Materials and Reagents

Mycolicibacterium smegmatis mc²155
Middlebrook 7H10 agar (BD Biosciences, catalog number: 262710)
Middlebrook 7H9 broth (BD Biosciences, catalog number: 271310)
Glycerol (Sigma-Aldrich, catalog number: G5516)
Tween 80 (Sigma-Aldrich, catalog number: P1754)
Ethanol (Sigma-Aldrich, catalog number: 51976)
Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626)
Bradford protein assay kit (Beyotime, catalog number: P0006)
Sodium nitrate (NaNO₃) (Sigma-Aldrich, catalog number: S5506)
Sodium nitrite (NaNO₂) (Sigma-Aldrich, catalog number: S2252)
Sodium phosphate dibasic (Na₂HPO₄) (Sigma-Aldrich, catalog number: 255793)
Sodium phosphate monobasic (NaH₂PO₄) (Sigma-Aldrich, catalog number: S3139)
Nicotinamide adenine dinucleotide phosphate (NADPH) (Sigma-Aldrich, catalog number: N7505)
Nicotinamide adenine dinucleotide (NADH) (Sigma-Aldrich, catalog number: 43420)
Methyl viologen (Sigma-Aldrich, catalog number: 856177)
Flavin mononucleotide (FMN) (Sigma-Aldrich, catalog number: F2253)
Flavin adenine dinucleotide (FAD) (Sigma-Aldrich, catalog number: F6625)
Sodium hydrosulfite (Na₂S₂O₄) (Sigma-Aldrich, catalog number: 71699)
Sodium bicarbonate (NaHCO₃) (Sigma-Aldrich, catalog number: S6014)
Sulfanilamide (Sigma-Aldrich, catalog number: S9251)
N-(1-naphthyl)ethylenediamine dihydrochloride (NED) (Sigma-Aldrich, catalog number: 222488)
7H10 agar (see Recipes)
7H9 medium (see Recipes)
MPLN medium (see Recipes)
0.5 M sodium phosphate buffer, pH 7.5 (see Recipes)
0.1 M sodium phosphate buffer, pH 7.5 (see Recipes)
0.1 M PMSF (see Recipes)
60 mM Na₂S₂O₄/NaHCO₃ (see Recipes)
1% (w/v) sulfanilamide solution (see Recipes)
0.01% (w/v) NED solution (see Recipes)

Equipment

Pipette kit (Gilson, catalog number: F167300)
High-speed centrifuge (Beckman, catalog number: AVANTI JXN-26)
Ultrasonic cell disruptor (Beyotime, catalog number: E0385)
Ultrasonic bath equipped with a degas mode (Beyotime, catalog number: E0439)
250-ml and 2-L Erlenmeyer flasks
Petri dish (90 mm diameter)
37°C incubator
Incubator shaker
Water bath
Spectrophotometer
Anaerobic chamber (glove box) filled with mixed gas (nitrogen, 95%; hydrogen, 5%)
Note: Place the pipette kit, ultrasonic cell disruptor, and water bath inside the anaerobic chamber.

