Kinetic plasticity of nitrite-oxidizing bacteria containing cytoplasmic nitrite oxidoreductase

Nitrite-oxidizing bacteria (NOB) use either periplasmic (pNXR) or cytoplasmic (cNXR) nitrite oxidoreductase to oxidize nitrite, and this distinction influences nitrite affinity and energy yield. cNXR-containing NOB have historically been considered low-affinity, copiotrophic nitrifiers adapted to high nitrite and neutral pH. Here, we report a previously uncharacterized pH- and substrate-dependent modulation of nitrite affinity in cNXR NOB i.e. not observed in pNXR NOB and is not a universal microbial trait. Nitrobacter winogradskyi Nb-255, grown at low nitrite (1 mM), had a high apparent affinity (Km(app) = 25.9 μM; specific affinity ao = 440.5 l g cells-1 h-1) comparable to oligotrophic pNXR NOB. However, when grown at high nitrite (10 mM), these cells showed a low affinity at pH 7.5 (Km(app) = 388.0 μM) but exhibited a rapid increase in affinity upon immediate exposure to pH 5.5 (Km(app) = 19.2 μM) without prior acid adaptation. In contrast, pNXR NOB exhibited consistent kinetic behavior across different pH conditions, underscoring that this kinetic plasticity is unique to cNXR NOB. Kinetic inhibition assays revealed that this plasticity is mechanistically underpinned by a shift from a low-affinity nitrite/nitrate antiporter (NarK) to a high-affinity nitrite channel (NirC), coupled with enhanced HNO2 diffusion at low pH, together increasing intracellular nitrite availability. These findings establish that cNXR NOB can dynamically tune nitrite affinity via transporter-level regulation in response to nitrite concentration and pH. This novel mechanism provides a mechanistic explanation for the unexpected prevalence of Nitrobacter in acidic, low-nitrite environments, highlighting its ecological relevance.