Distinct denitrification phenotypes in closely related bacteria: clues to understanding variations in nitrite accumulation among Stutzerimonas strains

Nitrite (NO 2 − ) is a key denitrification intermediate, formed from nitrate (NO 3 − ). Transient NO 2 − accumulation varies among denitrifiers, yet the underlying causes remain poorly understood, despite its potential toxicity and role in NO and N 2 O emissions. We profiled eighteen related Stutzerimonas strains, including the model S. perfectomarina ZoBell, and identified three phenotypic clusters (full, partial and low nitrite accumulators; FNA, PNA and LNA) based on the fraction of NO 3 − -N transiently accumulated as NO 2 − . LNA strains lack or express the membrane-bound nitrate reductase (NarG) late, relying on periplasmic NapA for NO 3 − reduction, possibly explaining their balanced NO 2 − production/reduction. FNA and PNA strains possess NapA and NarG but differ in their nitrite reductase (NirS) clades. Delayed nirS transcription as FNA strains transition to NO 3 − respiration likely accounts for some NO 2 − accumulation. However, addition of NO 3 − halted NO 2 − reduction in FNA strains, suggesting additional metabolic control. This may require the cytochromes NirTB, which are only found in FNA strains. The regulator DnrE was also unique to NO 2 − -accumulators, likely having a role in finetuning NO 2 − regulation. Our findings reveal diverse NO 2 − -handling phenotypes among denitrifiers and provide insights for optimizing wastewater nitrogen removal and soil bioaugmentation strategies to mitigate N 2 O emissions.