这项工作得到了国家卫生研究院授予GM118085的支持，部分得到了罗伯特·韦尔奇基金会的支持（授予R-1811R.M.H.）。

1. 在缺乏活动力的菌株中分离抑制突变体
注:使用此方法作为广泛的"捕获所有"，以确定动能缺陷的一般性质。
<ol><li>软加板准备 注:软阿加，也被称为运动或游泳的阿加，是一个低百分比的阿加（+0.2-0.35%w/v），长期用于检测化疗31，32。<ol>
<li>在 2 L 圆形底烧瓶中加入 3 克 bacto-agar（0.3% w/v） 和 20 克 LB。在烧瓶中加入 1 L 的 ddH2O（双蒸馏水），然后使用搅拌棒和磁搅拌板均匀地混合悬架。</li>
		<li>高压灭菌 20 分钟，121 °C。</li>
		<li>使用上面的杆/板，让温和的搅拌冷却。当温度达到约 50 °C 时，将 25 mL 倒入无菌的 Petri 盘（100 mm x 15 mm），并允许熔融的 agar 将盖子固定至少 1 小时，用于 16 小时内。</li>
	</ol></li>
	<li>抑制突变体的文化准备、接种和隔离 注: 大肠杆菌 在营养丰富的介质中心接种，用软醋凝固，在当地消耗营养物质，从而产生它们遵循的营养梯度。当它们向外移动时，定义的"环"出现（图1A），这与细菌对特定的化学作用有关。化疗系统或旗杆菌电机结构部件的缺陷可能会损害此检测中的性能。通常，具有活动优势的突变体在筛选过程中出现，并且可以看到从单点或环外围的多个点出现，从它们"燃烧"出来的地方（图1C）。人们会注意到，游泳前部最外侧的边缘很容易与未殖民的处女软阿加形成对比。<ol>
<li>在 5 mL 的伦诺克斯兄弟 （LB; 10 g/L 试剂酮， 5 克/升酵母提取物， 和 5 克/升 NaCl， 材料表） 在 30 °C 水平摇晃 （220 r.p.m.第二天，亚文化（1:100稀释）在新鲜LB，在相同的条件下增长到指数阶段（OD600 的0.6）。</li>
		<li>使用移液器将 6 μL 的培养物接种到软醋板 （1.1） 的中心，将装载的无菌尖端推入 Agar 以轻轻排出其内容。转移到30°C并孵育（图1B），直到运动"火焰"明显，从接种点或运动环的外围发出，通常在24-36小时（图1C）。 注:在动感分析中，在突变隔离物旁边接种野生菌株进行比较。这种野生型菌株将显示出典型的同心化学环（图1A），并在8-10小时内填充板。</li>
		<li>使用无菌线环将细胞从"火焰"区域和条纹中提升，将单个菌落纯化到 LB 硬阿加板上（如上所示，用 15 克/升的 bacto-agar 凝固）。</li>
		<li>使用无菌线环从条纹板中挑选单个菌落，并通过单个菌落的条纹重新净化，以确保隔离"纯"菌落隔离。</li>
	</ol></li>
	<li>抑制突变体的确认和特征
	<ol>
<li>确认孤立的抑制突变体已恢复活力。准备软加板（1.1），以及感兴趣的菌株（如1.2.1），包括野生类型和开始的"缺乏活力"菌株进行比较。</li>
		<li>在 30 °C 下接种板（如 1.2.2），在 8-10 小时孵育。</li>
		<li>记录最外环（圆的边缘）的直径，并比较确定哪些隔离物已显著恢复活力。 注:建议在整个实验过程中拍摄板材。为了获得最佳效果，请使用"光桶"设备30，其中数码相机安装在光源上方，以便更好地照明，以测量游泳殖民地的直径，并将其与未殖民化的偶发体区分开来。</li>
		<li>经验证的受试者可根据需要与 WGS 隔离，从而获得足够的"序列覆盖"，从而积极识别恢复野生类型功能的突变。</li>
	</ol></li>
</ol>2. 通过细胞系绳量化旗杆菌运动行为
注意:当正常运行翻滚行为（化学轴）出现受损时，使用此方法。
<ol><li>
文化准备和旗杆切口
	<ol>
<li>准备第 1.2.1 步中描述的兴趣应变的指数阶段文化。</li>
		<li>以 2，000 x g 离心方式将 10 mL 细胞颗粒 10 mL，在 3 分钟内再悬浮在 10 mL 的过滤灭菌运动缓冲器 （MB） 中: 10 mM 磷酸钾缓冲器 [0.0935 M K2HPO4， 0.0065 M KH2PO4， pH 7.0]， 0.1 mM EDTA [pH 7.0]， 10 mM NaCl， 75 mM KCl）. 注:MB支持 活性， 但不支持细菌生长</li>
		<li>重复步骤 2.1.2 两次，然后在 1 mL 的 MB 中重新暂停最后颗粒。</li>
		<li>将电池悬架转移到 1 mL 注射器中，并将 23G 针头连接到末端。组装相同的注射器/针具，并通过6英寸的聚乙烯管（内径0.58毫米）将两者连接在一起，紧紧地套在每根针尖上。</li>
		<li>通过轻轻地将细胞悬架从一个注射器来回传递到其他 50 倍，每 10 次通过之间暂停 1 分钟，从而剪切旗杆（它们很脆弱，很容易断裂）。</li>
		<li>将切变电池以 2，000 x g 离心 3 分钟，最后卷为 500 μL MB。</li>
	</ol>
</li>
	<li>
幻灯片制备和细胞系绳
	<ol>
