Shewanella oneidensis employs both β-lactam resistance and tolerance controlled by the PghKR system

ABSTRACT Antibiotic tolerance enables bacterial survival during antibiotic exposure and can promote the evolution of resistance, thereby threatening therapeutic efficacy. However, the molecular mechanisms underlying tolerance and its relationship with resistance remain incompletely understood. In this study, we show that a class D β-lactamase-deficient strain of Shewanella oneidensis (∆ blaA ) is highly susceptible to β-lactams, but retains the ability to survive lethal antibiotic concentrations. Under β-lactam stress, ∆ blaA cells form reversible spheroplasts that revert to rod-shaped morphology and resume growth upon antibiotic removal, confirming a tolerance phenotype. Genetic analysis revealed that this tolerance depends on the peptidoglycan damage-responsive two-component system PghKR, which positively regulates the marine sortase target protein SO2195. Mechanistically, SO2195 preserves cell envelope integrity, thereby preventing spheroplast swelling and lysis under cell wall stress. SO2195 homologs are widely distributed across multiple bacterial classes and are consistently located adjacent to a sortase gene, suggesting a conserved functional module. The PghKR-mediated regulatory pathway also governs tolerance to other cell wall-acting antibiotics, such as D-cycloserine. Together with our previous findings that PghKR controls blaA -mediated β-lactam resistance, these results establish PghKR as a central regulatory hub that coordinately controls both resistance and tolerance, allowing bacteria to dynamically balance distinct survival strategies under bactericidal antibiotic stress. IMPORTANCE Antibiotic treatment failure is attributed not only to resistance, where bacteria grow in the presence of drugs, but also to tolerance, defined as the ability to survive bactericidal antibiotics without a change in minimum inhibitory concentration (MIC) and to resume growth upon drug removal. Whether a single bacterial population can concurrently employ and coordinate both strategies remains unclear. Such a scenario could challenge widely used combination therapies, such as those combining β-lactams with β-lactamase inhibitors. Here, using the intrinsically β-lactam-resistant bacterium Shewanella oneidensis , we show that bacteria can indeed employ both resistance and tolerance, and that both are controlled by a single regulatory system. Remarkably, even when primary β-lactamase-mediated resistance is abolished by genetic deletion, a dedicated tolerance pathway sustains bacterial survival. These findings reveal an integrated bacterial defense strategy and highlight the need to target both resistance and tolerance to overcome antibiotic treatment failure.