Denitrifying metabolism of the methylotrophic marine bacterium Methylophaga nitratireducenticrescens strain JAM1

Bacterial growth conditions

M. nitratireducenticrescens JAM1 and the JAM1ΔnarG1narG2 double mutant were cultured in the American Type Culture Collection (ATCC, Manassas, VA, USA) Methylophaga medium 1403 (Villeneuve et al., 2013; Mauffrey, Martineau & Villemur, 2015). When required, ${\mathrm{NO}}_3^{-}$ (NaNO₃) or ${\mathrm{NO}}_2^{-}$ (NaNO₂) (Fisher Scientific Canada, Ottawa, ON, Canada) were added to the medium. Medium (40 or 60 mL) was dispensed into 720-mL bottles (680- or 660-mL head space) that were sealed with caps equipped with septum and which were then autoclaved. After autoclaving, the following filter-sterilized solutions were added to the bottles (40 mL volume): 120 µL methanol (final concentration 0.3% [vol/vol]; 74.3 mM), 800 µL solution T (per 100 mL: 0.7 g KH₂PO₄, 10 g NH₄Cl, 10 g Bis-Tris, 0.3 g ferric ammonium citrate (pH 8)), 400 µL Wolf’s mineral solution (pH 8) (ATCC), and 40 µL vitamin B₁₂ (stock solution 0.1 mg/mL). The Wolf mineral solution is composed of (per liter) 0.5 g EDTA, 3.0 g MgSO₄.7H₂O, 0.5 g MnSO₄.H₂O, 1.0 g NaCl, 0.1 g FeSO₄.7H₂O, 0.1 g Co(NO₃)₂.6H2O, 0.1 g CaCl₂ (anhydrous), 0.1 g ZnSO₄.7H₂O, 0.010 g CuSO₄.5H₂O, 0.010 g AlK(SO₄)₂ (anhydrous), 0.010 g H₃BO₃, 0.010 g Na₂MoO₄.2H2O, 0.001 g Na₂SeO₃ (anhydrous), 0.010 g Na₂WO₄.2H₂O, and 0.020 g NiCl₂.6H₂O. The final concentration of ammonium (${\mathrm{NH}}_4^{+}$) in the Methylophaga 1403 medium was measured as 21 mg-N vial⁻¹ (20.9 mg-N vial⁻¹ from NH₄Cl and 0.1 mg-N vial⁻¹ from ferric ammonium citrate). The amount of NO${}_3^{-}$ carried by the Wolf mineral solution (0.0038 mg-N vial⁻¹) was deemed negligible. For the anoxic cultures, bottles were flushed with nitrogen gas (N₂, purity >99.9%; Praxair, Mississauga, ON, Canada) or argon (purity 99.9%, Praxair) for 20 min prior to autoclaving. When necessary, N₂O (purity 99.9%, Praxair) and acetylene (10% [vol/vol] of headspace; Praxair) were injected into the headspace before autoclaving. Acetylene is an inhibitor of nitrous oxide reductase and has been extensively used in N₂O studies to observe N₂O production in cells (Klemedtsson et al., 1977). Inoculums were made from fresh culture cultivated under oxic conditions without ${\mathrm{NO}}_3^{-}$ to reach an optical density (OD₆₀₀) of 0.025. Culture bottles were incubated at 30 °C in the dark. For oxic cultures, bottles were shaken at 150 rpm.

The capacity for strain JAM1 to reduce NO was tested with sodium nitroprusside (sodium nitroprusside hypochloride ([SNP]; purity ≥99.0%, Sigma-Aldrich, St. Louis, MO, USA) as the NO source. To avoid SNP toxicity, strain JAM1 was first cultured in Methylophaga 1403 medium under oxic conditions without ${\mathrm{NO}}_3^{-}$ for 24 h. The cells were then centrifuged (8,000 g 5 min) and dispersed into fresh medium supplemented with 2 mM, 5 mM, or no SNP. Culture medium with 5 mM SNP and no biomass was also used as a control. Cells were incubated under oxic conditions at 30 °C in the dark, and N₂O production was monitored. To investigate the potential role of ${\mathrm{NH}}_4^{+}$ in N₂O production, NH₄Cl-free cultures were employed under oxic and anoxic conditions using solution T containing no NH₄Cl. Prior to inoculation, cells from start-up cultures were centrifuged and rinsed three times with saline solution to remove any residual traces of ${\mathrm{NH}}_4^{+}$.