Software

Microsoft Excel

Procedure

Preparation of M. smegmatis culture
1. Streak M. smegmatis frozen culture on a 7H10 agar plate and incubate aerobically at 37°C for 3 days until colonies are visible.
2. Inoculate a single colony into 50 ml 7H9 medium in a 250-ml Erlenmeyer flask and incubate aerobically at 37°C for 16 h with shaking at 180 rpm (overnight culture).
3. Collect cells from the overnight culture by centrifuging at 3,000 × g for 10 min. Wash the pellet twice with nitrogen-free MPLN medium and centrifuge as above. Resuspend the cells in 50 ml MPLN medium as the seeding culture.
4. Re-inoculate 10 ml seeding culture into 1 L MPLN medium supplemented with 10 mM nitrate in a 2-L Erlenmeyer flask and incubate aerobically at 37°C for 30 h with shaking to late-log phase (OD₆₀₀ 0.8–1.0).
Preparation of M. smegmatis crude extract
1. Collect cells by centrifuging at 5,000 × g for 10 min at 4°C.
2. Wash the cell pellet twice with 30 ml degassed 0.1 M sodium phosphate buffer (pH 7.5).
3. Transfer the cell pellet into an anaerobic chamber and resuspend in 20 ml degassed 0.1 M sodium phosphate buffer (pH 7.5) supplemented with 10% v/v glycerol and 1 mM PMSF.
4. Disrupt cells by sonication (pulse: 0.3 s, stop: 0.7 s) for 10 min on ice and transfer the lysate to a centrifuge tube inside the anaerobic chamber.
5. Centrifuge at 16,000 × g for 1 h at 4°C outside the anaerobic chamber.
6. Bring the centrifuge tube back into the anaerobic chamber and collect the supernatant (cytoplasmic fraction) as the crude enzyme extract.
  Note: Keep on ice during the assay and use within 24 h.
7. Determine the total protein concentration of the cytoplasmic fraction using the Bradford method.
Preparation of the nitrite standard curve
1. Prepare a nitrite dilution series using sodium nitrite (0, 3, 30, 60, 120, 240, and 300 μM) in deionized water.
2. Pipette 0.5 ml nitrite dilution series (Test sample) into 1.5-ml tubes. Use 0.5 ml 10 mM sodium nitrate as a control (Blank sample).
3. Add 0.5 ml sulfanilamide solution and 0.5 ml NED solution to all tubes.
4. Mix by swirling and incubate at 25°C for 10 min.
5. Transfer 1 ml assay mixture to a cuvette.
6. Measure the visible absorption at 540 nm using the spectrophotometer.
7. Calculate the absorbance change using the following equation:
  ΔA_{540 nm} (Sample) = A_{540 nm} (Test) - A_{540 nm} (Blank)
8. Plot the standard curve with the nitrite concentration on the horizontal axis and the absorbance change on the vertical axis (Figure 1).
  
  Figure 1. Nitrite standard curve. Error bars indicate the average value of three biological replicates ± SD (n = 3). According to the standard curve, the regression equation is x (µM) = (y-0.0204)/0.0092, where x stands for nitrite concentration and y stands for ΔA_{540 nm}(Sample).

In vitro NaR activity assay

Before preparing the reaction mixture for the enzyme assay, degas the solutions used in the assays and place in the anaerobic chamber before use.

Pipette the following reagents for the nitrate reduction reaction (Tables 1 and 2) into a 1.5-ml tube inside the anaerobic chamber:

Table 1. Assay mixtures for determining NAD(P)H-dependent NaR activity

	Assay sample
	Test	Blank
Deionized water	0.090 ml	0.100 ml
Sodium phosphate buffer (0.5 M)	0.100 ml	0.100 ml
NaNO₃(50 mM)	0.100 ml	0.100 ml
NADPH (2 mM) or NADH (2 mM)	0.100 ml	0.100 ml
FMN (0.25 mM)	0.050 ml	0.050 ml
FAD (0.25 mM)	0.050 ml	0.050 ml
Crude enzyme extract	0.010 ml	-
Step 1: Mix gently and incubate at 30°C for 10 min inside the anaerobic chamber
1% (w/v) sulfanilamide solution	0.500 ml	0.500 ml
0.01% (w/v) NED solution	0.500 ml	0.500 ml
Step 2: Mix gently and incubate at 25°C in the dark for 10 min inside the anaerobic chamber

Table 2. Assay mixtures for determining methyl viologen-dependent NaR activity

	Assay sample
	Test	Blank
Deionized water	0.090 ml	0.100 ml
Sodium phosphate buffer (0.5 M)	0.100 ml	0.100 ml
NaNO₃(50 mM)	0.100 ml	0.100 ml
Methyl viologen (0.75 mM)	0.100 ml	0.100 ml
Na₂S₂O₄/NaHCO₃(60 mM)	0.100 ml	0.100 ml
Crude enzyme extract	0.010 ml	-
Step 1: Mix gently and incubate at 30°C for 10 min inside the anaerobic chamber Step 2: Stop the reaction by vigorous stirring in the open air outside the anaerobic chamber until the blue color disappears
1% (w/v) sulfanilamide solution	0.500 ml	0.500 ml
0.01% (w/v) NED solution	0.500 ml	0.500 ml
Step 3: Mix gently and incubate at 25°C in the dark for 10 min outside the anaerobic chamber

Note: The assay mixture turns deep blue after adding Na₂S₂O₄/NaHCO₃, an indication of methyl viologen being reduced.

