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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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Relationships between mutation rates and physiologic conditions(a) Fold change in transversion (Tv), transition (Ts), and indel rate in response to ampicillin or norfloxacin according to MDS (for fluctuation assay results and raw substitution rates see Extended Data Fig. S9). (b) Fold change in mutation rate in a strain overexpressing catalase (KatG). (c) Fold change in mutation rate of mrcA in response to induction via IPTG promoter. Experiments are biological quadruplicates. Error bars are 95% CI.
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Figure 4: Relationships between mutation rates and physiologic conditions(a) Fold change in transversion (Tv), transition (Ts), and indel rate in response to ampicillin or norfloxacin according to MDS (for fluctuation assay results and raw substitution rates see Extended Data Fig. S9). (b) Fold change in mutation rate in a strain overexpressing catalase (KatG). (c) Fold change in mutation rate of mrcA in response to induction via IPTG promoter. Experiments are biological quadruplicates. Error bars are 95% CI.

Mentions: Exposing E. coli to sub-inhibitory doses of multiple classes of antibiotics increases the rate at which bacteria acquire resistance to rifampicin. Whether this increase is caused by nucleotide oxidation21, 22, downregulation of mismatch repair23, or an unrelated pathway24, has become a topic of immense interest. We investigated the effect of sub-inhibitory doses of ampicillin and norfloxacin—a beta lactam and fluoroquinolone respectively—on mutation rate using MDS of rpoB CDS and mrcA as well as detailed fluctuation assays16, 25 (Fig. 4A). Addition of ampicillin increased the rate of transition mutations in rpoB, a signature indicative of down-regulated mismatch repair3. In cells overexpressing catalase, basal mutation rate decreased by a factor of 8 (Fig. 4B), indicating that background oxidation contributes significantly to the basal mutation rate under non-stressed conditions. Addition of ampicillin during catalase overexpression did not increase this low rate (Fig. 4B). Overexpression of a catalase with inactivating point mutation H106Y did not confer similar mutagenic protection (Extended Data Fig. 7). These results together support a model in which ampicillin causes oxidative stress21, which acts upstream of downregulation of mismatch repair23 to increase mutation rate. Consistently, cells grown in anaerobic conditions did not display an increase in transition rate when challenged with ampicillin (Extended Data Fig. 8A). The same was true in aerobic conditions if mismatch repair gene mutS was knocked out (Extended Data Fig. 8B. See SI for further discussion).


Rates and Mechanisms of Bacterial Mutagenesis from Maximum-Depth Sequencing
Relationships between mutation rates and physiologic conditions(a) Fold change in transversion (Tv), transition (Ts), and indel rate in response to ampicillin or norfloxacin according to MDS (for fluctuation assay results and raw substitution rates see Extended Data Fig. S9). (b) Fold change in mutation rate in a strain overexpressing catalase (KatG). (c) Fold change in mutation rate of mrcA in response to induction via IPTG promoter. Experiments are biological quadruplicates. Error bars are 95% CI.
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Related In: Results  -  Collection

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Figure 4: Relationships between mutation rates and physiologic conditions(a) Fold change in transversion (Tv), transition (Ts), and indel rate in response to ampicillin or norfloxacin according to MDS (for fluctuation assay results and raw substitution rates see Extended Data Fig. S9). (b) Fold change in mutation rate in a strain overexpressing catalase (KatG). (c) Fold change in mutation rate of mrcA in response to induction via IPTG promoter. Experiments are biological quadruplicates. Error bars are 95% CI.
Mentions: Exposing E. coli to sub-inhibitory doses of multiple classes of antibiotics increases the rate at which bacteria acquire resistance to rifampicin. Whether this increase is caused by nucleotide oxidation21, 22, downregulation of mismatch repair23, or an unrelated pathway24, has become a topic of immense interest. We investigated the effect of sub-inhibitory doses of ampicillin and norfloxacin—a beta lactam and fluoroquinolone respectively—on mutation rate using MDS of rpoB CDS and mrcA as well as detailed fluctuation assays16, 25 (Fig. 4A). Addition of ampicillin increased the rate of transition mutations in rpoB, a signature indicative of down-regulated mismatch repair3. In cells overexpressing catalase, basal mutation rate decreased by a factor of 8 (Fig. 4B), indicating that background oxidation contributes significantly to the basal mutation rate under non-stressed conditions. Addition of ampicillin during catalase overexpression did not increase this low rate (Fig. 4B). Overexpression of a catalase with inactivating point mutation H106Y did not confer similar mutagenic protection (Extended Data Fig. 7). These results together support a model in which ampicillin causes oxidative stress21, which acts upstream of downregulation of mismatch repair23 to increase mutation rate. Consistently, cells grown in anaerobic conditions did not display an increase in transition rate when challenged with ampicillin (Extended Data Fig. 8A). The same was true in aerobic conditions if mismatch repair gene mutS was knocked out (Extended Data Fig. 8B. See SI for further discussion).

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