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

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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.