This work has been supported by Elizabeth E. Kennedy Research Award.

1，准备生产菌株的<ol><li>连胜肺炎球菌菌株对细菌生产的5％羊血的胰蛋白酶大豆琼脂从-80°C冰箱个股板块进行测试。 注：该菌株的抑制作用被测试的初期生长，使用血板大大增加了实验的可重复性。此外，其他能产生细菌素的非肺炎球菌菌株可使用虽然生长条件可能需要修改为特定的有机体。我们已经用缓症链球菌和乳酸乳球菌中的覆盖试验成功。 </li><li>在37℃下孵育平板过夜（O / N），用5％的CO 2。 </li><li>该生产菌株的培养后，吹打过氧化氢酶的3000至4000台准备胰酶大豆琼脂（TSA）平板上有25ml的TSA板，并使用无菌玻璃珠流传过氧化氢酶的解决方案。让板干燥下约10分钟生物安全柜。 </li><li>收集细菌的数量明显到一个枪头。刺枪头到TSA的干板。 注：不止一个的生产菌株可捅成一个单一的TSA板。它使得得到的光晕不重叠是很重要的，以空间出来的刺伤。 <ol><li>如果可能的话，包括已知的细菌素生产的，作为阳性对照，并在覆盖作为阴性对照使用的菌株。 注：阳性和阴性对照是此前已公布，并根据要求提供10。 </li></ol></li><li>孵育TSA板包含多个刺，在37℃，5％CO 2中6小时。 </li></ol>叠加应变2制作甘油股票<ol><li>备之前，覆盖测定当天甘油原液（培养物中加入甘油后生长至中期指数期，然后保存于-80℃）。接种肺炎球菌的海峡殖民地5-10AIN为敏感或信息素的分泌托德休伊特中有0.5％的酵母提取物（THY）进行测试。 注：BLPC诱导记者，施工已如前所述2株可根据要求提供。 </li><li>孵育5-10殖民地玻璃管用5毫升THY与盖紧盖子在37℃水浴中，不晃动。 </li><li>孵育培养物在37℃，直到它们达到的OD 0.3 620〜0.5。 注意：这将需要大约4-5小时，具体取决于所使用的菌株。 <ol><li>轻轻翻转试管前测量外径几次。 </li></ol></li><li>如果实验不能在同一天进行，或者在将来的应变会被多次使用，使甘油原液和恢复试验在稍后的日期。甘油股，加甘油文化的20％的最终浓度。分装到培养的1.5 ml离心管中，并储存在-80℃。注：样品可为stored几个月在-80℃。如果不是在甘油的股票，直接利用样本进行叠加分析。 </li></ol> 3，叠加分析<ol><li>之前叠加应用，混合叠加组成部分。在15 mL锥形管，加入5 ml祢，过氧化氢酶3,000-4,000 U和200微升这是不是长大的那一天，或者是在室温下解冻文化的甘油覆盖应变肉汤培养物。如果使用报告菌株，添加50μl的第X-gal的（40毫克/毫升），以覆盖混合物。用一个锥形管，每盘。 </li><li>在室温下平衡，然后加入熔融的TSA的覆盖混合物。 </li><li>从孵化器刺伤应变删除TSA板。熔化的固体TSA微波炉，并保持在55℃水浴中待用。加入熔融TSA 3毫升与1.5％琼脂冷却至55℃以叠加混合​​物。 <ol><li>立即移液之前，请准备叠加混合，混合物会开始凝固很快。注：如果叠加电镀开始之前固化，其结果将是无法解释的。 </li></ol></li><li>移液器向上和向下几次了500毫升吸管小心不要引入气泡到混合物混合叠加混合物。慢慢的吸液管直接覆盖混合到捅TSA板。保持在台式数分钟使琼脂变硬的板。 <ol><li>不要旋转盘来分发叠加混合，这将拉动从被刺的文化发展到覆盖。 </li></ol></li><li>小心地TSA板覆盖到含有5％的CO 2 O 2 / N（约16-18小时）37℃培养箱中培养。 </li><li>过夜生长后观察平板上。该板块应该会出现在覆盖株生长不透明的，除非发生抑制或信息素信号的地方的地方。这些将分别出现在左右应变刺伤清除或蓝色区域。 注：虽然测量电的蓝色或结算的直径对此语句可以用于定量评估来确定，在直径不同也可取决于细菌的被刺到板的量。覆盖法是一种定性分析，任何定量评估应谨慎解释。任何可见的结算相比于阴性对照被视为活性。 </li></ol>

