描述该项目由奖号5R01AI116895从过敏和传染病国家研究所的支持。内容完全是作者的责任，并不一定代表国家过敏和传染病研究所和美国国立卫生研究院的官方意见。

1.生成样本<ol><li>热激活C.难辨孢子在65℃下30分钟，并存储在冰上。 注:C.如先前7,9,10所述艰难梭菌芽孢可以产生并纯化。 </li><li>准备在X + 1毫升，其中X是在测定期间要采取的样本数萌发溶液。 注意:萌发溶液是特定细菌的物种孢子正在研究中。用于测定C.难辨孢子萌发，我们使用的10mM Tris（pH值7.5），150毫摩尔NaCl，在去离子水中的100mM甘氨酸和10mM牛磺胆酸的溶液。 </li><li>在开始测定前，画出萌发溶液中的1.0 ml样品无孢子作为皮质片段检测空白。 </li><li>加入孢子的外径〜3.0发芽解决方案，并迅速涡旋600。 注意:如果您在B.萌发研究枯草芽孢杆菌 ，我们使用相同的外径600孢子为C.难辨 。 注:孢子这一数额C.期间行之有效的监测皮质片段发布难辨孢子萌发。孢子在该试验中所使用的量应通过实验为每个细菌或应变来确定。 </li><li>在14000×g下加入孢子和离心机在室温下1分钟后，立即删除在1.2 ml样品。这是在零时间点。 </li><li>传送将1.0ml上清液从离心样品到新管用于随后皮质片段分析。 注意:如果DPA需要监控，取出一个单独的100微升样品在使用基于铽荧光9,18,19的测定溶液中的检测DPA的。 </li><li>冷冻在-80℃下收集的样品。 </li><li>定期重复步骤1.4和1.5，直到所有的时间点完成。 注:由于时间的关系所需的离心和取样时间点少次我们的过程期间的2分钟分开是不可能的，所以我们的采样间隔是一次14分钟每2分钟。 </li><li>至于还原糖的检测和定量阳性对照，包括以下少量的正 -acetylglucosamine（纳摩尔）的:0，12.5，25，50，100，250，500，5000。同时作为皮质样品制备这些样品，并与皮质样品，使得标准曲线是有关由萌芽孢子产生的数据一起运行。 </li><li>所有时间点样品的采集后，冻干样品。 注意:我们冻干我们的样品在2ml螺旋盖试管中。 </li></ol> 2.测量皮质碎片<ol><li>在3当量盐酸的120微升补充有1％的苯酚和0.5％β巯基乙醇悬浮冻干样品。 </li><li>传送再悬浮样本以2毫升螺旋盖试管中。 注意:对于重复性，确保所有的冻干样品重悬于和转让。 </li> &lt;利&gt;孵育再循环水浴将样品在95℃下4小时。 </li><li>孵育后，将样品置于冰上，直到他们都很酷。 </li><li>用120微升3 M氢氧化钠的中和样品。 </li><li>添加80μl的饱和碳酸氢钠溶液和80μl的5％的乙酸酐溶液于每个样品和旋涡。 </li><li>在室温下孵育样品10分钟。 </li><li>转移样本回95℃水浴正好3分钟。 注意:此步骤是时间敏感的和更长的时间能影响下游信号的发展。 </li><li>从水浴中取出样品，并在冰上冷却。 </li><li>添加6.54％K 2 B 4 O 7·4H 2 O 400微升，每管和旋涡。 </li><li>孵育在再循环水浴将样品在95℃EXACTLY 7分钟。 注意:此步骤是时间敏感的和更长的时间能影响下游信号的发展。 </li><li>去掉样品，并在冰上地方5分钟。 </li></ol> 3.显色剂<ol><li>虽然样本上冰（步骤2.12），备显色剂。溶解0.320克对 -dimethylaminobenzaldehyde的; 1.9毫升冰醋酸的（DMAB埃利希氏试剂）。 注:试剂是时间，温度和光敏感，并且不应提前进行。 </li><li>在DMAB完全溶解后，加入100微升10 N HCl和旋涡。 </li><li>加入5毫升冰醋酸和旋涡。 </li></ol> 4.显色反应<ol><li>转移100μl的每个冷却皮质样品到一个新的1.5 ml离心管。 </li><li>添加新鲜制备的色溶液至各样品的700微升。 </li><li>立即将样品转移到37℃水浴中孵育整整20分钟。 </li><li>转移200μl的各样品，以清晰的96孔板中。 </li><li>在量化波长585样本一盘读者。样本应开始以黄色和阳性反应将转向紫色。越皮质目前，较深的紫色。 </li></ol>