Bacterial growth was monitored by spectrophotometry (OD₆₀₀). Bacterial flocs were dispersed with a Potter-Elvehjem homogenizer prior to measurement. Oxygen concentrations in the headspace were monitored in cultures under oxic conditions by gas chromatography using a temperature conductivity detector (7890B series GC Custom, SP1 option 7890-0504/0537; Agilent Technologies, Mississauga, ON, Canada). Although vials were capped in the oxic cultures, O₂ concentrations in the headspace (680 ml) did not significantly decrease (T0 h = 20.4 ± 0.3%; T100 h = 19.7 ± 0.9%).

¹⁵N-labeling of N₂O

Strain JAM1 cultures were made with 22 mg-N vial⁻¹ Na¹⁵NO₃ (Sigma-Aldrich) in NH₄Cl-free medium or with 22 mg-N vial⁻¹ Na¹⁴NO₃ and 20.7 mg-N vial^{−1 15}NH₄Cl (Sigma-Aldrich). Both cultures were used under anoxic conditions, and 10% (vol/vol) acetylene was added to allow N₂O to accumulate. Cultures were made in triplicate. After 14 days of incubation, the headspace of each replicate was pooled, and 100 mL of the gaseous phase was sampled in Tedlar bags. N₂O-isotope measurements were performed at the Environmental Isotope Laboratory (Earth & Environmental Sciences; University of Waterloo, ON, Canada) via Trace Gas-GVI IsoPrime-Isotope Ratio Mass Spectrometry (TG-IRMS). ⁴⁵[N₂O]/⁴⁴[N₂O] and ⁴⁶[N₂O]/⁴⁴[N₂O] ratios were calculated according to the peak intensity measured for ⁴⁶[N₂O], ⁴⁵[N₂O] and ⁴⁴[N₂O]. The ¹⁵N/¹⁴N isotopic ratio was derived from the previous results from Eq. (1). (1)${\displaystyle Rs=\sum \left({}^{15}\mathrm{N}{\mathrm{vial}}^{-1}\right)∕\sum \left({}^{14}{\text{N vial}}^{-1}\right)=\left[{}^{15}\mathrm{N}45+2\left({}^{15}\mathrm{N}46\right)\right]∕\left[2\left({}^{14}\mathrm{N}44\right){+}^{14}\mathrm{N}45\right]}$ where Rs is the sample isotopic ratio. Calculated from the ⁴⁵[N₂O]/⁴⁴[N₂O] and ⁴⁶[N₂O]/⁴⁴[N₂O] isotopic ratios, ¹⁴N45 is the quantity of ¹⁴N in ⁴⁵[N₂O], ¹⁵N45 is the quantity of ¹⁵N in ⁴⁵[N₂O], ¹⁴N44 is the quantity of ¹⁴N in ⁴⁴[N₂O] and ¹⁵N46 is the quantity of ¹⁵N in ⁴⁶[N₂O]. We considered the isotope fractionation by denitrification enzymes as negligible in our calculations (delta values ranging from −10‰ to −40‰) (Snider, Schiff & Spoelstra, 2009).

Measurements of nitrogenous compounds

${\mathrm{NO}}_3^{-}$ and ${\mathrm{NO}}_2^{-}$ concentrations were determined by ion chromatography using the 850 Professional IC (Metrohm, Herisau, Switzerland) with a Metrosep A Supp 5 analytical column (250 mm × 4.0 mm).