<ol>
	<li>Dawid, S., Roche, A. M., Weiser, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blp+bacteriocins+of+Streptococcus+pneumoniae+mediate+intraspecies+competition+both+in+vitro+and+in+vivo.">The blp bacteriocins of Streptococcus pneumoniae mediate intraspecies competition both in vitro and in vivo.</a> Infect Immun. 75 (1), 443-451 (2007).</li><li>Sawa, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+enterocin+W,+a+novel+two-peptide+lantibiotic+produced+by+Enterococcus+faecalis+NKR-4-1.">Isolation and characterization of enterocin W, a novel two-peptide lantibiotic produced by Enterococcus faecalis NKR-4-1.</a> Appl Environ Microbiol. 78 (3), 900-903 (2012).</li><li>Kochan, T. J., Dawid, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+HtrA+protease+of+Streptococcus+pneumoniae+controls+density-dependent+stimulation+of+the+bacteriocin+blp+locus+via+disruption+of+pheromone+secretion.">The HtrA protease of Streptococcus pneumoniae controls density-dependent stimulation of the bacteriocin blp locus via disruption of pheromone secretion.</a> J Bacteriol. 195 (7), 1561-1572 (2013).</li><li>Lux, T., Nuhn, M., Hakenbeck, R., Reichmann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+bacteriocins+and+activity+spectrum+in+Streptococcus+pneumoniae.">Diversity of bacteriocins and activity spectrum in Streptococcus pneumoniae.</a> J Bacteriol. 189 (21), 7741-7751 (2007).</li><li>Reichmann, P., Hakenbeck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allelic+variation+in+a+peptide-inducible+two-component+system+of+Streptococcus+pneumoniae.">Allelic variation in a peptide-inducible two-component system of Streptococcus pneumoniae.</a> FEMS Microbiol Lett. 190 (2), 231-236 (2000).</li><li>Saizieu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray-based+identification+of+a+novel+Streptococcus+pneumoniae+regulon+controlled+by+an+autoinduced+peptide.">Microarray-based identification of a novel Streptococcus pneumoniae regulon controlled by an autoinduced peptide.</a> J Bacteriol. 182 (17), 4696-4703 (2000).</li><li>Barbour, A., Philip, K., Muniandy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+production,+purification,+characterization+and+mechanism+of+action+of+salivaricin+9+lantibiotic+produced+by+Streptococcus+salivarius+NU10.">Enhanced production, purification, characterization and mechanism of action of salivaricin 9 lantibiotic produced by Streptococcus salivarius NU10.</a> PLoS One. 8 (10), 77751 (2013).</li><li>Wladyka, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+biochemical+characterization,+and+cloning+of+a+bacteriocin+from+the+poultry-associated+Staphylococcus+aureus+strain+CH-91.">Isolation, biochemical characterization, and cloning of a bacteriocin from the poultry-associated Staphylococcus aureus strain CH-91.</a> Appl Microbiol Biotechnol. 97 (16), 7229-7239 (2013).</li><li>Shea, E. 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The authors have nothing to disclose.

这个覆盖测定法是一种快速的方式来确定用于细菌素生产活动的范围，并表明肺炎球菌菌株之间可能出现的竞争性相互作用。覆盖测定法可适于用于筛选多株为细菌素生产上的单个板。我们已通过使用一个48针复制器从一个半到96孔板的接种SBA板成功筛选大应变的集合与该测定。隔夜培养后，成长正在使用的复制和穿孔板与复制的引脚的琼脂表面转移到TSA的板块。 被调节在像?...

