Abstract

3-Nitropropionic acid (3-NPA) is a toxic nitroaliphatic compound, but the genomic basis of bacterial adaptation to 3-NPA remains incompletely resolved, especially in organisms lacking a canonical nitronate monooxygenase. Here, we used phenotype screening, whole-genome resequencing, targeted UHPLC-MS/MRM, and composite RNA-seq profiling to investigate 3-NPA utilization in Leclercia barmai EMC7. Following attempted Tn5-Mob mutagenesis and kanamycin-based recovery, two independently derived mutants, LTM01 and LTM14, showed impaired growth under 3-NPA-dependent minimal conditions. No stable transposon insertion was detected in either mutant. Comparative genome analysis instead revealed convergent loss-of-function (LOF) mutations affecting amino acid metabolism, nitrogen allocation, central carbon metabolism, cofactor-linked functions, transport, and metal-redox homeostasis, while the predicted flavin-dependent nitro-redox candidate and canonical nitrate/nitrite reduction loci were not disrupted. Targeted UHPLC-MS/MRM analysis showed that wild-type EMC7 depleted approximately 50% of 3-NPA within 36 h, whereas the mutants displayed poor 3-NPA-utilization phenotypes under the same growth framework. Wild-type RNA-seq under glucose-3NPA relative to glucose-KNO3 identified a broad expression profile involving flavin-redox functions, nitrogen assimilation, oxidative-stress response, envelope stress, efflux, cofactor metabolism, and iron homeostasis. Integration of mutant LOF profiles with wild-type gene expression data showed that several disrupted genes or functional counterparts belonged to modules showing expression changes during the wild-type glucose-3NPA response. These findings identify candidate adaptive modules associated with 3-NPA utilization in EMC7, including nitrogen redistribution, carbon entry, sulfur-redox support, cofactor supply, transport, envelope remodeling, and metal-redox control. These results extend the landscape of metabolic adaptation beyond canonical pathways in environmental bacteria.