Infections with Pseudomonas aeruginosa have become a real concern among hospital-acquired infections, especially in cystic fibrosis patients and immunocompromised individuals. Control of the pathogen is challenging due to antibiotic resistance.

Sodium-translocating NADH:quinone oxidoreductase (Na+-NQR) functions as a unique redox-driven sodium pump, generating membrane potential, which is related to aminoglycoside antibiotic resistance. However, whether it modulates other metabolisms to confer antibiotic resistance is unknown. The present study showed that loss of nqrA or nqrF led to differential metabolomes with elevated resistance to aminoglycoside antibiotics. Decreased alanine, aspartate, and glutamate metabolism and depressed abundance of alanine were characterized as the most impacted pathway and crucial biomarker, respectively. Further data showed that higher viability was detected in ΔnqrA and ΔnqrF mutant strains than their parent strain ATCC 33787 in the presence of gentamicin but recovered by exogenous l-alanine. It proceeds by the following events. The loss of nqrA or nqrF led to the decrease of membrane potential, ATPase activity, and then ATP and cyclic AMP (cAMP), which reduced the cAMP/CRP (cAMP receptor protein) complex. The reduced cAMP/CRP complex promoted l-alanine catabolism and inhibited l-alanine anabolism, causing reduced levels of alanine. Reduced alanine affected the expression of antiporter families Atp and Mnh genes. Our results suggest a novel mechanism by which the Na+-NQR system regulates antibiotic resistance via l-alanine metabolism in a cAMP/CRP complex-dependent manner. IMPORTANCE The Na+-NQR complex functions as a unique redox-driven sodium pump, generating membrane potential directly. However, whether it mediates generation of membrane potential indirectly is unknown. The present study shows that the Na+-NQR complex impacts membrane potential through other antiporter families Atp and Mnh. It proceeds by ATP and then cAMP/CRP regulon, which inhibits l-alanine catabolism and promotes l-alanine anabolism. When the Na+-NQR complex is reduced as in antibiotic-resistant bacteria, l-alanine is depressed, which is related to the antibiotic resistance phenotypes. However, exogenous l-alanine reverts the phenotype and promotes antibiotic-mediated killing. These findings suggest a novel mechanism by which the Na+-NQR system regulates antibiotic resistance via l-alanine metabolism in a cAMP/CRP complex-dependent manner.

The present study explored the cooperative effect of both alanine (Ala) and gentamicin (Gent) on metabolic mechanisms by which exogenous Ala potentiates Gent to kill antibiotic-resistant Vibrio alginolyticus. To test this, GC-MS-based metabolomics was used to characterize Ala-, Gent-and both-induced metabolic profiles, identifying nitric oxide (NO) production pathway as the most key clue to understand metabolic mechanisms. Gent, Ala and both led to low, lower and lowest activity of total nitric oxide synthase (tNOS) and level of NO, respectively. NOS promoter L-arginine and inhibitor N G -Monomethyl-L-arginine inhibited and promoted the killing, respectively, with the elevation and decrease of NOS activity and NO level. The present study further showed that CysJ is the enzyme-producing NO in V. alginolyticus. These results indicate that the cooperative effect of Ala and Gent causes the lowest NO, which plays a key role in Ala-potentiated Gent-mediated killing.