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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.

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Substitution rates and indel frequencies(a) Comparison of mutation rates calculated from fluctuation assays (FA) using either rifampicin (Rif) or nalidixic acid (Nal), long-term evolution (LTE), and mutation accumulation (MA). Rates calculated using MDS are boxed. All error bars are 95% CI. Note that number of generations is calculated according to population doubling time in Ref. 2 and 3 (see SI: Generation Time Models). (b) Frequency of indel mutations recovered at t=120 generations. Values are normalized for possible indel lengths considered in each category. Experiments are biological quadruplicates.
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Figure 2: Substitution rates and indel frequencies(a) Comparison of mutation rates calculated from fluctuation assays (FA) using either rifampicin (Rif) or nalidixic acid (Nal), long-term evolution (LTE), and mutation accumulation (MA). Rates calculated using MDS are boxed. All error bars are 95% CI. Note that number of generations is calculated according to population doubling time in Ref. 2 and 3 (see SI: Generation Time Models). (b) Frequency of indel mutations recovered at t=120 generations. Values are normalized for possible indel lengths considered in each category. Experiments are biological quadruplicates.

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
Substitution rates and indel frequencies(a) Comparison of mutation rates calculated from fluctuation assays (FA) using either rifampicin (Rif) or nalidixic acid (Nal), long-term evolution (LTE), and mutation accumulation (MA). Rates calculated using MDS are boxed. All error bars are 95% CI. Note that number of generations is calculated according to population doubling time in Ref. 2 and 3 (see SI: Generation Time Models). (b) Frequency of indel mutations recovered at t=120 generations. Values are normalized for possible indel lengths considered in each category. Experiments are biological quadruplicates.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4940094&req=5

Figure 2: Substitution rates and indel frequencies(a) Comparison of mutation rates calculated from fluctuation assays (FA) using either rifampicin (Rif) or nalidixic acid (Nal), long-term evolution (LTE), and mutation accumulation (MA). Rates calculated using MDS are boxed. All error bars are 95% CI. Note that number of generations is calculated according to population doubling time in Ref. 2 and 3 (see SI: Generation Time Models). (b) Frequency of indel mutations recovered at t=120 generations. Values are normalized for possible indel lengths considered in each category. Experiments are biological quadruplicates.
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