For cultures grown in microaerobic ammonium medium, neither emission of N2O nor N2 was found

in WT and the ΔMgfnr mutant, suggesting that the presence find more of nitrate is essential to activate denitrification. After growth in microaerobic nitrate medium, N2O emission rates from nitrate were similar in WT and ΔMgfnr mutant (Table 3). As estimated by N2 evolution, we found that N2O reductase activity was very low in both strains compared to nitrate, nitrite, and NO reductase activities, since the rate for N2O production from nitrate was 20-fold higher than the rate for N2 production. Due to the low values and the detection limit of the gas-mass spectrometer, the standard deviation is quite critical for evaluation of significance of the N2 emission values. However, in 8 independent experiments the N2 emission rates appeared lower for ΔMgfnr strain than for the WT (0.4 μM/min versus 0.7 μM/min). In addition, we also tested oxygen reduction in both WT and ΔMgfnr mutant grown under microaerobic conditions by determining the consumption rate of oxygen in cell suspension with the gas-mass spectrometer. WT and ΔMgfnr mutant cells consumed oxygen at similar rates (Table 3), which indicated that MLL inhibitor MgFnr is not involved in regulation of O2

respiration. Adavosertib in vitro Figure 4 Analysis of Δ Mgfnr mutant. (A) N2 production in WT, ΔMgfnr mutant, ΔMgfnr mutant plus pLYJ110, and ΔMgfnr mutant plus pLYJ153 cultures in oxygen gradient tubes with 0.3% agar. ΔMgfnr mutant plus pLYJ110, and ΔMgfnr mutant plus pLYJ153 cells contained respective fnr gene from MSR-1 and E. coli. Gas bubbles were indicated by white arrows. (B) Transcription of Mgfnr promoter fused to gusA in both WT and ΔMgfnr mutant under different conditions. Expression was measured by β-glucuronidase activity. Cultures were grown aerobically or microaerobically in nitrate and ammonium medium. (C) Heterologous transcomplementation of ΔEcfnr ALOX15 mutant harboring the plasmid pLYJ132

which contains Mgfnr. Cultures were anaerobically grown to stationary phase at 30°C in glucose minimal medium (black box) and lactate minimal medium (gray box). (D) Transcription of nosZ fused to gusA in Mgfnr variant strains under aerobic conditions in the presence of nitrate. Expression was measured by β-glucuronidase activity. Table 3 Rates of N2O and N2 emission in WT and ΔMgfnr mutant after nitrate addition and rates of O2 consumption during aerobic respiration Culture (2% oxygen) N2O emission N2emission O2consumption (μM/mina) (μM/min) (μM/min) WT without nitrate NDb ND 50.7 ± 10.0 ΔMgfnr mutant without nitrate ND ND 44.0 ± 2.0 WT with nitrate 14.1 ± 2.0 0.7 ± 0.5 41.3 ± 2.0 ΔMgfnr mutant with nitrate 12.0 ± 2.0 0.4 ± 0.2 44.0 ± 4.7 aThe values (in μM per min for a cell suspension of OD565 nm of 1) are the average of eight independent experiments. bND: not detectable.