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Zymogram Assay for the Detection of Peptidoglycan Hydrolases in Streptococcus mutans
采用酶谱分析法检测变异链球菌中的肽聚糖水解酶   

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Abstract

Peptidoglycan hydrolases or autolysins are enzymes capable of cleaving covalent bonds in bacterial peptidoglycan cell wall layer. They can participate in the cell division process, in the release of turnover products from peptidoglycan during cell growth, and in cell autolysis induced under particular conditions. The protocol for zymogram presented below should enable the identification of such enzymes through their separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis containing bacterial cells as substrate.

Keywords: Peptidoglycan(肽聚糖), Murein hydrolase(胞壁质水解酶), Streptococcus mutans(变形链球菌), Oral streptococci(口腔链球菌), Zymogram(酶谱)

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. 

简介

肽聚糖水解酶或自溶素是能够切割细菌肽聚糖细胞壁层中的共价键的酶。 它们可以参与细胞分裂过程,在细胞生长期间从肽聚糖释放周转产物,以及在特定条件下诱导的细胞自溶。 下面给出的酶谱方案应该能够通过含有细菌细胞作为底物的十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分离来鉴定这些酶。

关键字:肽聚糖, 胞壁质水解酶, 变形链球菌, 口腔链球菌, 酶谱

材料和试剂

  1. 细菌菌株(变异链球菌UA159野生型菌株或其他变异链球菌菌株)
  2. Todd-Hewitt肉汤(BD Biosciences)
  3. 酵母提取物(BioShop)
  4. Tris碱
  5. NaCl
  6. 十二烷基硫酸钠(SDS)
  7. Triton X-100
  8. KOH
  9. MgCl 2
  10. 甘氨酸
  11. 甘油
  12. 溴酚蓝
  13. 亚甲蓝
  14. 40%丙烯酰胺/双溶液(37.5:1丙烯酰胺:双丙烯酰胺)(BioShop)
  15. 过硫酸铵(Sigma-Aldrich)
  16. TEMED(BioBasic,Inc。)
  17. 乙醇
  18. 异丙醇
  19. dH 2 2 O
  20. 过滤纸
  21. Precision Plus蛋白质预染色标准品(Bio-Rad Laboratories)
  22. THYE汤(见食谱)
  23. 20mM Tris,100mM NaCl(pH7.4)(见Recipes)
  24. 1.5 M Tris(pH 8.8)(参见配方)
  25. 0.5 M Tris(pH 6.8)(参见配方)
  26. SDS-PAGE加载缓冲液(参见配方)
  27. Tris-甘氨酸SDS运行缓冲液(参见配方)
  28. Zymogram复性缓冲液(见配方)
  29. 染色溶液(见配方)
  30. 10%分离凝胶溶液(见配方)
  31. 4%堆积胶溶液(见配方)

设备

  1. 15毫升规范管
  2. 烧瓶
  3. 1.5 ml微量离心管
  4. 冷冻离心机
  5. 冷藏微量离心机
  6. CO <2>孵化器
  7. 分光光度计
  8. 一次性塑料比色杯
  9. 蛋白质微型凝胶盒
  10. 加热块模块
  11. 电源
  12. 轨道振动器
  13. 37°C温度室

程序

  1. 制备掺入凝胶中的细菌基质
    1. 开始5毫升的培养。 mutans UA159野生型菌株(或其它变异链球菌菌株)在THYE肉汤中转移到15ml规范管中,并在37℃下在含有5%CO 2的空气中温育过夜, sub> 2 。
    2. 将具有1%过夜预培养物的300ml新鲜THYE肉汤接种到500ml烧瓶中。
    3. 在37℃下在具有5%CO 2的空气中静置培养物直至达到600nm(OD 600)的光密度为0.2。
    4. 通过在4℃下以10,000×g离心10分钟收获细胞。
    5. 使用5ml的20mM Tris,100mM NaCl缓冲液(pH 7.4)洗涤细胞,并将细胞沉淀重悬在1.0ml的1.5M Tris缓冲液(pH 8.8)中。
    6. 保持细胞在-20°C,直到使用。

