Molecular Insights into the Oxygen-Tolerant Dissimilatory Nitrate Reduction to Ammonium by Sulfide Oxidation in <i>Mycobacterium</i>

Dissimilatory nitrate reduction to ammonium (DNRA), essential for nitrogen retention in ecosystems, is traditionally considered a strictly anaerobic process. Challenging this, emerging evidence revealed robust DNRA activity under aerobic conditions, yet the underlying mechanisms remain elusive. In this study, we elucidate the molecular basis of oxygen-tolerant DNRA driven by sulfide oxidation in a Mycobacterium-dominated system. Under fully aerobic conditions (dissolved oxygen >3 mg/L), this system converted 1.4–4.5 mmol of nitrate to ammonium per 100 mmol of sulfide oxidized, with efficiency increasing at higher sulfide loads. Transcriptomic analysis revealed strong upregulation of DNRA genes (narGHI, nirBD) in Mycobacterium in response to elevated sulfide. In contrast to canonical DNRA organisms, which rely on the oxygen-sensitive transcription factor Fnr, Mycobacterium lacks the fnr gene and instead uses the nitrite-responsive NarL to drive DNRA gene expression irrespective of oxygen availability. Structural and molecular-dynamics analysis of Mycobacterium NarGHI complex─initiating enzyme of DNRA─reveals an evolutionarily adapted, distinctly narrowed, and highly hydrophobic substrate channel. This confined architecture protects the catalytic site from oxidative damage by raising the O2 diffusion free-energy barrier and enhancing nitrate-specific induced-fit recognition. Our findings reveal metabolic plasticity of microbial nitrogen cycling, imply convergent evolutionary strategies for oxygen tolerance in other classically anaerobic pathways, and offer key practical insights for optimizing nitrogen management in both sustainable agriculture and wastewater treatment.