Name: quantifying bacterial surface swarming motility on inducer gradient plates
Uploaded: 2022-01-05T13:00:00
Description: Watch this Scientific Journal Video about Quantifying Bacterial Surface Swarming Motility on Inducer Gradient Plates at JoVE.com

该技术的开发得到了科技部国家重点研发计划（2018YF0902604）、国家自然科学基金国际青年科学家研究基金会（22050410270）和深圳先进技术研究院对外资金（DWKF20190001）的资助。我们衷心感谢陈心怡小姐在校对文件和实验室管理方面的帮助。

1. 梯度群板的制备
<ol><li>蜂群培养基的制备 注：有关不同介质粘度的简要比较，请参阅讨论部分;该协议中使用了0.7%（w / v）琼脂浓度的群体培养基。<ol>
<li>在两个锥形烧瓶中用琼脂制备溶菌汤（LB）粉末;每个烧瓶含有2克色蛋白酮，2克氯化钠，1克酵母提取物和1.4克琼脂。加入双蒸馏水（ddH 2 O），并使用磁力搅拌棒搅拌悬浮液。通过添加额外的ddH2 O将最终体积调节至20...

<ol>
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J., Ben Jacob, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Swarming+and+complex+pattern+formation+in+Paenibacillus+vortex+studied+by+imaging+and+tracking+cells.">Swarming and complex pattern formation in Paenibacillus vortex studied by imaging and tracking cells.</a> BMC Microbiology. 8, 36 (2008).</li><li>Shimada, H., 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=Dependence+of+local+cell+density+on+concentric+ring+colony+formation+by+bacterial+species+Bacillus+subtilis.">Dependence of local cell density on concentric ring colony formation by bacterial species Bacillus subtilis.</a> Journal of the Physical Society of Japan. 73 (4), 1082-1089 (2004).</li><li>Brahmachari, P. V., et al., Brahmachari, P. V., 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=Quorum+sensing+regulated+swarming+motility+and+migratory+behavior+in+bacteria.">Quorum sensing regulated swarming motility and migratory behavior in bacteria.</a> Implication of quorum sensing system in biofilm formation and virulence. , 49-66 (2018).</li><li>Lindum, P. W., 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=N-Acyl-L-homoserine+lactone+autoinducers+control+production+of+an+extracellular+lipopeptide+biosurfactant+required+for+swarming+motility+of+Serratia+liquefaciens+MG1.">N-Acyl-L-homoserine lactone autoinducers control production of an extracellular lipopeptide biosurfactant required for swarming motility of Serratia liquefaciens MG1.</a> Journal of Bacteriology. 180 (23), 6384-6388 (1998).</li><li>Wang, W. 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=Inhibition+of+swarming+and+virulence+factor+expression+in+Proteus+mirabilis+by+resveratrol.">Inhibition of swarming and virulence factor expression in Proteus mirabilis by resveratrol.</a> Journal of Medical Microbiology. 55, 1313-1321 (2006).</li><li>Zahringer, F., Massa, C., Schirmer, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+enzymatic+production+of+the+bacterial+second+messenger+c-di-GMP+by+the+diguanylate+cyclase+YdeH+from+E.+coli.">Efficient enzymatic production of the bacterial second messenger c-di-GMP by the diguanylate cyclase YdeH from E. coli.</a> Applied Biochemistry and Biotechnology. 163 (1), 71-79 (2011).</li><li>Kuchma, S. L., 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=Cyclic+di-GMP-mediated+repression+of+swarming+motility+by+Pseudomonas+aeruginosa+PA14+requires+the+MotAB+stator.">Cyclic di-GMP-mediated repression of swarming motility by Pseudomonas aeruginosa PA14 requires the MotAB stator.</a> Journal of Bacteriology. 197 (3), 420-430 (2015).</li><li>Heering, J., Alvarado, A., Ringgaard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+cellular+differentiation+and+single+cell+imaging+of+Vibrio+parahaemolyticus+swimmer+and+swarmer+cells.">Induction of cellular differentiation and single cell imaging of Vibrio parahaemolyticus swimmer and swarmer cells.</a> Journal of Visualized Experiments: JoVE. (123), e55842 (2017).</li><li>Bru, J. L., Siryaporn, A., H&#248;yland-Kroghsbo, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+imaging+of+bacterial+swarms+and+the+collective+stress+response.">Time-lapse imaging of bacterial swarms and the collective stress response.</a> Journal of Visualized Experiments: JoVE. (159), e60915 (2020).</li><li>H&#246;lscher, T., 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=Monitoring+spatial+segregation+in+surface+colonizing+microbial+populations.">Monitoring spatial segregation in surface colonizing microbial populations.</a> Journal of Visualized Experiments: JoVE. (116), e54752 (2016).</li><li>Yeung, A. 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研究半固体梯度板上细菌聚集的运动可能是一项具有挑战性的任务18，19，20，因为它涉及多种因素，如底物粘度，湿度和介质组分。在这些因素中，琼脂浓度在确定微生物恢复到游泳或聚集运动方面起着决定性作用。随着琼脂浓度从0.3%（w/ v）增加到1%（w / v），细菌游泳运动将受到限制并逐渐转变为表面聚集，高于1%（w / v）?...

