Distinct Denitrification Phenotypes in Closely Related Bacteria: Clues to Understanding Variations in Nitrite Accumulation Among Stutzerimonas Strains

Nitrite (NO2−) is a key denitrification intermediate, formed from nitrate (NO3−). Transient NO2− accumulation varies among denitrifiers, yet the underlying causes remain poorly understood, despite its potential toxicity and role in NO and N2O 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 NO3−-N transiently accumulated as NO2−. LNA strains lack or express the membrane-bound nitrate reductase (NarG) late, relying on periplasmic NapA for NO3− reduction, possibly explaining their balanced NO2− 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 NO3− respiration likely accounts for some NO2− accumulation. However, addition of NO3− halted NO2− 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 NO2−-accumulators, likely having a role in finetuning NO2− regulation. Our findings reveal diverse NO2−-handling phenotypes among denitrifiers and provide insights for optimizing wastewater nitrogen removal and soil bioaugmentation strategies to mitigate N2O emissions.