Journal Article

Single-molecule nanopore sequencing reveals extreme target copy number heterogeneity in arylomycin-resistant mutants

DuPai, Cory D., Durinck, Steffen, Skippington, Elizabeth, Smith, Peter A., Girgis, Hany S., Guillory, Joseph, Kaminker, Joshua, Liang, Yuxin, Lund, Jessica, Reeder, Janina

2021

Overview

Tandem gene amplification is a frequent and dynamic source of antibiotic resistance in bacteria. Ongoing expansions and contractions of repeat arrays during population growth are expected to manifest as cell-to-cell differences in copy number (CN). As a result, a clonal bacterial culture could comprise subpopulations of cells with different levels of antibiotic sensitivity that result from variable gene dosage. Despite the high potential for misclassification of heterogenous cell populations as either antibiotic-susceptible or fully resistant in clinical settings, and the concomitant risk of inappropriate treatment, CN distribution among cells has defied analysis. Here, we use the MinION single-molecule nanopore sequencer to uncover CN heterogeneity in clonal populations of Escherichia coli and Acinetobacter baumannii grown fromsingle cells isolated while selecting for resistance to an optimized arylomycin, a member of a recently discovered class of Gram-negative antibiotic. We found that gene amplification of the arylomycin target, bacterial type I signal peptidase LepB, is a mechanism of unstable arylomycin resistance and demonstrate in E. coli that amplification instability is independent of RecA. This instability drives the emergence of a nonuniform distribution of lepB CN among cells with a range of 1 to at least 50 copies of lepB identified in a single clonal population. In sum, this remarkable heterogeneity, and the evolutionary plasticity it fuels, illustrates how gene amplification can enable bacterial populations to respond rapidly to novel antibiotics. This study establishes a rationale for further nanopore-sequencing studies of heterogeneous cell populations to uncover CN variability at single-molecule resolution.

• Publisher: National Academy of Sciences
• Subject: Acinetobacter baumannii - genetics / Amplification / Anti-Bacterial Agents - pharmacology / Antibiotic resistance / Antibiotics / Bacteria / Biological Sciences / Cell culture / Copy number / DNA Copy Number Variations / DNA-Binding Proteins - metabolism / Drug Resistance, Microbial - genetics / E coli / Escherichia coli - genetics / Escherichia coli Proteins - metabolism / Evolution / Gene amplification / Gene Amplification - drug effects / Gene dosage / Genetic Heterogeneity / Heterogeneity / High-Throughput Nucleotide Sequencing / Membrane Proteins - genetics / Microbial Sensitivity Tests / Mutation / Nanopore Sequencing - instrumentation / Nanopore Sequencing - methods / Peptidases / Peptides, Cyclic - genetics / Population growth / Population studies / Porosity / Rec A Recombinases - metabolism / RecA protein / Resistant mutant / Serine Endopeptidases - genetics / Signal peptidase / Subpopulations