ABSTRACT
Nitric oxide (NO) inactivates iron-sulfur enzymes in bacterial amino acid biosynthetic pathways, causing amino acid auxotrophy. We demonstrate that exogenous supplementation with branched-chain amino acids (BCAA) can restore the NO resistance of hmp mutant Salmonella Typhimurium lacking principal NO-metabolizing enzyme flavohemoglobin, and of mutants further lacking iron-sulfur enzymes dihydroxy-acid dehydratase (IlvD) and isopropylmalate isomerase (LeuCD) that are essential for BCAA biosynthesis, in an oxygen-dependent manner. BCAA supplementation did not affect the NO consumption rate of S. Typhimurium, suggesting the BCAA-promoted NO resistance independent of NO metabolism. BCAA supplementation also induced intracellular survival of ilvD and leuCD mutants at wild-type levels inside RAW 264.7 macrophages that produce constant amounts of NO regardless of varied supplemental BCAA concentrations. Our results suggest that the NO-induced BCAA auxotrophy of Salmonella, due to inactivation of iron-sulfur enzymes for BCAA biosynthesis, could be rescued by bacterial taking up exogenous BCAA available in oxic environments.
Subject(s)
Amino Acids, Branched-Chain/metabolism , Nitric Oxide/metabolism , Salmonella typhimurium/growth & development , Salmonella typhimurium/metabolism , Aerobiosis , Amino Acids/metabolism , Amino Acids, Branched-Chain/biosynthesis , Animals , Bacterial Proteins/genetics , Cell Line , Hemeproteins/genetics , Hydro-Lyases/genetics , Iron/metabolism , Isomerases/genetics , Mice , Salmonella typhimurium/genetics , Stress, PhysiologicalABSTRACT
Cytotoxic nitic oxide (NO) damages various bacterial macromolecules, resulting in abnormal metabolism by mechanisms largely unknown. We show that NO can cause amino acid auxotrophy in Salmonella Typhimurium lacking major NO-metabolizing enzyme, flavohemoglobin Hmp. In NO-producing cultures, supplementation with amino acid pool restores growth of Hmp-deficient Salmonella to normal growth phases, whereas excluding Cys or BCAA Leu, Ile, or Val from amino acid pool reduces growth recovery. Data suggest that, without detoxification, NO might inactivate key enzymes in the biosynthesis pathway of amino acids essential for Salmonella replication in amino acid-limiting host environments.