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The authors have nothing to disclose.

在刺激时，进行发芽的过程孢子丧失其抗损伤性，而无需大分子合成。当孢子萌发被触发时，孢子释放核心DPA，以换取水2。由于休眠孢子的高DPA含量，孢子核心是在强烈渗透压和专门肽，皮质，有助于防止芯从通过作用，据推测，作为屏障，以扩张2肿胀。 上述的方法使用艾氏试剂（黄色溶液），以检测还原糖作为可测量的移位到紫色色调的存在。期间皮质水解，NAG和NAM糖解放（取决于水解酶的程度，糖类可以解放如二糖或多糖）。由盐酸（步骤2.1）的糖苷键的水解与自由还原端7产生单糖。 neutraliz后用等量的氢氧化钠（步骤2.5）ING酸，包围NAG醇类和NAM单糖是由乙酸酐酰化（步骤2.6）13。接着，未反应的乙酸酐是通过加热样品（步骤2.8）转化为乙酸。由于假设由摩根和埃尔森20，终端醛自发地转化为烯醇式。在碱性条件下（步骤2.10），此烯醇形式可以与相邻的乙酰反应以产生其中用Ehrlich试剂（步骤4.3），以产生紫色15,20反应的恶唑衍生物。 上述这种方法产生的检测皮质碎片一致的，可靠的结果，从萌芽孢子，无论是B.枯草和C.难辨 9,16,17。该试验需要特别注意以确保时间，温度和程序的精度进行该反应。如果没有适当关注细节，很容易在反应条件失败或不实验13之间再现。然而，在实验室环境之间的测定的应用中纳入NAG标准助剂。包括在任何跨实验变异性（ 例如，在反应时间的细微变化，或显色剂的有效性），每一个实验对照组的NAG阳性对照标准。仅使用缓冲液条件和孢子不能水解皮质两者的反应的反应，得到了无颜色变化。 这种方法的最常见的问题是跨实验变异或完全没有信号。为了控制的可变性，孢子制剂必须无任何营养细胞碎片。此细胞碎片将包含肽片段（NAG和NAM），并会导致对萌芽孢子高背景信号。以确保纯孢子制剂（&gt; 99.9％孢子），指的是literatu重新为方法，以在生物体准备纯孢子悬浮液进行研究7,9,21-23。 如果该测定没有产生任何可检测的信号，我们观察到，冻干步骤可以引入可变性。由于与该释放真空的力，我们观察到干燥后的样品的粉末可以吹出的样品管。为了减少/防止这种情况发生，使用一个小的注射器针头戳在样品管的顶部孔。引入孔的小区域将受到干扰（粉末形式现在）降低冻干样品的可能性。最后，如​​在上述方法中指出的那样，几个孵育步骤都非常时间敏感。确保所有定时步骤仔细监测。对于该测定的一个重要考虑因素是，皮质碎片必须从发芽孢子被释放以便被检测到。如果皮层被水解成较大的碎片，这些碎片可能太大是RELE通过芽孢外壳ASED。虽然一个重要的考虑因素的故障排除的实验不产生信号时，我们建议，以确认使用的条件发芽最此处列出在溶液中的孢子和其它故障排除之前考虑皮质片段大小作为一个潜在的问题化验。 不是所有的发芽缓冲器/条件是适合于这种检测。因为该测定法检测减少在发芽过程13，它包含还原糖或即，本身的任何开端，还原糖任何发芽缓冲解放糖的存在（ 例如 ，葡萄糖），将导致在该测定中的强烈信号。如果是这种情况，高压液相色谱（HPLC）可用于检测皮质片段24-26的存在。然而，因为不是所有的实验室都配备这样的仪器，本文描述的方法可以通过使用最多的实验室和提供简单且容易量化的结果。 通过发芽细菌孢子已经在很大程度上被研究芽孢杆菌和少数梭菌属2,8。该方法一般保守的，但是存在差异。重要的是，并不是所有的生物体发芽作为模型孢子形成剂9,27观察用孢子萌发的机制。因为发芽在更加多样的生物变得研究，上述测定可用于这些微生物孢子萌发期间检测皮质片段。通过应用该方法对这些新研究的生物体，研究人员能够确定皮质水解作用的各种蛋白质发芽玩。

