A novel ATP-generating machinery to counter nitrosative stress is mediated by substrate-level phosphorylation

Abstract

BackgroundIt is well-known that elevated amounts of nitric oxide and other reactive nitrogen species (RNS) impact negatively on the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. These perturbations severely compromise O2-dependent energy production. While bacteria are known to adapt to RNS, a key tool employed by macrophages to combat infections, the exact mechanisms are unknown. MethodsThe bacterium was cultured in a defined mineral medium and cell-free extracts obtained at the same growth phase were utilized for various biochemical studies Blue native polyacrylamide gel electrophoresis followed by in-gel activity assays, high performance liquid chromatography and co-immunoprecipitaton are applied to investigate the effects of RNS on the model microbe Pseudomonas fluorescens. ResultsCitrate is channeled away from the tricarboxylic acid cycle using a novel metabolon consisting of citrate lyase (CL), phosphoenolpyruvate carboxylase (PEPC) and pyruvate phosphate dikinase (PPDK). This metabolic engine comprising three disparate enzymes appears to transiently assemble as a supercomplex aimed at ATP synthesis. The up-regulation in the activities of adenylate kinase (AK) and nucleoside diphosphate kinase (NDPK) ensured the efficacy of this ATP-making machine. ConclusionMicrobes may escape the effects of nitrosative stress by re-engineering metabolic networks in order to generate and store ATP anaerobically when the electron transport chain is defective. General significanceThe molecular configuration described herein provides further understanding of how metabolism plays a key role in the adaptation to nitrosative stress and reveals novel targets that will inform the development of antimicrobial agents to counter RNS-resistant pathogens.