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 (NO3−-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 NO3−-N removal efficiency of napA mutant was 88.59% with comparable to the wild-type (87.24%), whereas the growth and NO3−-N reduction of the narG mutant were severely impaired. 15N isotope experiments demonstrated that narG is essential for both DNRA and ANRA pathways. napA- and narG-deficient mutants showed lower nitrite (NO2−-N) removal efficiencies (82.30 and 83.86%, respectively) than the wild-type (97.00%). Although both mutants completely removed ammonium (NH4+-N), less NH4+-N was oxidized into gaseous nitrogen (11.96 and 13.16%, respectively) than the wild-type (22.89%). Meanwhile, glnA (regulating NH4+-N assimilation) was significantly upregulated in both mutants. Hence, narG is responsible for the aerobic reduction of NO3−-N to NO2−-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 NO2−-N reduction but promotes NH4+-N assimilation. These findings provide the first genetic evidence that narG facilitates aerobic NO3−-N reduction in Pseudomonas spp., advancing our understanding of the molecular mechanisms of napA and narG in the nitrogen cycle.
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