芽孢是细菌代谢，使细菌在不利的环境中坚持的休眠形式。在许多孢子形成细菌，孢子形成是通过营养剥夺诱导但此过程可通过改变pH值，暴露于氧或其它应力1进行控制。而在其代谢休眠孢子形式，细菌抵抗紫外线辐射，干燥，更高的温度，和冷冻2。最上的孢子形成过程的知识来自在模式生物的研究， 枯草芽孢杆菌 。在孢子形成过程开始于DNA复制和轴向灯丝3,4的形成。非对称隔膜然后把细胞分成两个大小不等车厢。较大的隔间里，母细胞，吞噬较小的隔间里，forespore。无论是母细胞和forespore协调基因表达成熟的孢子母细胞裂解最终，释放多尔曼吨孢子到周围环境中1。 孢子的结构和组合物在许多细菌物种保守的。孢子芯具有比营养细胞低水含量和有丰富的吡啶二羧酸（DPA）1,2。期间形成孢子，DPA被泵入孢子芯换取水。围绕核心的是一种内在的孢子膜的地方，在多数孢子形成的细菌，其中认识到刺激发芽（germinants）的小分子的受体位于2。刚好位于孢子的内膜外是细胞壁肽聚糖的层。一个专门的肽聚糖层（皮层）围绕细胞壁肽聚糖，并且由许多相同的部件，细胞壁肽聚糖[交替的N-乙酰葡糖胺酶（NAG）和N-乙酰胞壁酸（NAM）]的。但是，在皮质的NAM残基的约50％已被转化为胞壁-δ内酰胺5,6- </s了&gt;。期间孢子萌发，这些胞壁-δ内酰胺残基被孢子皮质裂解酶（SCLEs）的认可，因此允许皮质被降解但不细胞壁（必需完成发芽过程）。周围皮质的外膜和外壳蛋白2几层。 控制发芽的方法对于形成孢子的细菌至关重要。当开端受体各自germinants 2交互萌发开始。在许多孢子形成的细菌，这些germinants是氨基酸，糖，核苷酸，离子或它们的2组合。C.难辨孢子萌发是由某些胆汁酸，[ 例如 ，牛磺胆酸（TA）]和氨基酸（ 例如 ，甘氨酸）7-10的组合引发。虽然有在C之间的差异难辨发芽途径和在所研究的途径其他孢子形成细菌，如B.枯草芽孢杆菌，共同所有的降解孢子皮质以允许营养细胞以从发芽孢子2,8成长的绝对要求。皮质降解可由SCLEs CwlJ / SleB来实现或SLEC（如在枯草芽孢杆菌中发现）（如在许多梭菌找到）。皮质水解降低了细胞壁和孢子的约束。这允许充分芯补液，在重新激活许多必需的细胞代谢2蛋白质的一个必要步骤。 当孢子发芽，从相亮状态到相暗状态，这个过程的休眠孢子的变化可以通过在600光密度（OD）的变化来测量 纳米11。先前的一份报告表明，在很多OD这一变化是由于DPA从12孢子释放。在我们最近的研究中，我们试图比较C的定时难辨孢子萌发和监测DPA和皮质碎片9的释放。在这项研究中是至关重要的监测皮质片段的释放，他们开始由萌芽孢子被释放。 此处所使用的比色测 ​​定法是基于与还原末端检测糖的方法开发Ghuysen 等 13。因为其他人描述协议检测还原糖14或修改这些协议15，关于这一问题的文献可能会造成混淆。在这里，我们详细地一步一步方法还原糖的比色检测从发芽C.解放艰难梭菌芽孢。虽然本研究采用C.艰难梭菌芽孢，从其他孢子形成的细菌孢子的发芽过程中释放的还原糖能够与该协议9,16,17进行检测。

