Functional and Genomic Evidence of L-Arginine-Dependent Bacterial Nitric Oxide Synthase Activity in Paenibacillus nitricinens sp. nov.

Although nitric oxide (NO) production in bacteria has traditionally been associated with denitrification or stress responses in model or symbiotic organisms, functionally validated L-arginine-dependent nitric oxide synthase (bNOS) activity has not been documented in free-living, non-denitrifying soil bacteria. This paper reports Paenibacillus nitricinens sp. nov., a bacterium isolated from rainforest soil capable of synthesizing NO via a bNOS under aerobic conditions. A bnos-specific PCR confirmed gene presence, while whole-genome sequencing (6.7 Mb, 43.79% GC) revealed two nitrogen metabolism pathways, including a bnos-like gene. dDDH (<70%) and ANI (<95%) values with related Paenibacillus strains support the delineation of this isolate as a distinct species. Extracellular and intracellular NO measurements under aerobic conditions showed a dose-dependent response, with detectable production at 0.1 µM L-arginine and saturation at 100 µM. The addition of L-NAME reduced NO formation, confirming enzymatic mediation. The genomic identification of a bnos-like gene strongly supports the presence of a functional pathway. The absence of canonical nitric oxide reductase (Nor) genes or other typical denitrification-related enzymes reinforces that NO production arises from an alternative, intracellular enzymatic mechanism rather than classical denitrification. Consequently, P. nitricinens expands the known repertoire of microbial NO synthesis and suggests a previously overlooked source of NO flux in well-aerated soils.