<li>通过将 18 mm x 18 mm 的盖片堆叠在 3 英寸 x 1 英寸 x 1 毫米玻璃显微镜滑动上，通过双面胶带（图 S1）分离，准备细胞固定室。</li>
		<li>通过应用到腔室顶部，用 0.01% （w/v） 多赖氨酸溶液冲洗腔室。将底部边缘（覆盖唇与显微镜滑动一起冲洗）倾斜到任务雨刷器（纸巾）上，以帮助通过腔室绘制溶液（图 S1）。然后在室温下孵育10分钟。</li>
		<li>使用第 2.2.2 步中描述的 40 μL MB 清洗腔室三次</li>
		<li>将 40 μL 的剪切细胞悬架（上图所示）添加到腔室顶部，并在室温下孵育 10 分钟，使细胞附着在盖唇上。</li>
		<li>轻轻冲洗腔室与 40 μL 的 MB 在 2.2.3 中删除未连接的细胞。</li>
	</ol>
</li>
	<li>
细胞旋转记录和量化
	<ol>
<li>将装有系绳细胞的显微镜滑梯转移到显微镜阶段。</li>
		<li>使用相对比显微镜和100倍目标，扫描人口，寻找固定到位的细胞，并在单轴上旋转，即在固定点上平稳旋转，而不是以细胞进出聚焦的角度呈现（视频1）。</li>
		<li>使用商用显微镜和相关摄像机。打开相关软件，确保感兴趣的单元格处于对焦状态，并单击 视频采集 以录制一分钟的单元格旋转（每秒 10 帧或更高）。</li>
		<li>从视频播放中，量化每分钟完整旋转次数和细胞改变方向（切换频率）的次数。 注:旋转速度和切换频率可能太快，无法用眼睛测量，因此建议使用视频软件提供慢/精细播放，或采用自动化软件系统来量化旋转模式33。另一种选择是使用甲基纤维素（或类似剂）增加MB的粘度，以帮助减缓和解决更快的细菌的旋转，或在使用帧速率低的相机时进行补偿。</li>
		<li>重复步骤 2.3.2 与生物复制，以编制感兴趣的人口的表示。</li>
	</ol>
</li>
</ol>3. 在过境分析中准备成群
注意:使用此方法评估突变或条件对群体活力的影响。沼泽-阿加是指通常高于软阿加的百分比的阿加。在软阿加（0.3%）中，细胞在亚加体内单独游泳。在群加（0.5%及以上）中，细胞在表面上作为一组移动。虽然蜂群板必须在这里详细使用，但游泳板的保质期更长，并且可能使用数天。我们的个人喜好是在1-2天内使用。
<ol><li>
准备群阿加
	<ol>
<li>在 2 L 圆形底烧瓶中加入 5 克 Eiken agar（0.5% w/v） 和 20 克 LB。在烧瓶中加入 1 L 的 ddH2O，然后使用搅拌棒和磁搅拌板均匀地混合悬架。</li>
		<li>高压灭菌 20 分钟，121 °C。</li>
		<li>允许用温和的搅拌冷却，以避免使用上面的杆/板的任何气泡。当大约 50 °C 时，添加过滤消毒葡萄糖，最终浓度为 0.5%。</li>
		<li>将 25 mL 倒入无菌的 Petri 菜肴（100 mm x 15 mm），并允许在室温下设置至少 14 小时且不超过 20 小时。不要存储供将来使用。</li>
	</ol>
</li>
	<li>
蜂盘的接种和孵化
	<ol>
<li>通过在 agar 顶部发现 6 μL 的中指数文化（如 1.2 中准备）接种。</li>
		<li>将盖子关闭 5-10 分钟，当接种管干燥到阿加表面时更换。</li>
		<li>在 30 °C 下孵育 8 小时。通过移除盖子来避免检查群进度的诱惑，因为这将有助于干燥的阿加和受损的蜂群。 注:孵化时间可能因应变表型而异。一些孤立的突变可能会阻碍成群的能力，需要降低加糖百分比或更长的潜伏期。</li>
	</ol>
</li>
	<li>
准备过境检测板 注:此检测使用改良的培养皿，其中塑料隔（边）创建两个腔室，而不是一个腔室（图 2A）。每个腔室可以独立于其他房间准备，在"连接"两个房间之前，为蜂群提供不同的条件。根据实验设计，第一个腔室（指定左）可以准备与游泳的阿加（0.3%w/v）或群阿加（0.5%w/v），从那里细菌可以越过边界迁移到右室含有蜂群阿加+/-任何必要的补充或挑战（例如，抗生素）。对任何一种阿加的迁移通常通过记录细菌殖民化（边缘到边缘）从原始接种点的最宽直径来测量/比较。<ol>
<li>如果需要，准备 1.1 中描述的游泳阿加。</li>
		<li>准备3.1中描述的群加。</li>
		<li>将 +30 mL 的蜂群阿加（如果需要，需要补充）倒入双隔间 Petri 盘（100 mm x 15 mm）的右室，使其与腔室之间的塑料分隔器平起平落，但不会溢出到左侧（图 2B）。</li>
		<li>在阿加变硬后，用+30ml的游泳或蜂群的阿加填充左室，再次到与塑料隔线接触点（图2C）。在设置之前，使用无菌移液器尖端轻轻地将阿加拖过边界，将两侧连接起来，连接横跨整个鸿沟长度的 +1 毫米高的阿加桥（图 2D）。</li>
		<li>让盘子在室温下干燥（3.1）。 注:创建桥梁的另一种方法是让左室醋干燥，然后沿着塑料分隔器缓慢移液器 +100 μL 熔融群醋，以连接两个腔室 （3.4）。在 30 °C 孵育 12-16 小时之前，或直到蜂群在右腔上取得足够进展，以便比较感兴趣的菌株之前，如上文所述，为左室的盘子接种疫苗（1.2.2）或群（3.2）agar。</li>
	</ol>
</li>
</ol>

