Distinct Denitrification Phenotypes in Closely Related Bacteria: Clues to Understanding Variations in Nitrite...

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 18 related Stutzerimonas strains, including the model Stutzerimonas 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.