Materials and Reagents

Pipette tips
50 ml sterile conical Falcon tubes (Nunc^® EZ Flip^TM, Thermo Fisher Scientific, catalog number: 362696 )
1.5 ml sterile Eppendorf centrifuge tubes (Eppendorf, catalog number: 022364111 )
Sonication device
Note: This was assembled in our laboratory by attaching four plastic tubes (3 cm diameter, 3 cm high) to a plastic box (Figure 1).

Figure 1. Sonication device
Paper towel (WypAll* X 60 Jumbo Roll, KCWW, Kimberly-Clark, catalog number: 30218593 )
Spatula
Pyrex tray (Pyrex^® Storage 13 x 18 cm)
P. aeruginosa strains
Note: PAO1, referred to as the wild-type, and catalase mutants PW8190 and PW8769 were obtained from the Washington Genome Center. Catalase mutants were constructed by insertion of IslacZ/hah transposon into katA (PW8190, hereinafter KatA-less strain) or katB (PW8769, hereinafter KatB-less strain) (Jacobs et al., 2003).
Distilled water
Tryptone (Oxoid, catalog number: LP0042 )
Yeast extract (Merck, catalog number: 103753 )
Sodium chloride (NaCl) (Biopack, catalog number: 1646.08 )
Albumin from bovine serum (Sigma-Aldrich, catalog number: A4378 )
Sodium phosphate dibasic (Na₂HPO₄) (Sigma-Aldrich, catalog number: S3264 )
Sodium phosphate (NaH₂PO₄) (Sigma-Aldrich, catalog number: S0751 )
Hydrogen peroxide (H₂O₂) 30% (Merck, catalog number: 107210 )
Bugbuster Protein Extraction Reagent (Merck, Novagen, catalog number: 70584-4 )
Sodium thiosulfate (Na₂S₂O₃) (Avantor Performance Materials, Macron^TM, catalog number: 8100-04 )
Ammonium persulfate ((NH₄)₂S₂O₈) (MP Biomedicals, catalog number: 04802811 )
TEMED (MP Biomedicals, catalog number: 02195516 )
Trizma base (Tris[hydroxymethyl]aminomethane) (C₄H₁₁NO₃) (Sigma-Aldrich, catalog number: T1503 )
Glycine (Sigma-Aldrich, catalog number: G7126 )
Hydrochloric acid fuming 37% (HCl) (Merck, catalog number: 100317 )
Acrylamide (Sigma-Aldrich, catalog number: A8887 )
Bisacrylamide N,N’-methylene-bis-acrylamide (Sigma-Aldrich, catalog number: M7256 )
EDTA (Merck, Calbiochem, catalog number: 324503 )
Sodium hydroxide (NaOH) (Avantor Performance Materials, Macron^TM, catalog number: 7708 )
Glycerol (Merck, catalog number: 104094 )
Bromophenol blue (VWR, DBH, catalog number: 20015 )
Ferric chloride (FeCl₃) (Avantor Performance Materials, Macron^TM, catalog number: 5029-04 )
Potassium ferricyanide (K₃Fe (CN)₆) (UCB, catalog number: b1599 )
LB medium (see Recipes)
4 M NaCl (see Recipes)
Saline solution (see Recipes)
50 mM sodium phosphate buffer, pH 7 (see Recipes)
30 mM H₂O₂ (see Recipes)
4 mM H₂O₂ (see Recipes)
10% ammonium persulfate (see Recipes)
1.5 M Tris-HCl buffer pH 8.8 (see Recipes)
1.5 M Tris-HCl buffer pH 6.8 (see Recipes)
30% acrylamide mix solution (acrylamide bisacrylamide ratio 37.5:1) (see Recipes)
6% resolving gel solution (see Recipes)
5% stacking gel solution (see Recipes)
1 M Tris-HCl buffer pH 8 (see Recipes)
0.5 M EDTA pH 8 (see Recipes)
Loading sample buffer (see Recipes)
Running buffer (see Recipes)
Ferric chloride/potassium ferricyanide solution (see Recipes)

