Zymogram Assay for the Detection of Peptidoglycan Hydrolases in Streptococcus mutans   

Materials and Reagents

1. Bacterial strain (S. mutans UA159 wild-type strain or other S. mutans strains)
   
2. Todd-Hewitt broth (BD Biosciences)
   
3. Yeast-Extract (BioShop)
   
4. Tris Base
   
5. NaCl
   
6. Sodium dodecyl sulfate (SDS)
   
7. Triton X-100
   
8. KOH
   
9. MgCl₂
   
10. Glycine
    
11. Glycerol
    
12. Bromophenol blue
    
13. Methylene blue
    
14. 40% Acrylamide/Bis solution (37.5:1 acrylamide:bisacrylamide) (BioShop)
    
15. Ammonium persulfate (Sigma-Aldrich)
    
16. TEMED (BioBasic, Inc.)
    
17. Ethanol
    
18. Isopropanol
    
19. dH₂O
    
20. Filter paper
    
21. Precision Plus Protein Prestained Standards (Bio-Rad Laboratories)
    
22. THYE broth (see Recipes)
    
23. 20 mM Tris, 100 mM NaCl (pH 7.4) (see Recipes)
    
24. 1.5 M Tris (pH 8.8) (see Recipes)
    
25. 0.5 M Tris (pH 6.8) (see Recipes)
    
26. SDS-PAGE loading buffer (see Recipes)
    
27. Tris-Glycine SDS running buffer (see Recipes)
    
28. Zymogram renaturing buffer (see Recipes)
    
29. Staining solution (see Recipes)
    
30. 10% separating gel solution (see Recipes)
    
31. 4% stacking gel solution (see Recipes)
    

Equipment

1. 15-ml canonical tubes
   
2. Flasks
   
3. 1.5 ml microcentrifuge tubes
   
4. Refrigerated centrifuge
   
5. Refrigerated microcentrifuge
   
6. CO₂ incubator
   
7. Spectrophotometer
   
8. Disposable plastic cuvettes
   
9. Protein mini gel cassettes
   
10. Heating block module
    
11. Power supply
    
12. Orbital shaker
    
13. 37 °C temperature chamber
    

Procedure

1. Preparation of the bacterial substrates incorporated into the gel
   1. Start 5 ml culture of S. mutans UA159 wild-type strain (or other S. mutans strains) in THYE broth into a 15-ml canonical tube and incubate overnight statically at 37 °C in air with 5% CO₂.
      
   2. Inoculate 300 ml of fresh THYE broth with 1% of the overnight preculture into a 500-ml flask.
      
   3. Incubate the culture statically at 37 °C in air with 5% CO₂ until an optical density at 600 nm (OD₆₀₀) of 0.2 is reached.
      
   4. Harvest the cells by centrifugation at 10,000 x g for 10 min at 4 °C.
      
   5. Wash the cells using 5 ml of 20 mM Tris, 100 mM NaCl buffer (pH 7.4) and resuspend the cell pellet in 1.0 ml of 1.5 M Tris buffer (pH 8.8).
      
   6. Keep the cells at -20 °C until used.
      
      
2. Preparation of bacterial whole-cell extracts
   1. Start 5 ml overnight culture of S. mutans UA159 wild-type strain (or other S. mutans strains) in THYE broth into a 15-ml canonical tube and incubate statically at 37 °C in air with 5% CO₂. Whole cell extract of a mutant strain deficient in the peptidoglycan hydrolase under study can also be analyzed concomitantly as negative control to confirm the specificity of the hydrolytic band(s) observed.
      
   2. Inoculate 10 ml of fresh THYE broth with 1% of the overnight preculture into a 15-ml canonical tube.
      
   3. Incubate the culture statically at 37 °C in air with 5% CO₂ until the desired OD₆₀₀ is reached. If the expression profile of the targeted peptidoglycan hydrolase is not known, we recommend to harvest cells at different optical densities corresponding to early log, mid-log, early stationary, and late stationary phase of growth.
      
   4. Harvest the cells by centrifugation at 10,000 x g for 10 min at 4 °C.
      
   5. Keep the cell pellet at -20 °C until used.
      
   6. Resuspend the cell pellet in 20 μl of SDS-PAGE loading buffer freshly prepared.
      
   7. Heat the samples at 95 °C for 10 min. Keep the samples on ice until loading.
      
      
3. Preparation of the zymogram gel
   1. Clean glass plates, spacers, and combs with ethanol and completely dry before use. Assemble the gel cassette following the manufacturer’s instructions.
      
   2. Prepare 10% separating gel solution (see Recipe 9).
      
   3. Transfer the separating gel solution (approx. 3.8 ml per small gel) to the casting chamber between the glass plates and fill up to about 0.7 cm below the bottom of the comb when the comb is in place.
      
   4. Add a small layer of isopropanol to the top of the gel prior to polymerization to straighten the level of the gel. Once the gel has polymerized, remove the isopropanol layer by several washes with dH₂O, and dry with filter paper.
      
   5. Prepare 4% stacking gel solution (see Recipe 10).
      
   6. Pour the stacking gel solution (approx. 2.5 ml per small gel) on top of the separating gel until the space is full, and then insert the appropriate comb. Once the gel has polymerized, carefully remove comb.
      
   7. Remove the gel cassette from the casting stand and place it in the electrode assembly as recommended by the manufacturer.
      
