Robustness of nitric oxide detoxification to nitrogen starvation in Escherichia coli requires RelA

Reactive nitrogen species and nutrient deprivation are two elements of the immune response used to eliminate pathogens within phagosomes. Concomitantly, pathogenic bacteria have evolved defense systems to cope with phagosomal stressors, which include enzymes that detoxify nitric oxide (^•NO) and respond to nutrient scarcity. A deeper understanding of how those defense systems are deployed under adverse conditions that contain key elements of phagosomes will facilitate targeting of those systems for therapeutic purposes. Here we investigated how Escherichia coli detoxifies ^•NO in the absence of useable nitrogen, because nitrogen availability is limited in phagosomes due to the removal of nitrogenous compounds (e.g., amino acids). We hypothesized that nitrogen starvation would impair ^•NO detoxification by E. coli because it depresses translation rates and the main E. coli defense enzyme, Hmp, is synthesized in response to ^•NO. However, we found that E. coli detoxifies ^•NO at the same rate regardless of whether useable nitrogen was present. We confirmed that the nitrogen in ^•NO and its autoxidation products could not be used by E. coli under our experimental conditions, and discovered that ^•NO eliminated differences in carbon and oxygen consumption between nitrogen-replete and nitrogen-starved cultures. Interestingly, E. coli does not consume measurable extracellular nitrogen during ^•NO stress despite the need to translate defense enzymes. Further, we found that RelA, which responds to uncharged tRNA, was required to observe the robustness of ^•NO detoxification to nitrogen starvation. These data demonstrate that E. coli is well poised to detoxify ^•NO in the absence of useable nitrogen and suggest that the stringent response could be a useful target to potentiate the antibacterial activity of ^•NO.