作者没有什么可透露的。

抑制剂突变的分离和定性成功地帮助识别了化疗系统35、36、37以及电机机械本身38、39、40的关键部件。在使用第1号议定书时，必须包括多个独立复制，以确保隔离大量可能的突变，以弥补活动性损失。通过将培养体划入一条线而不是一个点来增加细菌的数?...

细菌在其生态利基中利用许多附属物进行运动和分散。弗拉盖拉驱动的动能是其中最快的，促进有利地区响应环境信号的殖民化，并大大有助于一些物种2，3的致病能力。旗菌可以单独在散装液体中游泳，或集体聚集在半固体表面4。细胞外旗手附着在膜内，由嵌入膜的旋转电机驱动，该电机利用离子梯度的功率产生扭矩，导致旋转 1、2、4、5、6、7、8。在大肠杆菌中，电机以恒定的扭矩9运行，电机输出可以按旋转速度和转子在逆时针 （CCW） 和顺时针 （CW） 方向之间的切换进行分类。CCW 旋转促进形成一个连贯的旗杆束，推动细胞向前（运行），而旋转方向 （CW） 的瞬时开关导致束部分或完全拆解10，细胞重新定位其游泳方向（翻滚）。大肠杆菌通常运行一秒钟，跌落十分之一秒。转子或"翻滚偏置"的切换频率由化疗信号系统控制，其中转子切除器检测外部化学信号并通过磷化物将其传输到旗杆电机，以延长运行以响应吸引物，或抑制它们以响应有毒化学物质11，12。游泳运动在0.3%的软性啤酒中测定。
在蜂拥而至时，细菌在半固体表面作为密集的集体导航，细菌群在连续旋转运动中流淌2、13、14、15。大肠杆菌群表现出化学感官生理变化（较低的翻滚偏差），更高的速度，和更高的耐受性抗菌细胞在散装液体中游动16，17。沼泽人部署的策略，以帮助运动，包括表面活性剂生产，恶性通货膨胀，和细胞拉长2。沼泽为细菌提供了在生态和临床环境中的竞争优势18，19，20。有两类蜂群细菌:温带蜂群，它只能聚集在媒体凝固与0.5-0.8%的阿加，和强大的蜂群，它可以导航通过更高的阿加浓度21。
存在各种分析来询问游泳的活力及其规定。当受到突变或环境条件的损害时，活动本身为识别功能增益抑制剂突变提供了强有力的选择。这些抑制剂可以是原始突变的真正还原剂，也可以是伪反变剂，其中第二个突变会恢复功能。这种突变体可以通过全基因组测序（WGS）来识别。不偏不倚的抑制器选择的替代方案是偏颇的靶向突变策略（例如 PCR 突变）。这些方法往往揭示了动能装置的功能或环境调节。如果目标是研究运动功能，那么用软糖测量的野生型运动性的恢复不一定意味着野生型运动输出的恢复。细胞系绳检测，其中细胞通过单个旗杆连接到玻璃表面，随后对细胞体的旋转进行监测，可以作为评估运动行为的初始选择。虽然现在可以采用更复杂的方法来监控电机性能，但所需的高速摄像头设置和用于运动分析的软件包限制了其广泛使用 22、23、24、25。细胞系绳检测仅要求剪切旗杆，允许将短丝附着在玻璃滑梯上，然后录下细胞体的旋转。虽然记录的电机速度在本次测定中较低，因为细胞体对旗手施加了高负荷，但此测定还是有助于对化学反应 26、27、28、29 进行有价值的见解，并且仍然是下面讨论的有效调查工具。
沼泽的活力给研究人员带来了一系列不同的挑战。功能抑制器的选择只在产生大量表面活性剂的群中有效，并且容易地蜂拥而至。表面活性剂非生产者，如大肠杆菌是挑剔的选择，在阿加的选择，媒体组成和湿度的环境2，13，14，21。一旦蜂群条件建立，过境分析17是一个有用的方法来询问群导航新的/苛刻条件的能力。虽然下面提出的协议与大肠杆菌有关，但它们很容易适应其他物种的应用。