<table><tbody><tr><td>2.0 ml screw cap tube</td><td>USA scientific</td><td>1420-3700</td><td></td></tr><tr><td>p1000 Eppendorf pipet</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>p200 Eppendorf pipet</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>p20 Eppendorf pipet</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>100 - 1,250 &amp;#956;l pipet tips</td><td>VWR</td><td>89079-486</td><td></td></tr><tr><td>1 - 200 &amp;#956;ll pipet tips</td><td>VWR</td><td>89079-458</td><td></td></tr><tr><td>N-acetyl-D-glucosamine</td><td>Sigma</td><td>A3286-25G</td><td></td></tr><tr><td>Hydrochloric Acid - 10 N</td><td>BDH-Aristar</td><td>BDH3032-3.8LP</td><td></td></tr><tr><td>Acetic Anhydride</td><td>Alfa Aesar</td><td>L04295</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>BDH</td><td>BDH0280-500G</td><td></td></tr><tr><td>Potassium Tetraborate tetrahydrate</td><td>Alfa Aesar</td><td>39435</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>BDH</td><td>BDH8019-500G</td><td></td></tr><tr><td>4-(Dimethylamino)benzaldehyde</td><td>Sigma</td><td>156477-100G</td><td></td></tr><tr><td>Saturated Phenol</td><td>Fisher</td><td>BP1750-400</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Aldrich</td><td>M6250-100ML</td><td></td></tr><tr><td>SpectraMax M3</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Acetic Acid, Glacial</td><td>BDH</td><td>BDH3098-3.8LP</td><td></td></tr><tr><td>Heated, Circulating Water Bath</td><td>VWR Scientific</td><td>Model 1136</td><td></td></tr><tr><td>Microtest 96</td><td>Falcon</td><td>353072</td><td>96 well clear tissue culture plates</td></tr><tr><td>Culture tubes</td><td>VWR</td><td>89000-506</td><td></td></tr><tr><td>Lyophilizer</td><td></td><td></td><td></td></tr><tr><td>Heat Blocks</td><td></td><td></td><td></td></tr><tr><td>Vortex Machine</td><td></td><td></td><td></td></tr></tbody></table>

detecting cortex fragments during bacterial spore germination

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming bacterium, Bacillus subtilis. Germination is triggered by small molecule germinants and occurs without the need for macromolecular synthesis. Though differences exist between the mechanisms of spore germination in species of Bacillus and Clostridium, a common requirement is the hydrolysis of the peptidoglycan-like cortex which allows the spore core to swell and rehydrate. After rehydration, metabolism can begin and this, eventually, leads to outgrowth of a vegetative cell. The detection of hydrolyzed cortex fragments during spore germination can be difficult and the modifications to the previously described assays can be confusing or difficult to reproduce. Thus, based on our recent report using this assay, we detail a step-by-step protocol for the colorimetric detection of cortex fragments during bacterial spore germination.

