mutation in rpoB obtained with MDS indicates a rate uninfluenced by negative selection, phenotypic lag, or imperfect plating efficiency5.

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