Comparative physiological and genomic characterization of a novel Nitrobacter vulgaris strain from a...

Nitrite-oxidizing bacteria (NOB) represent a crucial node in the global nitrogen cycle. By catalyzing the second step of nitrification-the oxidation of nitrite to nitrate to generate energy for growth-NOB activity controls the fate of nitrite (NO₂^-) in aerobic environments. Despite thriving in diverse environments, including soils, freshwater, marine ecosystems, subsurface habitats, and water treatment systems, organisms capable of nitrite oxidation are confined to Nitrobacter, Nitrospira, Nitrospina, Nitrotoga, and a few other specific lineages. The genus Nitrobacter, recognized for its facultative heterotrophic metabolism, is often associated with high-nitrogen environments. Here, we report the physiological characterization of a novel strain, Nitrobacter vulgaris strain MLSD-S22, isolated from a nitrate- and heavy-metal-contaminated subsurface. Growth inhibition experiments revealed that strain MLSD-S22 and the N. vulgaris type strain Z exhibited similar sensitivities to nitrite and nitrate, with nitrite being the most inhibitory. Microrespirometry demonstrated that the two N. vulgaris strains and Nitrobacter winogradskyi Nb-255 possessed higher affinities for nitrite and oxygen than previously reported for Nitrobacter, suggesting potential to compete in low-substrate environments. Long-read DNA sequencing provided a complete genome for strain MLSD-S22, revealing two plasmids and an intact nitrous oxide (N₂O) reduction operon-an unexpected feature for Nitrobacter. While N₂O reduction activity was not observed under the tested conditions, this discovery raises questions about the contribution of Nitrobacter NOB to the N₂O sink. These findings broaden the physiological and genomic diversity of Nitrobacter, offering new insights into their adaptation strategies and providing a framework for future evaluation of their potential roles in nitrogen loss.IMPORTANCENitrobacter species play a key role in nitrogen cycling; however, their physiological adaptations and genomic diversity remain largely underexplored. This study characterizes Nitrobacter vulgaris strain MLSD-S22, isolated from a nitrate- and heavy-metal-contaminated subsurface, revealing unexpected genomic and metabolic traits. Higher nitrite and oxygen affinities compared to previous reports indicate that some Nitrobacter are more competitive in low-substrate environments than previously believed. The presence of a complete nitrous oxide (N₂O) reduction operon highlights the genomic potential related to N₂O reduction within this genus; however, we did not observe N₂O reduction under the conditions tested. By expanding Nitrobacter ecophysiology, this work provides a foundation for future research on the metabolic flexibility of Nitrobacter and their contributions to nitrogen transformations in diverse environments.