Materials and Reagents

Bacterial strain (S. mutans UA159 wild-type strain or other S. mutans strains)
Todd-Hewitt broth (BD Biosciences)
Yeast-Extract (BioShop)
Tris Base
NaCl
Sodium dodecyl sulfate (SDS)
Triton X-100
KOH
MgCl₂
Glycine
Glycerol
Bromophenol blue
Methylene blue
40% Acrylamide/Bis solution (37.5:1 acrylamide:bisacrylamide) (BioShop)
Ammonium persulfate (Sigma-Aldrich)
TEMED (BioBasic, Inc.)
Ethanol
Isopropanol
dH₂O
Filter paper
Precision Plus Protein Prestained Standards (Bio-Rad Laboratories)
THYE broth (see Recipes)
20 mM Tris, 100 mM NaCl (pH 7.4) (see Recipes)
1.5 M Tris (pH 8.8) (see Recipes)
0.5 M Tris (pH 6.8) (see Recipes)
SDS-PAGE loading buffer (see Recipes)
Tris-Glycine SDS running buffer (see Recipes)
Zymogram renaturing buffer (see Recipes)
Staining solution (see Recipes)
10% separating gel solution (see Recipes)
4% stacking gel solution (see Recipes)

Equipment

15-ml canonical tubes
Flasks
1.5 ml microcentrifuge tubes
Refrigerated centrifuge
Refrigerated microcentrifuge
CO₂ incubator
Spectrophotometer
Disposable plastic cuvettes
Protein mini gel cassettes
Heating block module
Power supply
Orbital shaker
37 °C temperature chamber

Procedure

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.
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.
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.
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

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
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
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
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
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
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
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
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
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
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

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.
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.