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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.


Comparing substitution rate and indel rate across 5 ROIs reveals a positive correlation (Pearson correlation coefficient = 0.76).
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Figure 10: Comparing substitution rate and indel rate across 5 ROIs reveals a positive correlation (Pearson correlation coefficient = 0.76).

Mentions: We calculated short (≤12bp) indel rates in mrcA, rpoB UTR, ropZ, and cspE ROIs (Fig. 2B). Indel rate varies widely by position and size. As might be expected19, 100% of the observed 1bp indels occur at a site adjacent to a homopolymer. The frequency of 1-bp indels also increases with homopolymer length, suggesting why cspE, with an 8-bp T homopolymer, has the highest 1-bp indel rate. Longer indels are not localized to homopolymers and are positively correlated with substitution rates across all ROIs (Extended Data Fig. 6), supporting previous work suggesting that indels and substitutions spatially cluster in comparisons of genomes from divergent bacterial species20. In all ROIs deletions were detected at >10-fold frequency of insertions.


Rates and Mechanisms of Bacterial Mutagenesis from Maximum-Depth Sequencing
Comparing substitution rate and indel rate across 5 ROIs reveals a positive correlation (Pearson correlation coefficient = 0.76).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 10: Comparing substitution rate and indel rate across 5 ROIs reveals a positive correlation (Pearson correlation coefficient = 0.76).
Mentions: We calculated short (≤12bp) indel rates in mrcA, rpoB UTR, ropZ, and cspE ROIs (Fig. 2B). Indel rate varies widely by position and size. As might be expected19, 100% of the observed 1bp indels occur at a site adjacent to a homopolymer. The frequency of 1-bp indels also increases with homopolymer length, suggesting why cspE, with an 8-bp T homopolymer, has the highest 1-bp indel rate. Longer indels are not localized to homopolymers and are positively correlated with substitution rates across all ROIs (Extended Data Fig. 6), supporting previous work suggesting that indels and substitutions spatially cluster in comparisons of genomes from divergent bacterial species20. In all ROIs deletions were detected at >10-fold frequency of insertions.

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.