肺炎链球菌 ，鼻咽部的多种微生物社会的共同殖民者，能够通过生产细菌产生抗菌肽（细菌素）与其他肺炎球菌和密切相关的物种竞争。每一个完全测序肺炎球菌菌株检测到包含最新版本的B acteriocin- 升 IKE p eptide轨迹，BLP的。细菌素生产由肺炎球菌已被证明是既在体外的结果，并在体内竞争1-4重要。竞争的动力学是由沿与同源免疫蛋白多样的细菌素的表达响应于一个特定的肽信息素的影响。感应的BLP轨迹是由一个群体感应系统，其中的肽信息素，BLPC，结合到传感器激酶，BlpH控制，并开始导致信号级联转录BLP轨迹5,6。有BLPC四个等位基因变异，每绑定到其同源BlpH 6。对于细胞生存其自身细菌素的影响，其编码同源免疫蛋白的基因被转录于同一操纵子作为各细菌的基因。如果竞争者应变是不能够上调BLP轨迹响应于外源信息素，或如菌株缺少所需的保护的特定免疫基因发生杀伤。该转运体复合物，BlpAB是所必需的肽信息素的分泌，加工和细菌的肽2,4。最近，它已被证明，肺炎球菌菌株的显著一部分是“骗子”，这意味着他们在blpA基因，使它们无法分泌信息素和细菌素1,2保守突变。这些菌株能够外感BLPC 2所分泌的嘶鸣回应无聊株允许生产的免疫蛋白。 虽然细菌素，从上清液粗制剂可以从生产者菌株用生化方法步骤，以达到纯度来制备，这种方法是不实用的筛选分离的大的集合，如果细菌素的表达水平是低的肉汤生长培养2,7- 9。覆盖测定法作为一种快速的方法来调查可能存在的在体外的菌株之间的竞争动态。要做到这一点，肺炎球菌菌株是预先接种到琼脂平板，让它生长，以达到局部细菌浓度足以诱导BLP轨迹。第二应变，然后接种到熔融的软琼脂溶液中，然后施加在预接种菌株来测试灵敏度。以评估为BLP轨迹（独立抑制活性）的活性，菌株可与一系列日来覆盖稀土前面所述BLPC记者株。这些菌株被构造成包含三个不同blpH等位基因是由肺炎链球菌分泌的三种最常见的BLPC信息素响应之一。记者菌株具有一个集成的lacZ基因，该基因是一个BlpH依赖的启动子的控制下，并携带的缺失在BLPC基因，防止自励10,11。

<table border="0" cellpadding="0" cellspacing="0" width="753">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:351px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">Trypticase Soy Agar with 5% Sheep Blood</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">B21261X&#160;</td>
			<td>For growth from freezer cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Catalase</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">C100-500mg</td>
			<td>4,741 Units per plate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">Todd Hewitt Broth + Yeast Extract (THY)</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">DF0492-17-6&#160;</td>
			<td>30 g of THB, 5 g yeast extract, 1 L ddH20 autoclave</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">Trypticase Soy Broth + Agar plates (TSA)</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">B11768&#160;</td>
			<td>30 g of TS, 15 g agar, 1 L ddH20 autoclave</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">5-Bromo-4-chloro-3-indolyl &#946;-D-galactopyranoside (X-gal)</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">B4252-50MG</td>
			<td>Dissolve in dimethylformamide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Petri dishes</td>
			<td style="width:71px;">Fisher</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Spectrophometer</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Measures the OD620 of cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">37 &#176;C water bath</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">37 &#176;C incubator with 5% CO2</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">15 ml conical tube</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

using the overlay assay to qualitatively measure bacterial production of and sensitivity to pneumococcal bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

中有重叠测定两种可能的结果。它应当能够看到被刺菌株生长过夜孵育后。在图1A中，覆盖层的应变是敏感的正在生产的细菌素。间隙的区域可以显现周边刺伤应变。此外，被刺应变到覆盖混合物的旋涡是可能的，并且可以在图1A中可以看出。在图1B中，覆盖应变都难被分泌所指出的不存在包围短截卤代的细菌素。它也有可能是在被刺应变不分泌细菌素。对于?...

Watch this Scientific Journal Video about Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins at JoVE.com

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

使用复分析定性测量细菌生产和敏感性，以肺炎球菌细菌素

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

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JoVE Journal

Immunology and Infection

13.4K 次观看.  University of Michigan. Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.  Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. 