表1示出了使用NAG标准的典型结果。数据被用于产生标准曲线。 表2示出从发芽缓冲器萌发测定补充有100mM的甘氨酸和10mM TA（发芽促进条件）或100mM的甘氨酸只（非发芽条件）的典型结果。在不存在TA，C的艰难梭菌芽孢不发芽并且在皮质片段在溶液中存在的小的变化。然而，在这两个TA和甘氨酸，C的存在艰难梭菌芽孢发芽并释放皮质片段在溶液中。这被观察为在反应样品的时间的增加的OD 585。该测定过程中释放皮质片段（纳摩尔）的总量，使用类似于图1B的标准曲线来计算。在该试验中，大部分的皮质的水解（或释放）为15分钟后开端曝光，这表明大部分孢子在这个时间帧已发芽（ 图2;与先前出版物7一致）。 显影在彩色试剂的样品后，大多数样品可能不会显示紫色肉眼。 图1示出了从N-乙酰氨基葡糖产生的有代表性的样品。许多样品不显示可见紫色。然而，250纳摩尔，500纳摩尔和5000纳摩尔样品出现从阴性对照组（0纳摩尔）目视不同。当在OD 585处测量，所产生的信号最适合的线性回归（R 2 = 0.999）。 <img alt="图1" src="/files/ftp_upload/54146/54146fig1.jpg" /> 图 1: 检测N-乙酰葡糖胺 （A）的N-乙酰氨基葡糖的样品（0纳摩尔，12.5纳摩尔，25纳摩尔，50纳摩尔，100纳摩尔，250纳摩尔，500纳摩尔，5000纳摩尔）按照所述步骤冻干并反应。 （B）的样品加入到96孔板中并在585处检测到的反应材料的量。该信号被用来产生标准曲线。 <img alt="图2" src="/files/ftp_upload/54146/54146fig2.jpg" /> 图 2: 萌发结果的图形表示在表2中的计算纳摩尔皮质与时间的关系。 （●）100毫米甘氨酸+ 10毫米TA; （■）100毫米甘氨酸。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54146/54146fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> NAG浓度（纳摩尔） </td><td> OD 585 </td></tr><tr><td> 0 </td><td> 0.047 </td></tr><tr><td> 12.5</td><td> 0.054 </td></tr><tr><td> 25 </td><td> 0.066 </td></tr><tr><td> 50 </td><td> 0.084 </td></tr><tr><td> 100 </td><td> 0.128 </td></tr><tr><td> 250 </td><td> 0.285 </td></tr><tr><td> 500 </td><td> 0.556 </td></tr><tr><td> 5000 </td><td> 4 </td></tr><tr><td>空白= </td><td> 0.046 </td></tr></tbody></table> 表1:NAG标准。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td colspan="4"> 艰难梭菌孢子萌发</td></tr><tr><td></td><td colspan="4">发芽溶液（10mM的Tris（pH值7.5），150mM的NaCl）中</td></tr><tr><td></td><td colspan="2"> 100毫米甘氨酸+ 10毫米TA </td><td colspan="2"> 100毫米甘氨酸</td></tr><tr><td>时间（分钟） </td><td> OD 585 </td><td>计算（纳摩尔） </td><td> OD 585 </td><td>计算（纳摩尔） </td></tr><tr><td> 0 </td><td> 0.046 </td><td> 0.00 </td><td> 0.047 </td><td> 0.00 </td></tr><tr><td> 2 </td><td> 0.048 </td><td> 2.54 </td><td> 0.048 </td><td> 1.27 </td></tr><tr><td> 4 </td><td> 0.05 </td><td> 5.07 </td><td> 0.049 </td><td> 2.54 </td></tr><tr><td> 6 </td><td> 0.055 </td><td> 11.41 </td><td> 0.05 </td><td> 3.80 </td></tr><tr><td> 8 </td><td> 0.059 </td><td> 16.49 </td><td> 0.05 </td><td> 3.80 </td></tr><tr><td> 10 </td><td> 0.06 </td><td> 17.76 </td><td> 0.051 </td><td> 5.07 </td></tr><tr><td> 12 </td><td> 0.061 </td><td> 19.02 </td><td> 0.05 </td><td> 3.80 </td></tr><tr><td> 14 </td><td> 0.063 </td><td> 21.56 </td><td> 0.051 </td><td> 5.07 </td></tr></tbody></table> 表2:样品孢子Germinati对结果的。