  2. 细菌全细胞提取物的制备
    1. 开始5ml过夜培养的S. mutans UA159野生型菌株(或其它变异链球菌菌株)在THYE肉汤中转移到15ml规范管中,并在37℃下在具有5%CO > 2 。在研究中缺乏肽聚糖水解酶的突变体菌株的全细胞提取物也可以作为阴性对照同时分析以确认观察到的水解带的特异性。
    2. 将10ml新鲜THYE肉汤与1%的过夜预培养物接种到15ml经典管中。
    3. 在37℃下在具有5%CO 2的空气中静置培养物直至达到所需的OD 600。如果目标肽聚糖水解酶的表达谱是未知的,我们建议收获细胞在不同的光密度对应的生长早期对数,中对数,早稳定和晚稳定期。
    4. 通过在4℃下以10,000×g离心10分钟收获细胞。
    5. 保持细胞沉淀-20℃,直到使用。
    6. 重悬细胞沉淀在20μl的新鲜制备的SDS-PAGE加载缓冲液中。
    7. 将样品在95℃下加热10分钟。 保持样品在冰上,直到加载
  3. 酶谱凝胶的制备
    1. 用乙醇清洁玻璃板,隔板和梳子,使用前完全干燥。 按照制造商的说明装配凝胶盒。
    2. 制备10%分离胶溶液(见配方9)。
    3. 将分离的凝胶溶液(每个小凝胶约3.8ml)转移到玻璃板之间的浇铸室中,并在梳子就位时填充到梳子底部下方约0.7cm处。
    4. 在聚合之前向凝胶顶部加入一小层异丙醇以使凝胶水平变直。 一旦凝胶聚合,通过用dH 2 O 2洗涤几次除去异丙醇层,并用滤纸干燥。
    5. 制备4%堆积胶溶液(见配方10)。
    6. 将堆积的凝胶溶液(约2.5ml /小凝胶)倒在分离凝胶顶部,直到空间充满,然后插入适当的梳子。 一旦凝胶聚合,小心取出梳子。
    7. 从铸造台上取下凝胶盒,并将其放置在制造商推荐的电极组件中。
    8. 将Tris-甘氨酸SDS运行缓冲液倒入凝胶盒之间的铸造框架的开口中。 加入足够的缓冲液填充凝胶的孔。 也填充框架外部的区域。
    9. 将样品(从第II节,步骤B-7)装入每个孔以及5μl的Precision Plus蛋白质预染色标准品。
    10. 将电泳槽连接到电源。 在125-200伏之间的恒定电压下运行凝胶,直到染料前沿接近凝胶底部
  4. 肽聚糖水解酶检测
    1. 从电泳室中取出凝胶,使凝胶剥离并轻轻地落入容器中。 在室温下在持续搅拌下在100ml dH 2 O中洗涤凝胶两次30分钟。
    2. 在室温下在持续搅拌下将凝胶在100ml的酶谱复性缓冲液中孵育30分钟。该步骤是复性肽聚糖水解酶所必需的。
    3. 用新鲜的酶谱复性缓冲液替换酶谱复性缓冲液,并在恒温搅拌下在37℃下在恒温箱中孵育凝胶,直到出现清晰的水解带,通常在16小时和48小时之间。蛋白水解活性表现为针对白色背景的清晰条带。
    4. 任选染色步骤:倾析缓冲液并加入100 ml染色溶液,并在室温下在15分钟至2小时之间的恒定搅拌下温育凝胶。没有染色的区域指示裂解(图1)。蛋白水解活性表现为针对蓝色背景的清晰条带。


      图1.在亚甲基蓝染色后的酶谱活性凝胶。将 突变体的热灭活细胞用作底物,并掺入10 %SDS-PAGE凝胶。 (1)分子大小标记(Precision Plus Protein Prestained Standards)。 (2)S的全细胞提取物。 mutans UA159野生型菌株。观察到的两个水解带(箭头)对应于AtlA自溶素的未处理(上)和处理(下)形式。