N₂O and N₂ concentrations were determined by gas chromatography. Headspace samples (10 mL) were collected using a Pressure Lok gastight glass syringe (VICI Precision Sampling Inc., Baton Rouge, LA, USA) and were injected through the injection port of a gas chromatograph equipped with a thermal conductivity detector and electron-capture detector (7890B series GC Custom, SP1 option 7890-0504/0537; Agilent Technologies). The reproducibility of the N₂O was assessed before each set of measurements via the repeated analysis of certified N₂O standard gas with standard deviations <5%. N₂O standards (500 ppmv and 250 ppmv) were created based on dilutions from the 10,000 ppmv N₂O stock standard. The 10,000 ppmv stock standard was obtained by injecting 1% pure N₂O (Praxair) into a 720 mL gastight bottle. The detection limit of the N₂O was set to <10 ppbv, corresponding to the 0.3 nmol/vial composition of our bioassays. No significant N₂O production patterns were observed through our blank experiments involving sterile media and empty glass bottles. The total quantity of N₂O in the culture bottle (aqueous phase and headspace) (X_N2O in µmole vial⁻¹) was calculated according to Eq. (2). (2)${\displaystyle {\mathrm{X}}_{\mathrm{N2O}}={\left[{\mathrm{K}}_{\mathrm{cpH30sw}}\ast {\mathrm{A}}_{\mathrm{N2O}}\ast \mathrm{P}\ast {\mathrm{V}}_1\right]}_{\mathrm{aq}}+{\left[A_{\mathrm{N2O}}\ast {\mathrm{V}}_{\mathrm{g}}∕{\mathrm{V}}_{\mathrm{n}}\right]}_{\mathrm{gaz}}}$ where A_N2O: the N₂O mixing ratio measured in the headspace (µmole_N2O mole⁻¹); P: 1 atm; V₁ and V_g: volume of the aqueous (0.04 or 0.06 L vial⁻¹) and gaseous phases (0.68 or 0.66 L vial⁻¹), respectively; and V_n: molar volume (RT (gas constant): 0.08206 L atm K⁻¹ mol⁻¹ * 303 K = 24.864 L mol⁻¹). K_H30sw is the corrected Henry’s constant for seawater at 30 °C (0.01809 mol L⁻¹ atm⁻¹) according to Weiss and Price (1980). X_N2O was then converted (Eq. (3)) into mg-N vial⁻¹ for an easier calculation of mass balances using the other nitrogenous compounds: (3)${\displaystyle {\mathrm{X}}_{\mathrm{N}-\mathrm{N2O}}=X_{\mathrm{N2O}}\ast \left[2\mathrm{N}∕{\mathrm{N}}_2\mathrm{O}\right]\ast \left[0.014\mathrm{mg}-\mathrm{N}\mathrm{\mu }{\mathrm{mole}}^{-1}\right].}$ The reproducibility of the N₂ was assessed before each set of measurements via a repeated analysis of N₂ (purity >99.99%, Praxair) diluted in a 720 mL gastight bottle (0 and 500 ppmv) flushed with argon (purity >99.99%, Praxair). The total quantity of N₂ in the culture bottles was only considered for the headspace, as the quantity of dissolved N₂ in the aqueous phase was considered to be negligible in our experimental design based on Henry’s constant (0.0005 mol L⁻¹ atm⁻¹) and was thus calculated according to Eq. (4). (4)${\displaystyle {\mathrm{X}}_{\mathrm{N}\text{-}\mathrm{N2}}\left(\mathrm{mg}\text{-}\mathrm{N}{\mathrm{vial}}^{-1}\right)={\left[{\mathrm{A}}_{\mathrm{N2}}\ast {\mathrm{V}}_{\mathrm{g}}∕{\mathrm{V}}_{\mathrm{n}}\right]}_{\mathrm{gaz}}\ast \left[2\mathrm{N}∕{\mathrm{N}}_2\right]\ast \left[0.014\mathrm{mg}-\mathrm{N}\mathrm{\mu }{\mathrm{mole}}^{-1}\right].}$

RNA extraction

Anoxic cultures of strain JAM1 were created in an NH₄Cl-free 1403 medium supplemented with 22 mg-N vial⁻¹ ${\mathrm{NO}}_3^{-}$. Cells were harvested at specific times, and RNA was immediately extracted using the PureLink RNA mini kit (Ambion Thermo Fisher Scientific, Burlington, ON, Canada). RNA extracts were treated twice with TurboDNase (Ambion), and RNA quality was verified by agarose gel electrophoresis. The absence of remaining DNA was checked via the end-point polymerase chain reaction (PCR) amplification of the 16S rRNA gene using RNA extracts as the template.