Watch this Scientific Journal Video about Detecting Cortex Fragments During Bacterial Spore Germination at JoVE.com

Detecting Cortex Fragments During Bacterial Spore Germination

Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

检测皮质片段在细菌孢子萌发

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming ...

detecting-cortex-fragments-during-bacterial-spore-germination

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

9.3K 次观看.  Texas A&M University. Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

视频: Detecting Cortex Fragments During Bacterial Spore Germination

Physical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells

Endospore formation is a survival strategy found among some bacteria from the phylum Firmicutes. During endospore formation, these bacteria enter a morpho-physiological resting state that enhances survival under adverse environmental conditions. Even though endospore-forming Firmicutes are one of the most frequently enriched and isolated bacterial groups in culturing studies, they are often absent from diversity studies based on molecular methods. The resistance of the spore core is considered one of the factors limiting the recovery of DNA from endospores. We developed a method that takes advantage of the higher resistance of endospores to separate them from other cells in a complex microbial community using physical, enzymatic and chemical lysis methods. The endospore-only preparation thus obtained can be used for re-culturing or to perform downstream analysis such as tailored DNA extraction optimized for endospores and subsequent DNA sequencing. This method, applied to sediment samples, has allowed the enrichment of endospores and after sequencing, has revealed a large diversity of endospore-formers in freshwater lake sediments. We expect that the application of this method to other samples will yield a similar outcome.

A method to single out bacterial endospores from complex microbial communities was developed to perform tailored culture or molecular studies of this group of bacteria.

从环境样品中的内生孢子通过无性细胞定向裂解物理隔离

Endospore formation is a survival strategy found among some bacteria from the phylum Firmicutes. During endospore formation, these bacteria enter a ...

科研

环境科学

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

检测使用荧光脱氧核酶的细菌

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

免疫与感染

Improvement of Bacillus subtilis Spore Enumeration and Label Analysis in Flow Cytometry

The spores of Bacillus subtilis have already been proposed for different biotechnological and immunological applications; however, there is an increasing need for the development of methodologies that improve the detection of antigens immobilized on the surface of spores together with their quantification. Flow cytometry-based analyses have been previously proposed as fast, reliable, and specific approaches for detecting labeled cells of B. subtilis. Herein, we propose the use of flow cytometry to evaluate the display efficiency of a fluorescent antibody (FA) on the surface of the spore and quantify the number of spores using counting beads. 
For this, we used ethidium bromide as a DNA marker and an allophycocyanin (APC)-labeled antibody, which was coupled to the spores, as a surface marker. The quantification of spores was performed using counting beads since this technique demonstrates high accuracy in the detection of cells. The labeled spores were analyzed using a flow cytometer, which confirmed the coupling. As a result, it was demonstrated that DNA labeling improved the accuracy of quantification by flow cytometry, for the detection of germinated spores. It was observed that ethidium bromide was not able to label dormant spores; however, this technique provides a more precise determination of the number of spores with fluorescent protein coupled to their surface, thus helping in the development of studies that focus on the use of spores as a biotechnological platform in different applications.

Improvement of Bacillus subtilis Spore Enumeration and Label Analysis in Flow Cytometry

This protocol focuses on the use of flow cytometry and counting beads to quantify bacterial spores labeled with ethidium bromide. The method is also efficient for analyzing the covalent coupling of proteins on the surface of intact spores.

枯草芽孢杆菌孢子计数和标记分析在流式细胞术中的改进

The spores of Bacillus subtilis have already been proposed for different biotechnological and immunological applications; however, there is an increasing ...