细菌聚集运动是指细菌细胞在物质表面的集体迁移。除了在实验室中专门制备的半固体琼脂平板1外，在一些软基质上也观察到这种表型，例如动物组织2，水合表面3和植物根4。虽然半固体表面被认为是细菌聚集的基本条件之一，但一些物种也需要富含能量的培养基来支持它们的聚集运动5。鞭毛旋转为游泳和聚集运动提供动力 - 游泳描述了液体环境中的单细胞运动，而聚集是微生物种群在半固体表面上的同步运动。
底物粘度影响细菌运动;对致病微生物（如 幽门螺杆菌）的研究表明，病原体的运动性根据粘蛋白层粘度而变化，这受到人类宿主中环境酸化的影响6。为了复制这些环境，早期使用琼脂浓度高于0.3%（w / v）的研究限制了细菌游泳运动，以逐渐转变为表面聚集。使用高于1%（w / v）的琼脂浓度可以防止许多物种的聚集运动7。在表面上形成的蚁群模式是多种多样的，包括无特征的垫子8，牛眼9，树突10和涡旋11。
虽然这些模式的相关性尚不清楚，但这些模式似乎取决于环境和化学线索12。环境线索涵盖不同的方面，包括温度、盐度、光照和pH值，而化学线索包括微生物群体感应分子、生化副产物和营养物质的存在。自动诱导群体传感信号分子，如AHL（N-己酰-L高丝氨酸内酯）可以通过调节表面活性剂的产生来影响表面聚集13，14。白藜芦醇是一种植物抗毒素化合物，可以限制细菌聚集的运动15.
本文研究了白藜芦醇梯度浓度对野生型 大肠 杆菌K12菌株的影响，并研究了工程 大肠 杆菌K12-YdeH和 铜绿假单胞菌 PAO1-YdeH物种的阿拉伯糖诱导群运动。阿拉伯糖通过 araBAD 启动子诱导YdeH酶的产生，导致细胞c-di-GMP扰动并影响细菌群运动16，17。使用阿拉伯糖梯度群板与 大肠杆菌 K12-YdeH和 铜绿假单胞 菌PAO1-YdeH菌株研究这种诱导的群体行为。
梯度群板通过连续固化双层介质制备（图1B）。底层包括与诱导剂一起加入的培养基，倒在支撑的培养皿的一侧。在底层固化后，培养皿返回到平坦的表面，其中含有没有诱导剂的介质的上层从板的另一侧加入。在群板完全固化后，通过按照固定布局在群板上钻孔（图1C）或通过在介质固化过程中使用包含销子的板盖的3D打印模型印印孔来产生等距排列的保持井（补充图S1）。凝胶成像系统用于捕获不同时间点的聚集形态（图2）。使用ImageJ软件分析表面群（补充图S2）提供了表面聚集过程的定量结果（图3）。
因此，我们提出了一种简单的方法来测试诱导剂浓度范围内的表面聚集运动。该方法可用于通过将诱导剂混合到底层培养基中来测试其他诱导剂的多种浓度反应，并且可以应用于其他活动物种（例如， 枯草芽孢杆菌， 肠沙门氏菌， 奇异变形杆菌， 肠溶性耶尔森菌）。这种方法可以为筛选单一化学诱导剂提供可靠的定性和定量结果，并且可以使用单独的板来评估更多的化学诱导剂。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agar</td>
			<td>Sigma-Aldrich</td>
			<td>V900500</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Solarbio</td>
			<td>A8180</td>
			<td>25 g, &#8805; 85% (GC)</td>
		</tr>
		<tr>
			<td>Centrifuge tube</td>
			<td>Corning</td>
			<td>430790</td>
			<td>15 mL</td>
		</tr>
		<tr>
			<td>Cryogenic vial</td>
			<td>Corning</td>
			<td>430488</td>
			<td>2 mL</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Aladdin</td>
			<td>D103272</td>
			<td>AR, &#62; 99% (GC)</td>
		</tr>
		<tr>
			<td>L(+)-Arabinose</td>
			<td>Aladdin</td>
			<td>A106195</td>
			<td>98% (GC), 500 g</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Bkman</td>
			<td>B-SLPYM90-15</td>
			<td>Plastic Petri dishes,circular,90 mm x 15 mm</td>
		</tr>
		<tr>
			<td>Resveratrol</td>
			<td>Aladdin</td>
			<td>R107315</td>
			<td>99% (GC), 25 g</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Macklin</td>
			<td>S805275</td>
			<td>AR, 99.5% (GC), 500 g</td>
		</tr>
		<tr>
			<td>Square Petri dishes</td>
			<td>Bkman</td>
			<td>B-SLPYM130F</td>
			<td>Plastic Petri dishes, square, 13 mm x 13 mm</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Thermo Scientific Oxoid</td>
			<td>LP0042</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Thermo Scientific Oxoid</td>
			<td>LP0021</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biochemical incubator</td>
			<td>Blue pard</td>
			<td>LRH-70</td>
			<td></td>
		</tr>
		<tr>
			<td>Tanon 5200multi imaging system</td>
			<td>Tanon</td>
			<td>5200CE</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermostatic water bath</td>
			<td>Jinghong</td>
			<td>DK-S28</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantifying bacterial surface swarming motility on inducer gradient plates