Gene expression

cDNAs samples were generated from the RNA using hexameric primers and the Reverse Transcription System developed by Promega (Madison, WI, USA) with 1 µg of RNA and quantified by spectrophotometry. Real-time quantitative PCR (qPCR) assays were performed using the Faststart SYBR Green Master (Roche Diagnostics, Laval, QC, Canada) according to the manufacturer’s instructions. All reactions were performed in a Rotor-Gene 6000 real-time PCR thermocycler (Qiagen Inc. Toronto, ON, Canada), and each reaction contained 25 ng of cDNA and 300 nM of primers (Table 1). Genes tested included cnorB1, cnorB2, nnrS, nosZ, norR and nr, and the reference genes dnaG, rpoD and rpoB (Mauffrey, Martineau & Villemur, 2015) and the PCR began with an initial denaturation step of 10 min at 95 °C followed by 40 cycles of 10 s at 95 °C, 15 s at 60 °C, and 20 s at 72 °C. To confirm the purity of the amplified products, a melting curve analysis was performed by increasing the temperature from 65 °C to 95 °C at increments of 1 °C per step with a pause of 5 s included between each step. All genes for each sample and standard were tested in a single run. The amplification efficiency level was tested for each set of primer pairs by qPCR using a dilution of strain JAM1 genomic DNA as the template. The amplification efficiencies for all primer pairs varied between 0.9 and 1.1. The copy number of each gene was calculated according to standard curves using dilutions of strain JAM1 genomic DNA. The gene expression levels of the targeted genes were standardized with the three reference genes. The RNA extraction and qPCR were performed with three to four independent biological replicates. The significance of differential expression levels was tested for each phase against the pre-culture phase using One-way ANOVA tests with Tukey post hoc tests.

M. nitratireducenticrescens JAM1 grows on N₂O under anoxic conditions

Strain JAM1 was cultured under anoxic conditions with either ${\mathrm{NO}}_3^{-}$ in the medium or with N₂O injected in the headspace as the sole electron acceptor. Both types of culture received the same electron equivalent of ${\mathrm{NO}}_3^{-}$ or N₂O (1.3 mmole vial⁻¹ or 18.2 and 36.4 mg-N vial⁻¹, respectively) according to: (5)${\displaystyle {\mathrm{NO}}_3^{-}+2{\mathrm{e}}^{-}+2{\mathrm{H}}^{+}\to {\mathrm{NO}}_2^{-}+{\mathrm{H}}_2\mathrm{O}}$ (6)${\displaystyle {\mathrm{N}}_2\mathrm{O}+2{\mathrm{e}}^{-}+2{\mathrm{H}}^{+}\to {\mathrm{N}}_2+{\mathrm{H}}_2\mathrm{O}.}$ In N₂O-amended cultures, N₂O decrease was apparent from the start and consumption continued for 48 h (Fig. 1A). The N₂O decrease paralleled strain JAM1 growth with almost complete N₂O consumption. The ${\mathrm{NO}}_3^{-}$-amended cultures showed complete ${\mathrm{NO}}_3^{-}$ consumption and equivalent ${\mathrm{NO}}_2^{-}$ accumulation after 24 h (Fig. 1B). However, slower growth than that recorded for the N₂O cultures was observed. Such growth kinetics could be related to the toxicity of ${\mathrm{NO}}_2^{-}$ that accumulated in the medium. Both types of culture reached equivalent biomass concentration (t test on the last 4-time points, P > 0.05).

M. nitratireducenticrescens JAM1 consumes N₂O under oxic conditions

In a previous study, Mauffrey, Martineau & Villemur (2015) demonstrated that strain JAM1 can consume ${\mathrm{NO}}_3^{-}$ under oxic growth conditions with equivalent accumulation of ${\mathrm{NO}}_2^{-}$. We tested if this was also the case with N₂O. Culturing strain JAM1 under oxic conditions with N₂O (3.5 mg-N vial⁻¹) showed a complete N₂O consumption within 24 h (Fig. 2). Growth patterns illustrated in Fig. 2 were similar between oxic cultures amended with or without either N₂O or ${\mathrm{NO}}_3^{-}$. In the presence of O₂, cultures reached higher (4–5 times) biomass concentration than the anoxic cultures.