生物化学

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence microscopy. Super-resolution three-dimensional Structured Illumination Microscopy (3D-SIM) is a promising tool to overcome this hurdle and reveal the molecular details of the process of germination of Bacillus subtilis (B. subtilis) spores. Here, we describe the use of a modified SIMcheck (ImageJ)-assistant 3D imaging process and fluorescent reporter proteins for SIM microscopy of B. subtilis spores&#8217; germinosomes, cluster(s) of germination proteins. We also present a (standard)3D-SIM imaging procedure for FM4-64 staining of B. subtilis spore membranes. By using these procedures, we obtained unsurpassed resolution for germinosome localization and show that &#62;80% of B. subtilis KGB80 dormant spores obtained after sporulation on defined minimal MOPS medium have one or two GerD-GFP and GerKB-mCherry foci. Bright foci were also observed in FM4-64 stained spores&#8217; 3D-SIM images suggesting that inner membrane lipid domains of different fluidity likely exist. Further studies that use double labeling procedures with membrane dyes and germinosome reporter proteins to assess co-localization and thus get an optimal overview of the organization of Bacillus germination proteins in the inner spore membrane are possible.

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

Germinant receptor proteins cluster in &#8216;germinosomes&#8217; in the inner membrane of Bacillus subtilis spores. We describe a protocol using super resolution microscopy and fluorescent reporter proteins to visualize germinosomes. The protocol also identifies spore inner membrane domains that are preferentially stained with the membrane dye FM4-64.

枯草芽孢杆菌中细菌体和内膜的可视化

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence ...

生物工程

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of antibiotics and other secondary metabolites. Streptomyces coelicolor is a well-characterized, non-pathogenic species that is amenable to a variety of analyses in the lab. The phenotyping methods described here use S. coelicolor as a model streptomycete; however, the methods are applicable to all members of this large genus as well as some closely related actinomycetes. Phenotyping is necessary to characterize new species of Streptomyces identified in the environment, and it is also a vital first step in characterizing newly isolated mutant strains of Streptomyces. Proficiency in phenotyping is important for the many new researchers who are entering the field of Streptomyces research, which includes the study of bacterial development, cell division, chromosome segregation, and second messenger signaling. The recent crowdsourcing of antibiotic discovery through the isolation of new soil microbes has resulted in an increased need for training in phenotyping for instructors new to the field of Streptomyces research and their college or high school students. This manuscript describes methods for bacterial strain propagation, storage, and characterization through visual and microscopic examination. After reading this article, new researchers (microbiology education laboratories and citizen scientists) should be able to manipulate Streptomyces strains and begin visual characterization experiments.

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Here we present protocols for novice researchers to initiate phenotyping for the pharmacologically important bacterial genus Streptomyces.

链霉菌发育突变体的视觉和显微评价

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of ...

Sexual Development and Ascospore Discharge in Fusarium graminearum

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces fruiting bodies, called perithecia, on carrot agar. Perithecia contain numerous tissue types, produced at specific stages of perithecium development. These include (in order of appearance) formation of the perithecium initials (which give rise to the ascogenous hyphae), the outer wall, paraphyses (sterile mycelia which occupy the center of the perithecium until the asci develop), the asci, and the ascospores within the asci14. The development of each of these tissues is separated by approximately 24 hours and has been the basis of transcriptomic studies during sexual development12,8. Refer to Hallen et al. (2007) for a more thorough description of development, including photographs of each stage. Here, we present the methods for generating and harvesting synchronously developing lawns of perithecia for temporal studies of gene regulation, development, and physiological processes. Although these methods are written specifically to be used with F. graminearum, the techniques can be used for a variety of other fungi, provided that fruiting can be induced in culture and there is some synchrony to development. We have recently adapted this protocol to study the sexual development of F. verticillioides. Although individual perithecia must be hand picked in this species, because a lawn of developing perithecia could not be induced, the process worked well for studying development (Sikhakolli and Trail, unpublished).
The most important function of fungal fruiting bodies is the dispersal of spores. In many of the species of Ascomycota (ascus producing fungi), spores are shot from the ascus, due to the generation of turgor pressure within the ascus, driving ejection of spores (and epiplasmic fluid) through the pore in the ascus tip2,7. Our studies of forcible ascospore discharge have resulted in development of a "spore discharge assay", which we use to screen for mutations in the process. Here we present the details of this assay.
F. graminearum is homothallic, and thus can form fruiting bodies in the absence of a compatible partner. The advantage of homothallism is that crossing is not necessary to generate offspring homozygous for a particular trait, a facet that has facilitated the study of sexual development in this species14,7. However, heterothallic strains have been generated that can be used for crossing5,9. It is also possible to cross homothallic strains to obtain mutants for several genes in one strain1. This is done by coinoculating one Petri dish with 2 strains. Along the meeting point, the majority of perithecia will be recombinant (provided a mutation in one of the parent strains does not inhibit outcrossing). As perithecia age, they exude ascospores en masse instead of forcibly discharging them. The resulting spore exudate (called a cirrhus) sits at the tip of the perithecium and can easily be removed for recovery of individual spores. Here we present a protocol to facilitate the identification of recombinant perithecia and the recovery of recombinant progeny.