食谱

  1. THYE汤液
    将15g Todd-Hewitt和1.5g酵母提取物溶解在400ml dH 2 O中。
    一旦溶解,使用dH 2 O·v/v使最终体积达到500ml 在120℃下高压灭菌20分钟
    在室温下贮存
  2. 20mM Tris,100mM NaCl缓冲液(pH7.4) 将2.42g Tris碱和5.84g NaCl溶于800ml dH 2 O中。
    一旦溶解,将pH调节至7.4,然后用dH 2 O使最终体积达到1L。
    存储在4°C
  3. 1.5M Tris pH8.8缓冲液
    将181.71g Tris碱溶解在800ml dH 2 O中 一旦溶解,将pH调节至8.8,然后用dH 2 O升至1L的最终体积。
    存储在4°C
  4. 0.5M Tris pH 6.8缓冲液
    将60.57g Tris碱溶解在600ml dH 2 O中 一旦溶解,将pH调节至6.8,然后用dH 2 O使最终体积达到1L。
    存储在4°C
  5. SDS-PAGE上样缓冲液(0.25M Tris pH6.8,2%SDS,10%甘油,溴酚蓝) 将0.3g Tris碱,0.2g SDS,1.0ml甘油和痕量溴酚蓝溶于7ml dH 2 O中。
    一旦溶解,使用dH 2 O,使最终体积达到10ml
  6. Tris-甘氨酸SDS运行缓冲液(25mM Tris,192mM甘氨酸,0.1%SDS) 将3.03g Tris碱,14.4g甘氨酸和1g SDS溶于800ml dH 2 O中。
    一旦溶解,用dH 2 O 2/v升至最终体积为1L 存储在4°C
  7. Zymogram复性缓冲液(20mM Tris,50mM NaCl,20mM MgCl 2,0.5%Triton X-100,pH7.4)
    在800ml dH 2 O中溶解2.42g Tris碱,2.92g NaCl和4.06g MgCl 2。 将pH调节至7.4
    加入5ml Triton X-100,用dH 2 O使最终体积达到1L。
    存储在4°C
  8. 染色溶液(0.1%亚甲蓝,0.01%KOH) 将0.1g亚甲基蓝和0.01g KOH溶于100ml dH 2 O中。
    在室温下贮存
  9. 10%分离凝胶溶液
    将以下试剂混合在干净的烧瓶中(4个小凝胶的总体积):
    7.4ml dH 2 O 2 / 3.7ml 40%丙烯酰胺/bis
    将4ml细菌底物(来自A部分,步骤A-6)在刚使用前煮沸10分钟
    100μl10%SDS
    50μl10%过硫酸铵
    5μlTEMED
  10. 4%堆积胶溶液
    将以下试剂混合在干净的烧瓶中(4个小凝胶的总体积):
    6毫升dH 2 O 2 / 2.5ml 0.5M Tris(pH 6.8)
    1.0ml 40%丙烯酰胺/bis
    100μl10%SDS
    100μl10%过硫酸铵
    25μlTEMED

参考文献

  1. Berg,K.H.,Ohnstad,H.S。和Havarstein,L.S.(2012)。 LytF,一种在链球菌属中的新型竞争性调节的木霉素水解酶。 J Bacteriol 194(3):627-635。
  2. Dufour,D。和Lévesque,C.M。(2013)。 由群体感应肽诱导的变异链球菌的细胞死亡通过 a conserved streptococcal autolysin。 J Bacteriol 195(1):105-114。 
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Dufour, D. and Lévesque, C. M. (2013). Zymogram Assay for the Detection of Peptidoglycan Hydrolases in Streptococcus mutans. Bio-protocol 3(16): e855. DOI: 10.21769/BioProtoc.855.
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Veronica Larmour
University of Manitoba
I would like to find a method to preserve the gel. We normally soak our acrylamide gels in a glycerol/ethanol solution before drying between sheets of cellophane, but with the zymograms what happens is the bands disappear and everything becomes completely washed out.
I am looking for a way to preserve the gel as it is when the clearing is complete and the stain is still nice and blue.
If you have any suggestions for this it would be much appreciated. up to now, the only way we can record the freshly developed gel is to scan it, which can look great but has its own drawbacks.
1/8/2016 10:15:15 AM Reply
Delphine Dufour
Dental Research Institute, Faculty of Dentistry, University of Toronto, Canada

Hello,
It seems that we are doing as you do: scan the gel and then dry it between cellophane sheets. Unfortunately we do not have another way to keep it.
Thanks for your interest.
Best regards,
Delphine

1/8/2016 10:35:11 AM