N₂O production in ${\mathrm{NO}}_3^{-}$-amended cultures

During the first assays to test the capacity of strain JAM1 to reduce N₂O under anoxic conditions, cultures were performed with N₂O (3.5 mg-N vial⁻¹) but with the addition of ${\mathrm{NO}}_3^{-}$ (20 mg-N vial⁻¹) to make sure that growth would occur. Although N₂O was completely consumed within 24 h, a net production of N₂O was observed after 48 h. To further investigate this observation, strain JAM1 was cultured under anoxic conditions with ${\mathrm{NO}}_3^{-}$, and ${\mathrm{NO}}_3^{-}$, ${\mathrm{NO}}_2^{-}$ and N₂O were measured (Fig. 3A). Complete ${\mathrm{NO}}_3^{-}$ reduction (19.3 ± 0.3 mg-N vial⁻¹) was performed within 55 h. The ${\mathrm{NO}}_2^{-}$ level reached 17.5 ± 0.2 mg-N vial⁻¹ over this period and decreased slowly to 15.9 ± 0.5 mg-N vial⁻¹. N₂O production initiated when ${\mathrm{NO}}_3^{-}$ was nearly reduced and reached 0.70 ± 0.21 mg-N vial⁻¹ after 55 h of incubation (Fig. 3A). N₂O was completely reduced after 127 h. In parallel, for cultures in which the headspace was flushed with argon, N₂ production was also measured. The corresponding results show an increase of N₂ in the headspace (Fig. 3A) by 1.14 ± 0.54 mg-N vial⁻¹ after 127 h, which represent 6.0 ± 2.9% of the N input. As the Methylophaga 1403 medium contains ferrous chloride (216 µmole vial⁻¹), N₂O production could originate from the abiotic reaction between ${\mathrm{NO}}_2^{-}$ that accumulated in the cultures and the ferrous ion (Klueglein et al., 2014). An abiotic control was performed with 18.2 mg-N vial⁻¹ ${\mathrm{NO}}_2^{-}$. N₂O was detected in the abiotic control after 20 h and reached 0.00172 ± 0.00012 mg-N vial⁻¹ after 114 h, which is 407 times lower than the N₂O concentration measured in the anoxic cultures. This results showed that the abiotic reaction generated negligible amount of N₂O.

Under oxic conditions, ${\mathrm{NO}}_3^{-}$ reduction (17.4 ± 2.1 mg-N vial⁻¹) was complete after 24 h with equivalent ${\mathrm{NO}}_2^{-}$ accumulation (17.1 ± 1.3 mg-N vial⁻¹). N₂O production started after complete ${\mathrm{NO}}_3^{-}$ reduction (Fig. 3B) and increased to reach 0.31 ± 0.32 mg-N vial⁻¹ after 96 h of incubation (1.7% of N input). Unlike trends observed for the anoxic cultures, N₂O concentration did not decrease in the oxic cultures. N₂O production and consumption could have reached an equilibrium and loss of nitrogen would occur by N₂ production.

${\mathrm{NO}}_3^{-}$, ${\mathrm{NO}}_2^{-}$ and N₂O dynamics in NH₄Cl-free cultures

The original 1403 medium recommended by the ATCC for culturing Methylophaga spp. contains 20.9 mg-N vial⁻¹ NH₄Cl and 0.1 mg-N vial⁻¹ ferric ammonium citrate (see ‘Material and Methods’). Based on the deduced nitrogen metabolic pathways from strain JAM1 genome (Fig. S1), N-assimilation into the biomass should proceed directly from ${\mathrm{NH}}_4^{+}$ and minimal ${\mathrm{NO}}_3^{-}$ reduction to ${\mathrm{NH}}_4^{+}$ would be occurring. For the next set of experiments, we aimed to determine the effect of removing NH₄Cl, which provides most of the ${\mathrm{NH}}_4^{+}$ (99.5%), on the dynamics of ${\mathrm{NO}}_3^{-}$, ${\mathrm{NO}}_2^{-}$ and N₂O. We hypothesized that forcing strain JAM1 to reroute some ${\mathrm{NO}}_3^{-}$ for N assimilation would affect denitrification and thus growth rates. Strain JAM1 was cultured with ca. 20 mg-N vial⁻¹ ${\mathrm{NO}}_3^{-}$ under anoxic or oxic conditions in NH₄Cl-free medium (Fig. 4). Growth pattern observed under anoxic conditions was similar between the regular and NH₄Cl-free cultures, as also the growth pattern under oxic conditions between the regular and NH₄Cl-free cultures.

