narG, rather than napA, mediates aerobic nitrate reduction process in Pseudomonas putida Y-9

• napA in the aerobic NO 3 − -N reduction of P. putida Y-9 is redundant. • narG mediates ANRA, DNRA, and denitrification in strain Y-9 under aerobic conditions. • Mutation of napA or narG in strain Y-9 significantly inhibited the removal of NO 2 − -N. • NH 4 + -N assimilation was enhanced after knocking out napA or narG in strain Y-9. Pseudomonas putida Y-9 simultaneously performs assimilatory nitrate reduction (ANRA), dissimilatory nitrate reduction to ammonium (DNRA), and denitrification under aerobic conditions, among which ANRA and DNRA enhance nitrogen retention in ecosystems. However, the key genes involved in the process of nitrate (NO 3 − -N) reduction remain elusive. In this study, the nitrate reductase-encoding genes napA- or narG- knockout mutants of P. putida Y-9 were constructed to elucidate their roles in nitrogen transformation. The NO 3 − -N removal efficiency of napA mutant was 88.59% with comparable to the wild-type (87.24%), whereas the growth and NO 3 − -N reduction of the narG mutant were severely impaired. 15 N isotope experiments demonstrated that narG is essential for both DNRA and ANRA pathways. napA - and narG -deficient mutants showed lower nitrite (NO 2 − -N) removal efficiencies (82.30 and 83.86%, respectively) than the wild-type (97.00%). Although both mutants completely removed ammonium (NH 4 + -N), less NH 4 + -N was oxidized into gaseous nitrogen (11.96 and 13.16%, respectively) than the wild-type (22.89%). Meanwhile, glnA (regulating NH 4 + -N assimilation) was significantly upregulated in both mutants. Hence, narG is responsible for the aerobic reduction of NO 3 − -N to NO 2 − -N in P. putida Y-9, thereby influencing ANRA, DNRA, and denitrification, whereas napA shows functional redundancy in these pathways. Mutation of either napA or narG impairs NO 2 − -N reduction but promotes NH 4 + -N assimilation. These findings provide the first genetic evidence that narG facilitates aerobic NO 3 − -N reduction in Pseudomonas spp., advancing our understanding of the molecular mechanisms of napA and narG in the nitrogen cycle.