Equipment

50, 125 and 150 ml sterile Erlenmeyer flasks (DWK Life Sciences, Duran^®, catalog numbers: 21 216 17 , 21 216 28 , 21 990 27 )
2-20 µl, 20-100 µl, 100-1,000 µl Kartell pluripet micropipettes (Kartell LABWARE, catalog numbers: 13000 , 13210 , 13220 ) and 1-10 ml Acura^® manual micropipette (Socorex, model: Acura^® manual 825 / Acura^® manual 835 )
50, 100 and 1,000 ml borosilicate measuring cylinders (VILABO, catalog number: 3501114 , 3501115 , 3501118 )
Conventional incubator shaker (New Brunswick Scientific, model: G25 )
Gyratory water bath shaker (New Brunswick Scientific, model: G76 )
Ice maker (Brema, model: TB 551 )
UV-Vis Spectrophotometer (Biotraza, model: 752 )
Refrigerated centrifuge (Hanil Scientific, model: Combi 514R )
Vibra-Cell sonicator (Sonics & Materials, model: VC500 )
Electrophoresis cell (Bioamerica, model: DYCZ-24DNBA )
Power supply (Bioamerica, model: DYY-6CBA )
Freezer ultra-low temperature (Sanyo, model: MDF-U76VC )
Autoclave (HIRAYAMA, HICLAVETM, model: HVE-50 )
Hot air oven sterilizer (Dalvo Intrumentos, model: OHR/T )

Procedure

Preparation of cell extracts
Non-inducing conditions
1. Grow Pseudomonas aeruginosa strains (PAO1, KatA-less and KatB-less) overnight in 30 ml of LB medium in 150 ml Erlenmeyer flasks at 37 °C with shaking (200 rpm).
2. Centrifuge the cultures (20 ml) in 50 ml Falcon tubes for 10 min, 10,000 x g, 4 °C. Discard the supernatants.
3. Resuspend the cells with 20 ml of cold saline solution and keep them on ice.
4. Centrifuge for 10 min, 10,000 x g, 4 °C. Discard the supernatants.
5. Resuspend the cells in ice-cold 50 mM sodium phosphate buffer, pH 7 up to OD₆₅₀ 1 (about 7 ml).
6. Sonicate 5 ml of concentrated cells in plastic tubes in an ice-water bath (Figure 1) under the following conditions: 2 min in pulsed mode, 18 mm tip diameter, 50% duty cycle, microtip limit 2. Keep the samples on ice.
7. Centrifuge the extracts for 10 min, 10,000 x g, 4 °C. Discard the pellets carrying unlysed cells and cellular debris. Keep the supernatants on ice for a few hours until using them or store at -80 °C.
8. Determine protein concentration of the extracts according to Lowry et al. (1951); bovine serum albumin is used as a standard.
Inducing conditions
1. Grow Pseudomonas aeruginosa strains (PAO1 and KatB-less) overnight in 30 ml of LB medium in 150 ml Erlenmeyer flasks at 37 °C with shaking (200 rpm).
2. Dilute overnight cultures in 30 ml of fresh LB medium in 125 ml Erlenmeyer flasks to OD₆₅₀ 0.01 and grow in a gyratory water bath shaker at 37 °C until the cultures reach an OD₆₅₀ 0.3. This OD is reached in about 2 h.
3. Divide the cultures into two fractions of 10 ml each in 50 ml Erlenmeyer flasks. Maintain one of them untreated (control) and add 2.26 µl of 30 mM H₂O₂ to the other fraction every 10 min for 1 h under sterile conditions, while shaking both fractions at 37 °C.
4. Take 5 ml of the treated cultures at the end of 1 h and add 10 μl of 1 mg/ml sodium thiosulfate to neutralize the effect of H₂O₂; it is not necessary to keep the samples on ice.
5. Centrifuge the neutralized cultures and 5 ml of the control cultures for 10 min, 10,000 x g, 4 °C. Discard the supernatants.
6. Resuspend the cells with 10 ml of ice-cold 50 mM sodium phosphate buffer, pH 7.
7. Centrifuge for 10 min, 10,000 x g, 4 °C. Discard the supernatants.
8. Resuspend the cells in 1 ml of BugBuster Protein Extraction Reagent and keep for 15 min at room temperature.
9. Centrifuge for 10 min, 16,000 x g, 4 °C. Keep the supernatants on ice for a few hours until using them or store at -80 °C.
10. Determine protein concentration of the extracts according to Lowry et al. (1951); bovine serum albumin is used as a standard.
The protocol for inducing conditions is schematized in Figure 2.
Notes:
1. Antibiotics are not added to culture media.
2. BugBuster Protein Extraction Reagent was employed instead of sonication to detect KatB activity. This reagent is capable of cell wall perforation without denaturing soluble proteins. It provides an alternative to mechanical methods such as French Press or sonication for releasing proteins.
Figure 2. Schematic diagram of the experimental procedure
PAGE
1. Set the two glass plates with the special wedge frames in the gel casting stand (see Figure 3).
  