Sexual Development and Ascospore Discharge in Fusarium graminearum

Sexual crosses and isolation of recombinant progeny are important research tools for the filamentous fungus, Fusarium graminearum, The techniques necessary successfully carry out these processes are presented.

性发展和有性放电禾谷镰刀菌</em

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces ...

生物学

Spore Adsorption as a Nonrecombinant Display System for Enzymes and Antigens

The bacterial spore is a metabolically quiescent cell, formed by a series of protective layers surrounding a dehydrated cytoplasm. This peculiar structure makes the spore extremely stable and resistant and has suggested the use of the spore as a platform to display heterologous molecules. So far, a variety of antigens and enzymes have been displayed on spores of Bacillus subtilis and of a few other species, initially by a recombinant approach and, then, by a simple and efficient nonrecombinant method. The nonrecombinant display system is based on the direct adsorption of heterologous molecules on the spore surface, avoiding the construction of recombinant strains and the release of genetically modified bacteria in the environment. Adsorbed molecules are stabilized and protected by the interaction with spores, which limits the rapid degradation of antigens and the loss of enzyme activity at unfavorable conditions. Once utilized, spore-adsorbed enzymes can be collected easily with a minimal reduction of activity and reused for additional reaction rounds. In this paper is shown how to adsorb model molecules to purified spores of B. subtilis, how to evaluate the efficiency of adsorption, and how to collect used spores to recycle them for new reactions.

This protocol focuses on the use of bacterial spores as a &#34;live&#34; nanobiotechnological tool to adsorb heterologous molecules with various biological activities. The methods to measure the efficiency of adsorption are also shown.

作为酶和抗原的非重组显示系统的孢子吸附

The bacterial spore is a metabolically quiescent cell, formed by a series of protective layers surrounding a dehydrated cytoplasm. This peculiar structure ...

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

采用共培养，以检测化学介导的种间相互作用

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

用活细胞显微镜研究低压等离子体灭菌对枯草芽孢杆菌孢子存活的不利影响

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are thick-walled dispersal agents. Teliospores can remain dormant for decades. The dormancy is characterized by low metabolic rates, paused macromolecular biosynthesis and greatly reduced levels of respiration. Upon receiving required environmental signals, teliospores germinate to produce haploid cells, which can initiate new rounds of infection. Teliospore germination is characterized by the resumption of macromolecular biosynthesis, increased respiration and dramatic morphological changes. In order to precisely measure changes in cellular respiration during the early stages of germination, we have developed a simple protocol employing a Clark-type respirometer. The later stages of germination are distinguished by specific morphological changes, but germination is asynchronous. We developed a microdissection technique that enables us to collect teliospores at distinct germination stages.

Smut fungi cause many devastating agricultural diseases. They are dispersed as dormant teliospores that germinate in response to environmental cues. We outline two methods to investigate molecular changes during germination: measuring respiration increase to detect metabolic activation and assessing changing molecular events by isolating teliospores at distinct morphological stages.

利用 Microrespiration 分析和显微解剖研究 Teliospore 萌发

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are ...