   8. Pour Tris-Glycine SDS running buffer into the opening of the casting frame between the gel cassettes. Add enough buffer the fill the wells of the gel. Fill also the region outside of the frame.
      
   9. Load the samples (from Section II, step B-7) into each well as well as 5 μl of the Precision Plus Protein Prestained Standards.
      
   10. Connect the electrophoresis tank to the power supply. Run the gel at a constant voltage between 125-200 volts until the dye front is near the bottom of the gel.
       
       
4. Peptidoglycan hydrolase detection
   1. Remove the gel from the electrophoresis chamber, allow the gel to peel away and gently drop into a container. Wash the gel twice in 100 ml of dH₂O for 30 min at room temperature under constant agitation.
      
   2. Incubate the gel in 100 ml of zymogram renaturing buffer for 30 min at room temperature under constant agitation. This step is necessary to renature the peptidoglycan hydrolases.
      
   3. Replace the zymogram renaturing buffer with fresh zymogram renaturing buffer and incubate the gel at 37 °C in a temperature chamber under constant agitation until clear hydrolytic band(s) appear, usually between 16 h and 48 h. The proteolytic activity appears as clear bands against a white background.
      
   4. Optional staining step: Decant the buffer and add 100 ml of staining solution, and incubate the gel at room temperature under constant agitation between 15 min and 2 h. Regions without staining are indicative of lysis (Figure 1). The proteolytic activity appears as clear bands against a blue background.
      
      
      Figure 1. Zymogram activity gel after methylene blue staining. Heat-killed cells of S. mutans were used as substrate and were incorporated into a 10% SDS-PAGE gel. (1) Molecular size marker (Precision Plus Protein Prestained Standards). (2) Whole-cell extract of S. mutans UA159 wild-type strain. The two hydrolytic bands (arrows) observed correspond to the unprocessed (upper) and processed (lower) forms of the AtlA autolysin.

Recipes

1. THYE broth
   Dissolve 15 g of Todd-Hewitt and 1.5 g of Yeast Extract in 400 ml of dH₂O
   Once dissolved, bring up to a final volume of 500 ml with dH₂O
   Autoclave for 20 min at 120 °C
   Store at room temperature
   
2. 20 mM Tris, 100 mM NaCl buffer (pH 7.4)
   Dissolve 2.42 g of Tris Base, and 5.84 g NaCl in 800 ml of dH₂O
   Once dissolved, adjust the pH to 7.4, and then bring up to a final volume of 1 L with dH₂O
   Store at 4 °C
   
3. 1.5 M Tris pH 8.8 buffer
   Dissolve 181.71 g of Tris Base in 800 ml of dH₂O
   Once dissolved, adjust the pH to 8.8, and then bring up to a final volume of 1 L with dH₂O
   Store at 4 °C
   
4. 0.5 M Tris pH 6.8 buffer
   Dissolve 60.57 g of Tris Base in 600 ml of dH₂O
   Once dissolved, adjust the pH to 6.8, and then bring up to a final volume of 1 L with dH₂O
   Store at 4 °C
   
5. SDS-PAGE loading buffer (0.25 M Tris pH 6.8, 2% SDS, 10% glycerol, bromophenol blue)
   Dissolve 0.3 g of Tris Base, 0.2 g SDS, 1.0 ml glycerol, and traces of bromophenol blue in 7 ml of dH₂O
   Once dissolved, bring up to a final volume of 10 ml with dH₂O
   
6. Tris-Glycine SDS running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS)
   Dissolve 3.03 g of Tris Base, 14.4 g glycine, and 1 g SDS in 800 ml of dH₂O
   Once dissolved, bring up to a final volume of 1 L with dH₂O
   Store at 4 °C
   
7. Zymogram renaturing buffer (20 mM Tris, 50 mM NaCl, 20 mM MgCl2, 0.5% Triton X-100, pH 7.4)
   Dissolve 2.42 g of Tris Base, 2.92 g NaCl, and 4.06 g MgCl₂ in 800 ml of dH₂O
   Adjust the pH to 7.4
   Add 5 ml of Triton X-100, and bring up to a final volume of 1 L with dH₂O
   Store at 4 °C
   
8. Staining solution (0.1% methylene blue, 0.01% KOH)
   Dissolve 0.1 g of methylene blue and 0.01 g KOH in 100 ml of dH₂O
   Store at room temperature
   
9. 10% separating gel solution
   Mix the following reagents in a clean flask (total volume for 4 small gels):
   7.4 ml dH₂O
   3.7 ml 40% acrylamide/bis
   4 ml of bacterial substrate (from Section A, step A-6) boiled for 10 min just prior use
   100 μl 10% SDS
   50 μl 10% ammonium persulfate
   5 μl TEMED
   
10. 4% stacking gel solution
    Mix the following reagents in a clean flask (total volume for 4 small gels):
    6 ml dH₂O
    2.5 ml 0.5 M Tris (pH 6.8)
    1.0 ml 40% acrylamide/bis
    100 μl 10% SDS
    100 μl 10% ammonium persulfate
    25 μl TEMED
    

References

1. Berg, K. H., Ohnstad, H. S. and Havarstein, L. S. (2012). LytF, a novel competence-regulated murein hydrolase in the genus Streptococcus. J Bacteriol 194(3): 627-635.
   
2. Dufour, D. and Lévesque, C. M. (2013). Cell death of Streptococcus mutans induced by a quorum-sensing peptide occurs via a conserved streptococcal autolysin. J Bacteriol 195(1): 105-114.