细菌聚集运动是一种常见的微生物表型，细菌群落用它来在半固体表面上迁移。在诱导聚集运动的研究中，诱导剂的特定浓度可能无法报告在最佳浓度范围内发生的事件，以引起物种的所需反应。含有多种浓度的半固体板通常用于研究诱导剂浓度范围内的反应。然而，由于不均匀的凝固时间，单独的半固体板会增加每个板内介质粘度和水分含量的变化。
本文描述了一种在单个梯度板上同时测试表面聚集运动的一步法，其中等距排列的测试孔允许同时采集多重度响应。在目前的工作中，评估了 大肠 杆菌K12和 铜绿假单胞菌 PAO1的表面聚集，以响应白藜芦醇和阿拉伯糖等诱导剂的浓度梯度。定期使用成像系统对群体形态进行成像，以捕获整个表面集群过程。
使用ImageJ软件获取了群体形态的定量测量，提供了群体区域的可分析信息。本文提出了一种简单的梯度群板方法，该方法提供了诱导剂对表面聚集的影响的定性和定量信息，可以扩展到研究其他诱导剂对更广泛的运动细菌物种的影响。

由梯度群板的制备，接种和孵育组成的工作流程如图 1B所示。为了产生梯度游板，将底层介质从水平面以~4.3°的支撑皿中倒入支撑皿中（补充图S3），然后在底层完全固化后倒入上层介质。双层介质的组成如 表1所示。然后，将培养过夜的细菌培养物移液到测试孔中并在37°C下孵育，保持适当的湿度水平。在具有两个或三个重复的梯度群板中设置多个...

Watch this Scientific Journal Video about Quantifying Bacterial Surface Swarming Motility on Inducer Gradient Plates at JoVE.com

Quantifying Bacterial Surface Swarming Motility on Inducer Gradient Plates

在这里，我们描述了使用诱导剂梯度板来评估细菌聚集运动，同时获得多种浓度响应。

量化诱导剂梯度板上细菌表面聚集运动

Bacterial swarming motility is a common microbiological phenotype that bacterial communities use to migrate over semisolid surfaces. In investigations of ...

