NasR2 promoter-driven bioluminescent detection of bioavailable nitrogen

This study developed Klebsiella oxytoca W6-nasR2, a bioluminescent bioreporter containing the nasR2 promoter fused to the luxCDABE operon for detecting bioavailable nitrogen. The 267-bp nasR2 open reading frame (ORF) encodes an 88-amino acid protein with regulatory elements for nitrogen metabolism and stress responses, including NtrC/σ54, FNR-type motifs, GlnR, and SOS motifs. COBALT analysis identified conserved nitrate-regulation motifs (DLRQQLVDRIDGQQPC, FDSFQALAEAPQT). The nasR2 promoter, with -10 (TTTTCA) and -35 (CATATT) boxes, coordinates nitrogen metabolism and stress adaptation. Predicted phosphorylation sites at serine residues 49 and 75 likely enhance nasR2 stability and sensitivity, making it a robust nitrate reductase. Wild-type K. oxytoca W6 reduced 92.39% of 1.2 g NO₃-N/L in 27 h, with peak nitrate reductase activity (600 U/mL) observed at 168 h during extended enzymatic monitoring. The W6-nasR2 bioreporter exhibited a 186-fold increase in bioluminescence in response to peptone, with optimal activity at pH 6–7, retaining ~ 56% activity across pH 4–10. Strongest responses were observed with organic nitrogen sources (yeast extract > peptone > casein > skim milk), moderate responses with inorganic nitrogen (NaNO₃ > NH₄Cl > NaNO₂), and amino acids (lysine > asparagine). Urea and NaCN showed minimal or inhibitory effects. Field applications detected bioavailable nitrogen in municipal wastewater (45,000–62,000 CPS), industrial wastewater (41,000–42,000 CPS), desert soil (4,000 CPS), and agricultural soil (25,000 CPS), correlating with total nitrogen levels of 0.045–0.058 mg/L. These findings highlight the utility of the W6-nasR2 bioreporter for monitoring bioavailable nitrogen in real-world environments and position NasR2 as a potential novel nitrate reductase.