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Our investigations show that nonlethal concentrations of nitric oxide (NO) abrogate the antibiotic activity of β-lactam antibiotics against Burkholderia pseudomallei, Escherichia coli and nontyphoidal Salmonella enterica serovar Typhimurium. NO protects B. pseudomallei already exposed to β-lactams, suggesting that this diatomic radical tolerizes bacteria against the antimicrobial activity of this important class of antibiotics. The concentrations of NO that elicit antibiotic tolerance repress consumption of oxygen (O2), while stimulating hydrogen peroxide (H2O2) synthesis. Transposon insertions in genes encoding cytochrome c oxidase-related functions and molybdenum assimilation confer B. pseudomallei a selective advantage against the antimicrobial activity of the β-lactam antibiotic imipenem. Cumulatively, these data support a model by which NO induces antibiotic tolerance through the inhibition of the electron transport chain, rather than by potentiating antioxidant defenses as previously proposed. Accordingly, pharmacological inhibition of terminal oxidases and nitrate reductases tolerizes aerobic and anaerobic bacteria to β-lactams. The degree of NO-induced β-lactam antibiotic tolerance seems to be inversely proportional to the proton motive force (PMF), and thus the dissipation of ΔH+ and ΔΨ electrochemical gradients of the PMF prevents β-lactam-mediated killing. According to this model, NO generated by IFNγ-primed macrophages protects intracellular Salmonella against imipenem. On the other hand, sublethal concentrations of imipenem potentiate the killing of B. pseudomallei by NO generated enzymatically from IFNγ-primed macrophages. Our investigations indicate that NO modulates the antimicrobial activity of β-lactam antibiotics. Author Summary: β-lactam drugs that inhibit peptidoglycan biosynthesis are often used in the treatment of bacterial infections, including melioidosis. Independent of their antibiotic activity, we have noted that submicromolar concentrations of β-lactams potentiate the killing of intracellular B. pseudomallei supported by NO generated by IFNγ-primed macrophages. The production of NO can nonetheless be a double-edged sword, as indicated by our observations that sublethal concentrations of nitric oxide (NO), a diatomic radical produced by phylogenetically diverse organisms to regulate neurotransmission, vascular tone and host defense, tolerize B. pseudomallei, nontyphoidal Salmonella and E. coli against the antimicrobial activity of β-lactams. Accordingly, NO produced in the inflammatory response of macrophages protects nontyphoidal Salmonella against β-lactam antibiotics. NO mediates bacterial tolerance to β-lactam antibiotics by inhibiting the electrochemical gradient supported by terminal cytochrome oxidases of the respiratory chain, rather than by decreasing oxidative stress as previously thought. [ABSTRACT FROM AUTHOR]