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Significance Genetic heterogeneity is a significant driver of antibiotic resistance in bacteria. Understanding copy number (CN) heterogeneity is important because minority subclones with increased CN can drive resistance during antibiotic exposure, but revert and escape detection during clinical susceptibility testing. Despite its clinical relevance, CN variation has eluded quantification at single-molecule resolution. Here, we report nanopore sequencing of arylomycin-resistant mutants carrying tandem repeats ranging in size from 4.8 to 50.0 kb and encompassing the arylomycin target gene lepB. Reads spanning individual repeat arrays show vast differences in CN, underscoring the importance of amplifications in driving the emergence of genetic heterogeneity. This is a direct observation of cell-to-cell CN differences in an antibiotic-resistant bacterial population., 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 from single 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.

Multidrug-resistant bacteria are spreading at alarming rates, and despite extensive efforts no new class of antibiotic with activity against Gram-negative bacteria has been approved in over fifty years. Natural products and their derivatives have a key role in combating Gram-negative pathogens. Here we report chemical optimization of the arylomycins-a class of natural products with weak activity and limited spectrum-to obtain G0775, a molecule with potent, broad-spectrum activity against Gram-negative bacteria. G0775 inhibits the essential bacterial type I signal peptidase, a new antibiotic target, through an unprecedented molecular mechanism. It circumvents existing antibiotic resistance mechanisms and retains activity against contemporary multidrug-resistant Gram-negative clinical isolates in vitro and in several in vivo infection models. These findings demonstrate that optimized arylomycin analogues such as G0775 could translate into new therapies to address the growing threat of multidrug-resistant Gram-negative infections.

Phenotypic heterogeneity displayed by a clonal bacterial population permits a small fraction of cells to survive prolonged exposure to antibiotics. Although first described over 60 y ago, the molecular mechanisms underlying this behavior, termed persistence, remain largely unknown. To systematically explore the genetic basis of persistence, we selected a library of transposon-mutagenized Escherichia coli cells for survival to multiple rounds of lethal ampicillin exposure. Application of microarray-based genetic footprinting revealed a large number of loci that drastically elevate persistence frequency through null mutations and domain disruptions. In one case, the C-terminal disruption of methionyl-tRNA synthetase (MetG) results in a 10,000-fold higher persistence frequency than wild type. We discovered a mechanism by which null mutations in transketolase A ( tktA ) and glycerol-3-phosphate (G3P) dehydrogenase ( glpD ) increase persistence through metabolic flux alterations that increase intracellular levels of the growth-inhibitory metabolite methylglyoxal. Systematic double-mutant analyses revealed the genetic network context in which such persistent mutants function. Our findings reveal a large mutational target size for increasing persistence frequency, which has fundamental implications for the emergence of antibiotic tolerance in the clinical setting.