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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.


Related in: MedlinePlus

Rates of rpoB and mrcA substitutions in the presence of antibiotics as calculated by MDS. *=Grown separately and prepared with Phusion rather than Q5. Although not shown, we note that only in-frame (3×) indels were observed in rpoB in fluctuation assays, as expected since frameshift indels would be deleterious. These increased in frequency by a factor of 10 on addition of norfloxacin.
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Figure 13: Rates of rpoB and mrcA substitutions in the presence of antibiotics as calculated by MDS. *=Grown separately and prepared with Phusion rather than Q5. Although not shown, we note that only in-frame (3×) indels were observed in rpoB in fluctuation assays, as expected since frameshift indels would be deleterious. These increased in frequency by a factor of 10 on addition of norfloxacin.


Rates and Mechanisms of Bacterial Mutagenesis from Maximum-Depth Sequencing
Rates of rpoB and mrcA substitutions in the presence of antibiotics as calculated by MDS. *=Grown separately and prepared with Phusion rather than Q5. Although not shown, we note that only in-frame (3×) indels were observed in rpoB in fluctuation assays, as expected since frameshift indels would be deleterious. These increased in frequency by a factor of 10 on addition of norfloxacin.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4940094&req=5

Figure 13: Rates of rpoB and mrcA substitutions in the presence of antibiotics as calculated by MDS. *=Grown separately and prepared with Phusion rather than Q5. Although not shown, we note that only in-frame (3×) indels were observed in rpoB in fluctuation assays, as expected since frameshift indels would be deleterious. These increased in frequency by a factor of 10 on addition of norfloxacin.

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