该协议同时跟踪半固体琼脂上不同诱导剂浓度的细菌聚集反应。这是以具有成本效益的方式实现的。这种高通量筛选技术消除了具有各种诱导剂浓度的琼脂观察微化学反应的需要。该技术旨在用于药物筛选以及观察微病原体SD反应。此外，它还可用于研究人类宿主中对各种生化线索的微反应。这种方法可以提供对细菌趋化性的洞察。细菌会对各种浓度的化学引诱剂（称为正趋化性）或化学驱虫剂（称为阴性趋化性）作出反应。精心准备的设置对于避免下层凝胶角度之间的变化至关重要，以防止再现性问题。首先，在13×13厘米见方的培养皿上贴上污渍和诱导剂名称。然后将盘子撑在盖子的边缘。将阿拉伯糖溶液加入温暖的底层培养基中。加入40毫升温底层培养基。让底层介质在层流罩内裸露固化一小时，不受干扰。一旦底层完全凝固，取下盖子并将培养皿放在层流罩内。使用电动移液器过滤器加入40毫升温热的上层介质。固化双层板覆盖且不受干扰一小时，然后在四摄氏度下储存24小时。将标记的A4纸放在凝固良好的梯度板下。将100微升移液器吸头的较宽侧推入半固体介质表面的标记位置，然后按压移液器吸头，直到其到达上层介质的底部。当尖端接触底部时，停止对尖端施加任何垂直力，并轻轻旋转尖端以隔离圆柱形孔的内容物。沿一小段距离水平移动移液器吸头，让气流进入预留的狭窄位置。用食指按压吸头以阻挡吸头内的气体流。垂直拉出尖端，在将其拉出时将孔内容物保持在尖端中。将80微升的过夜生长培养物加入每孔中。每12小时将蜂群板从培养箱中取出一个，并将其置于凝胶成像系统中。选择凝胶成像模式，将蜂群板暴露在白光下，并调整焦距以获得群的清晰视图。通过将曝光时间调整为300毫秒，增强群体的亮度以进行精确观察。调整阈值以最大程度地减少来自背景光的干扰。保存图像文件以供进一步分析。在txt文件中记录成像时间，诱导剂类型，梯度方向和菌株。单击“处理”，然后单击“阴影”以增强图像的清晰度。单击“处理”，然后单击“批处理”以处理图像。然后通过单击“处理”、“阴影”和“南”将图像作为参考进行处理。单击“处理”、“批处理”、“宏”以打开“批处理”窗口。查找窗口中显示的命令。在输出文件地址中键入原始图像的文件夹地址，方法是单击“批处理”窗口中的“处理”。使用魔杖追踪工具单独选择群，然后双击魔杖追踪工具以调整容差，直到生成的线正确拟合群边界。单击“分析”，然后单击“测量”以导出区域值。在阿拉伯糖梯度板上测试大肠杆菌K12 YdeH，用于证明相邻井之间发生重叠的表面聚集过程。具有三个测试井的板能够形成非重叠边界。铜绿假单胞菌PAO1 YdeH聚集运动随着阿拉伯糖浓度从最低浓度的增加而得到促进，但逐渐限制为较高浓度的阿拉伯糖浓度。在0至400微升浓度范围内的白藜芦醇梯度板上测试的大肠杆菌K12野生型菌株显示，随着白藜芦醇浓度的增加，聚集运动受到适度限制。Swarm区域由ImageJ软件量化。群动曲线显示了多重浓度响应。该过程最关键的部分是制造具有特定浓度范围的诱导剂的双层蜂群板。这种方法使研究人员能够在特定浓度范围内研究诱导的表面聚集，并量化其他诱导剂和组分对群体的影响。

Research

JoVE Journal

Biochemistry

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

科研

生物化学

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

一种简单的高通量化学筛选方法在秀丽线虫中的应用

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

体外肠道宿主-微生物界面合成细菌联合的可行性评价

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

肾小球的分离及肾小球细胞表面蛋白的活体标记

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and ...

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

使用莱迪思光片显微镜可视化表面 T-Cell 受体动力学四维

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

A Planarian Motility Assay to Gauge the Biomodulating Properties of Natural Products

测量天然产品生物调节特性的平面运动分析

A straightforward, controllable means of using the non-parasitic planarian, Dugesia tigrina, a free-living aquatic flatworm, to study the stimulant and ...

Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

通过等密度梯度纯化对U937细胞进行细胞分级分离

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and ...

Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays

体外荧光显微镜基于动感检测的肌素特异性适应

Myosin proteins bind and interact with filamentous actin (F-actin) and are found in organisms across the phylogenetic tree. Their structure and enzymatic ...

Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells

密度梯度超速离心研究哺乳动物细胞内吞循环

Endosomal trafficking is an essential cellular process that regulates a broad range of biological events. Proteins are internalized from the plasma ...

Polysome Profiling without Gradient Makers or Fractionation Systems

无需梯度测定器或分馏系统的多聚体分析

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs ...