Abstract
Benzoic acid (BA) is a widely used weak organic acid preservative with antimicrobial activity, yet the metabolic basis of its antibacterial action and the determinants of bacterial sensitivity remain incompletely understood. Here we combined growth assays with (1)H NMR metabolomics to characterize BA-induced metabolic responses in Escherichia coli BW25113 and to examine metabolic changes associated with impaired glutamine transport. Wild-type BW25113 and its BA-sensitive isogenic ΔglnP mutant, lacking the membrane-bound glutamine permease of the high-affinity GlnHPQ transport system, were exposed to sublethal BA concentrations. BA slowed growth and significantly altered the levels of 42 metabolites in the wild-type and 38 in ΔglnP, with the mutant showing stronger growth inhibition and reduced BA tolerance. Both strains exhibited metabolic changes consistent with cellular responses to oxidative and acid stress, including alterations in central carbon metabolism, lysine degradation, cysteine and methionine metabolism, pyrimidine metabolism, and one-carbon pool by folate. However, several metabolic responses differed between the two strains. In wild-type cells, BA exposure was associated with changes in glycerolipid metabolism, glycerophospholipid metabolism, nicotinate and nicotinamide metabolism, lysine biosynthesis, glycine, serine and threonine metabolism, and purine metabolism. In contrast, ΔglnP cells showed distinct alterations in D-amino acid metabolism, arginine biosynthesis, and other carbon fixation pathways. In addition, the mutant displayed substantial baseline differences relative to the wild-type, including altered nucleotide and amino acid pools. Together, these results indicate that both BA exposure and deletion of glnP induce broad metabolic adjustments in Escherichia coli. Loss of glnP is associated with distinct metabolic states and altered responses to BA stress, highlighting the importance of glutamine transport in adaptation to weak organic acid stress. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11274-026-04971-5.