Defects in Efflux (), β-Lactamase (), and Lipopolysaccharide Transport () Genes Mediate Antibiotic Hypersusceptibility of Strain Z61

Antibiotic hypersensitive bacterial mutants (e.g., ) are used to investigate intrinsic resistance and are exploited in antibacterial discovery to track weak antibacterial activity of novel inhibitor compounds. Z61 is one such drug-hypersusceptible strain generated by chemical mutagenesis, although the genetic basis for hypersusceptibility is not fully understood. Genome sequencing of Z61 revealed nonsynonymous single-nucleotide polymorphisms in 153 genes relative to its parent strain, and three candidate mutations (in , , and ) predicted to mediate hypersusceptibility were characterized. The contribution of these mutations was confirmed by genomic restoration of the wild-type sequences, individually or in combination, in the Z61 background. Introduction of the mutation or genetic inactivation of and genes alone or together in the parent strain recapitulated drug sensitivities. This showed that disruption of (which encodes a major outer membrane efflux pump channel) increased susceptibility to pump substrate antibiotics, that inactivation of the inducible β-lactamase gene contributed to β-lactam susceptibility, and that mutation of the lipopolysaccharide transporter gene strongly altered the outer membrane permeability barrier, causing susceptibility to large antibiotics such as rifampin and also to β-lactams.