Transfer 1 ml assay mixture to a cuvette.
Measure the visible absorption at 540 nm for both the Test and Blank samples.
Calculate the NaR specific activity using the following equation:

NaR specific activity (nmol/min/mg) = C_nitriteDF/T/C_enzyme/V_enzyme

where,
C_nitrite(µM) = [A_{540 nm} (Test) - A_{540 nm} (Blank)-0.0204]/0.0092, the concentration of nitrite formed in the assay
DF: The dilution factor
T (min): The reaction time for nitrate reduction in the assay
Note: An incubation time of 10 min is sufficient for measuring the in vitro nitrate reductase activity.
C_enzyme(mg/ml): The protein concentration of the crude enzyme extract
V_enzyme(ml): The volume of the crude enzyme extract used in the assay

Recipes

7H10 agar
Dissolve 19 g 7H10 powder in 1 L deionized water
Add 4 ml 50% (v/v) glycerol and 5 ml 10% (v/v) Tween 80
Autoclave at 121°C for 15 min
7H9 medium
Dissolve 4.7 g 7H9 powder in 1 L deionized water
Add 4 ml 50% (v/v) glycerol and 5 ml 10% (v/v) Tween 80
Autoclave at 121°C for 15 min
Nitrogen-free MPLN medium (pH 6.6)
36.7 mM KH₂PO₄
7.7 mM sodium citrate
5 mM MgSO₄
60 µM FeCl₃
20 µM NaMoO₄
3.4 µM CaCl₂
3.5 µM ZnSO₄
4 µM CuSO₄
2% (v/v) glycerol
0.05% (v/v) Tween 80
Autoclave at 121°C for 15 min.
0.5 M sodium phosphate buffer (pH 7.50)
Prepare 0.5 M solutions of NaH₂PO₄ and Na₂HPO₄
Mix 20% (v/v) NaH₂PO₄ and 80% (v/v) Na₂HPO₄
Adjust pH to 7.5
0.1 M sodium phosphate buffer (pH 7.50)
Dilute 0.5 M sodium phosphate buffer 1:5 with deionized water
Store at room temperature
0.1 M PMSF
Dissolve 0.174 g PMSF in 10 ml ethanol
Store at -20°C
60 mM Na₂S₂O₄/NaHCO₃
Prepare 120 mM solutions of Na₂S₂O₄and NaHCO₃
Mix 50% (v/v) Na₂S₂O₄and 50% (v/v) NaHCO₃
Note: Prepare fresh
1% (w/v) sulfanilamide solution
Dissolve in 3 M HCl
Note: Prepare fresh and store at 4°C in the dark
0.01% (w/v) NED solution
Dissolve in deionized water
Note: Prepare fresh and store at 4°C in the dark

Acknowledgments

This protocol was adapted from a previously published paper by Tan et al. (2020). This research was supported by the National Natural Science Foundation of China (31430004, 31830002, 81671988, 91751000, 91951000 to G.-P. Z.), the Research Unit Fund of Li Ka Shing Institute of Health Sciences (7103506 to G.-P. Z.), and the Hong Kong Health and Medical Research Fund (12110622 to G.-P. Z.).

Competing interests

The authors declare that they have no conflicts of interest.

References

Kuypers, M. M. M., Marchant, H. K., and Kartal, B. (2018). The microbial nitrogen-cycling network. Nat Rev Microbiol 16(5): 263-276.
Morozkina, E. V., and Zvyagilskaya, R. A. (2007). Nitrate reductases: structure, functions, and effect of stress factors. Biochemistry (Mosc) 72(10): 1151-1160.
Tan, W., Liao, T. H., Wang, J., Ye, Y., Wei, Y. C., Zhou, H. K., Xiao, Y., Zhi, X. Y., Shao, Z. H., Lyu, L. D. and Zhao, G. P. (2020). A recently evolved diflavin-containing monomeric nitrate reductase is responsible for highly efficient bacterial nitrate assimilation. J Biol Chem 295(15): 5051-5066