Under anoxic NH₄Cl-free conditions, full ${\mathrm{NO}}_3^{-}$ reduction (19.1 ± 0.6 mg-N vial⁻¹) occurred within 48 h (Fig. 4A). The N₂O profile found was similar to that observed in regular cultures (Fig. 3A), though lower N₂O concentrations were detected during the accumulating phase. The ${\mathrm{NO}}_2^{-}$ level reached 18.5 ± 0.8 mg-N vial⁻¹ after 24 h and then slowly decreased to 12.8 ± 0.5 mg-N vial⁻¹ after 96 h. Cultures flushed with argon showed an increase of N₂ in the headspace (Fig. 4A) by 0.90 ± 0.28 mg-N vial⁻¹ after 127 h, which is similar to N₂ production in the regular culture medium. Nitrogen assimilation by the biomass could account for the difference in nitrogen mass balance (28.3%).

Unlike the cultures in regular medium (Fig. 3B), NO${}_3^{-}$ (21.3 ± 1.0 mg-N vial⁻¹) was not completely reduced under oxic NH₄Cl-free conditions, and it stopped after 24 h at 2.9 ± 2.7 mg-N vial⁻¹ (Fig. 4B). In conjunction with ${\mathrm{NO}}_3^{-}$ reduction, ${\mathrm{NO}}_2^{-}$ levels stopped accumulating at 13.0 ± 2.6 mg-N vial⁻¹ after 24 h. N₂O was observed after 48 h of incubation (Fig. 4B), after which it slowly accumulated and reached a concentration of 0.043 ± 0.048 mg-N vial⁻¹. This level is seven times lower than that of the regular culture medium (Fig. 3B).

To assess whether N₂O could have been generated through ${\mathrm{NH}}_4^{+}$, strain JAM1 was cultured under anoxic conditions with 22 mg-N vial⁻¹ ${\mathrm{NO}}_3^{-}$, 20.7 mg-N vial^{−1 15}${\mathrm{NH}}_4^{+}$, and acetylene to prevent the reduction of N₂O to N₂. If ${\mathrm{NH}}_4^{+}$ is involved in N₂O production, high proportion of labelled N₂O is expected. If ${\mathrm{NH}}_4^{+}$ is not involved in N₂O production, we expected the production of labeled N₂O to be derived from ¹⁵${\mathrm{NO}}_3^{-}$ naturally present in Na${\mathrm{NO}}_3^{-}$ at a natural ¹⁵N/¹⁴N isotopic ratio of 0.0036765. In the ¹⁵${\mathrm{NH}}_4^{+}$-amended cultures, the ⁴⁵[N₂O]/⁴⁴[N₂O] and ⁴⁶[N₂O]/⁴⁴[N₂O] ratios measured were 0.008 and 0.0165, respectively, with an ¹⁵N/¹⁴N isotopic ratio of 0.020418. As a control, strain JAM1 cultured under anoxic conditions with ¹⁵${\mathrm{NO}}_3^{-}$ in NH₄Cl-free medium with acetylene showed, as was expected, all N₂O recovered in ⁴⁶[N₂O]. Because low ¹⁵N/¹⁴N isotopic ratio were found in the ¹⁵${\mathrm{NH}}_4^{+}$-amended cultures, our results suggest that N₂O do not proceed through ${\mathrm{NH}}_4^{+}$.

NO reduction by M. nitratireducenticrescens JAM1

To verify NO reduction by strain JAM1, N₂O generation was monitored in cultures without ${\mathrm{NO}}_3^{-}$ and supplemented with sodium nitroprusside hypochloride (SNP) used as an NO donor (Moore et al., 2004). Because N₂O is quickly reduced under anoxic conditions but accumulates under oxic conditions, these assays were performed under oxic conditions (Fig. 5). N₂O started to accumulate in both 2 mM and 5 mM SNP-supplemented media after 24 h of incubation, reaching 7.9 ± 0.5 µg-N vial⁻¹ and 14.5 ± 0.4 µg-N vial⁻¹, respectively, after 168 h. No N₂O production was observed in strain JAM1 cultures without SNP or in the controls with non-inoculated culture medium supplemented with SNP or inoculated with autoclaved biomass.

Role of Nar systems in NO/N₂O production

In the absence of NirK or NirS, N₂O could have been generated via NO by the Nar system (see ‘Discussion’). We used the JAM1ΔnarG1narG2 double mutant, which lacks functional Nar-type nitrate reductases and which cannot grow under anoxic conditions (Mauffrey, Martineau & Villemur, 2015). Strain JAM1 and the JAM1ΔnarG1narG2 were cultured with 16.8 mg-N vial⁻¹ ${\mathrm{NO}}_3^{-}$ under oxic conditions. The growth of strain JAM1 and the mutant was similar (Mauffrey, Martineau & Villemur, 2015). After 96 h of incubation, strain JAM1 completely reduced ${\mathrm{NO}}_3^{-}$ to ${\mathrm{NO}}_2^{-}$ and produced 0.14 mg-N vial⁻¹ of N₂O (Table 2). As was expected, ${\mathrm{NO}}_3^{-}$ was not reduced, and ${\mathrm{NO}}_2^{-}$ was not produced by JAM1ΔnarG1narG2. Contrary to the wild type strain, the mutant did not produce N₂O.

The influence of ${\mathrm{NO}}_2^{-}$ was also tested. As the toxicity of ${\mathrm{NO}}_2^{-}$ has been attested from 0.36 mM (0.2 mg-N vial⁻¹) (Auclair et al., 2010), strain JAM1 and the mutant were cultured without ${\mathrm{NO}}_3^{-}$ under oxic conditions to allow for biomass growth. After 24 h, 4.7 mg-N vial⁻¹ NO${}_2^{-}$ was added to the cultures and was incubated for another 72 h. Strain JAM1 and the mutant produced 0.11 mg-N vial⁻¹ and 0.18 mg-N vial⁻¹ of N₂O, respectively, reflecting N₂O concentrations produced by strain JAM1 under oxic conditions with ${\mathrm{NO}}_3^{-}$ (Table 2). Our results show that ${\mathrm{NO}}_2^{-}$ and not ${\mathrm{NO}}_3^{-}$ is directly involved in N₂O production, and the Nar systems are not involved in N₂O production via NO.

Relative expression levels of denitrification genes in M. nitratireducenticrescens JAM1

We assessed whether variations in the expression levels of denitrification genes correlate with the N₂O accumulation and consumption cycles of strain JAM1 cultures. Strain JAM1 was cultured in NH₄Cl-free medium with 22 mg-N vial⁻¹ ${\mathrm{NO}}_3^{-}$ under anoxic conditions. RNA was extracted from cells harvested over four different phases (Fig. 4): (1) at T0 for the pre-cultures (oxic cultures with no ${\mathrm{NO}}_3^{-}$), (2) during the growth phase with ${\mathrm{NO}}_3^{-}$ reduction and no N₂O accumulation, (3) during the N₂O accumulation phase, and (4) during the N₂O consumption phase. The transcript levels of cnorB1, cnorB2 and nosZ, which encode the catalytic subunits of the corresponding NO and N₂O reductases, and nnrS and norR, were measured by RT-qPCR. nnrS and norR encode NO-sensitive regulators and were used as an indicator of the presence of NO in the cultures. Because the assimilatory nitrate reductase is involved in the re-routing of ${\mathrm{NO}}_3^{-}$ to the biomass, RT-qPCR assays were also performed on the gene encoding this reductase (named here nr). The expression levels were calculated relative to the transcript levels measured during the preculture phase (set to one) (Fig. 6).

The relative cnorB1 transcript levels showed an 18.5-fold increase during the growth phase. cnorB1 expression was still upregulated during the N₂O accumulation and consumption phases (5.5 and 6.9-fold increases, respectively). The relative cnorB2 transcript levels had a 1.6-fold increase during the growth phase. These levels returned nearly to the same levels of those in the preculture phase. Significant increases (5.5- and 6.0-fold) of the relative expression levels of nnrS were observed in the N₂O accumulation and consumption phases. norR was upregulated (2.3-fold increase) during the growth phase. The nosZ expression levels had a 5.4-fold increase during the growth phase relative to the preculture phase, and decreased to the preculture levels during the N₂O accumulation phase. No significant difference was observed in the relative transcript levels of the nr gene between all phases.