  Figure 3. Electrophoresis cell (Bioamerica)
2. Pipet the 6% resolving gel solution into the gap between the glass plates.
3. Wait for 15-30 min until it solidifies.
4. Pipet the 5% stacking gel solution until overflow.
5. Insert the comb without trapping air under the teeth. Wait for 15-30 min until it solidifies.
6. Take the glass plates out of the gel casting stand and set them into the electrophoresis cell.
7. Pour running buffer into the electrophoresis cell until the buffer level is higher than the top of the (shorter) inner gel plate (i.e., until the buffer covers the wells).
8. Mix 50 µl of the cell extracts with 2 µl of loading buffer in 1.5 ml Eppendorf tubes and heat them in boiling water for 10 min.
9. Load 10 µg of protein per sample into each well. Cover the cell with the lid and connect the electrodes to the power supply.
10. Run the electrophoresis at 15 mA at room temperature (non-inducing conditions, KatA activity) or at 4 °C (inducing conditions, KatB activity).
  Note: Electrophoresis is run at 4 °C for detecting KatB activity. This can be done in a cold room or in a refrigerator. However, this is not necessary for the detection of KatA activity.
11. Stop PAGE running when the dye front almost reaches the foot line of the glass plate. The run generally takes 4 h.
Gel development
1. Remove the glass plates from the electrophoresis tank and place them on a paper towel. Separate the plates by using a spatula.
2. Soak the gel in distilled water in a Pyrex tray for 5 min at room temperature. Discard the water.
3. Incubate the gel with 100 ml of a solution containing 4 mM H₂O₂ for 10 min at room temperature.
4. Remove the solution and wash the gel with 100 ml of distilled water at room temperature.
5. Soak the gel in 100 ml of a solution containing 1% (w/v) ferric chloride and 1% (w/v) potassium ferricyanide at room temperature.
6. As soon as the gel turns dark green, remove the ferric chloride/potassium ferricyanide solution and rinse with distilled water to prevent overdevelopment.
7. Once the dye has been removed, photograph the gel immediately. Storage in distilled water for a few days at 4 °C is possible. Areas of catalase activity show up as clear bands.
Note: It is not necessary to use a shaker or a nutator for the washing and staining steps.

Data analysis

According to this procedure described above, Figure 4 shows representative images of non-denaturing polyacrylamide gels stained for catalase activity employing non-inducing conditions (A) or inducing conditions with H₂O₂ (B).

Figure 4. Catalase native PAGE analysis of P. aeruginosa wild type (PAO1) and its derivatives KatA-less and KatB-less strains. A. Extracts of PAO1 (wild-type), KatA-less and KatB-less strains grown under non-inducing conditions. The only activity detected corresponds to KatA since KatB is not expressed under this condition. B. Cultures of PAO1 and KatB-less strains, untreated (-) or treated with sublethal concentrations of H₂O₂ (+), were analyzed for catalase activity to demonstrate detection of KatB activity in non-denaturing polyacrylamide gels.
Note: The images were originally reported in Pezzoni et al., (2014). Protective role of extracellular catalase (KatA) against UVA radiation in Pseudomonas aeruginosa biofilms. J Photochem Photobiol B: Biol 131, 53-64.

Recipes

LB medium
Dissolve:
10 g tryptone
5 g yeast extract
5 g NaCl
Bring the volume up to 1,000 ml in distilled water
Autoclave at 1 atm for 20 min
4 M NaCl
Dissolve 46.7 g NaCl in 200 ml of distilled water
Autoclave at 1 atm for 20 min
Saline solution
Mix 7.5 ml sterile 4 M NaCl with 300 ml of sterile distilled water
50 mM sodium phosphate buffer, pH 7
Solution A: dissolve 70.99 g Na₂HPO₄ in 500 ml of distilled water
Solution B: dissolve 59.98 g NaH₂PO₄ in 500 ml of distilled water
Mix 10.6 ml of solution A with 14.4 ml of solution B and add 475 ml distilled water
30 mM H₂O₂
Add 0.340 ml of 30% H₂O₂ to 100 ml 50 mM sodium phosphate buffer, pH 7
Prepare fresh for every activity assay
The solution can be kept at room temperature during the experiment
4 mM H₂O₂
Add 0.045 ml of 30% H₂O₂ to 100 ml distilled water
Prepare fresh for every activity assay
10% ammonium persulfate
Dissolve 1 g ammonium persulfate in 10 ml distilled water
Store at -20 °C (shelf life 3 months)
1.5 M Tris-HCl buffer pH 8.8
Dissolve 18.5 g Trizma base in 80 ml distilled water
Adjust to pH 8.8 with concentrated HCl and make up the volume to 100 ml
1 M Tris-HCl buffer pH 6.8
Dissolve 12.114 g Trizma base in 80 ml distilled water
Adjust to pH 6.8 with concentrated HCl and make up the volume to 100 ml
30% acrylamide mix solution
Dissolve 60 g acrylamide and 1.6 g bis acrylamide in 200 ml distilled water (acrylamide bisacrylamide ratio 37.5:1)

Notes:

Avoid directly contacting with polyacrylamide, ferricyanide and gels; they need to be handled with care.
The order of adding solutions in the resolving and stacking gels solutions is important to avoid an early polymerization before pouring them between the glass plates.

6% resolving gel solution
5.4 ml distilled water
2 ml 30% acrylamide mix
2.5 ml 1.5 M Tris (pH 8.8)
0.1 ml 10% ammonium persulfate
0.008 ml TEMED
5% stacking gel solution
2.1 ml distilled water
0.5 ml 30% acrylamide mix
0.38 ml 1.5 M Tris (pH 6.8)
0.03 ml 10% ammonium persulfate
0.003 ml TEMED
1 M Tris-HCl buffer pH 8
Dissolve 12.114 g Trizma base in 80 ml distilled water
Adjust to pH 8 with concentrated HCl and make up the volume to 100 ml
0.5 M EDTA pH 8
Dissolve 47 g EDTA in 200 ml distilled water
Adjust to pH 8 with concentrated NaOH and make up the volume to 250 ml
Loading sample buffer
1 ml 1 M Tris-HCl (pH 8)
4 ml 0.5 M EDTA (pH 8)
4 ml glycerol
25 mg bromophenol blue
1 ml distilled water
Store at -20 °C
Running buffer
Dissolve 3.03 g Trizma base and 14.4 g glycine in 1,000 ml distilled water
Store at 4 °C (shelf life 3 months)
Ferric chloride/potassium ferricyanide solution
Dissolve 1 g ferric chloride and 1 g potassium ferricyanide in 100 ml distilled water
Prepare fresh for every activity assay

Acknowledgments

The excellent technical assistance of Ms. P. Pereyra Schuth is gratefully acknowledged. This protocol is based on the work by Wayne and Díaz (1986). Financial support for this research was received from the Comisión Nacional de Energía Atómica (Argentina). M.P. is investigator at Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina).

Competing interests

The authors declare no conflict of interest.

References

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