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Bacto Dehydrated Agar</td><td>Fisher Scientific</td><td>DF0140-15-4</td><td></td></tr><tr><td>EDTA Disodium Salt, Dihydrate</td><td>Fisher Scientific</td><td>02-002-786</td><td></td></tr><tr><td>Eiken agar</td><td>Eiken Chemical Co. Japan</td><td>E-MJ00</td><td>Essential for E. coli swarming</td></tr><tr><td>Glucose D (+)</td><td>Fisher Scientific</td><td>410955000</td><td></td></tr><tr><td>LB (Lennox) Broth</td><td>Fisher Scientific</td><td>BP1427-500</td><td></td></tr><tr><td>Poly-L-lysine Solution (0.1%)</td><td>Sigma-Aldrich</td><td>P8920</td><td></td></tr><tr><td>Potassium chloride (KCl)</td><td>Fisher Scientific</td><td>18-605-496</td><td></td></tr><tr><td>Potassium Phosphate monobasic (KH2PO4)</td><td>Fisher Scientific</td><td>BP362-500</td><td></td></tr><tr><td>Potassium Phosphate dibasic (K2HPO4)</td><td>Fisher Scientific</td><td>BP363-500</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Fisher Scientific</td><td>S271-500</td><td></td></tr><tr><td>&lt;strong&gt;Materials and Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CellSense microscope imaging software (V. 1.6)</td><td>Olympus</td><td></td><td>Or equivalent software for microscope used</td></tr><tr><td>Electron Microscopy Sciences Scotch 666 Doube Sided Tape</td><td>Fisher</td><td>50-285-28</td><td></td></tr><tr><td>Frosted microscope slides 3x1x1mm</td><td>Fisher</td><td>12-550-343</td><td></td></tr><tr><td>Olympus BX53 microscope</td><td>Olympus</td><td>BX53</td><td>Any upright or inverted phase microscope can be used</td></tr><tr><td>Petri dishes (100 mm diameter)</td><td>Fisher Scientific</td><td>FB0875712</td><td>For soft-agar assays</td></tr><tr><td>Polyethylene Nebulizer Capillary Tubing (0.58mm x 99mm 3.0m)</td><td>Perkin Elmer</td><td>9908265</td><td></td></tr><tr><td>Round Petri Dish with 2 Compartments</td><td>VWR</td><td>89200-944</td><td>For border-crossing assays</td></tr><tr><td>Safety Hypodermic Needles (23G)</td><td>Fisher Scientific</td><td>14-826A</td><td></td></tr><tr><td>Sterile Syringe - 1 mL</td><td>Fisher scientific</td><td>14-955-450</td><td></td></tr><tr><td>Task/Tissue wipes</td><td>Fisher scientific</td><td>06-666</td><td>Or equivalent single use tissue wipes</td></tr><tr><td>VWR micro cover-glass 18x18mm</td><td>VWR</td><td>48366205</td><td></td></tr><tr><td>XM10 camera</td><td>Olympus</td><td>XM10</td><td>Or equivalent microscope camera</td></tr></tbody></table>

investigating flagella-driven motility in escherichia coli by applying three established techniques in a series

活力对许多细菌物种的生存和成功至关重要。存在许多方法来利用动感来理解信号通路，阐明旗手部件的功能和组装，并检查和理解运动模式。在这里，我们演示了其中三种方法的组合。软性阿加的活力是最古老的，为分离体弱化菌株中的功能增益抑制剂突变提供了强有力的选择，通过第二次突变恢复活力。细胞系绳技术首先用于演示旗手电机的旋转性质，可用于评估信号效应器对电机速度的影响及其切换旋转方向的能力。"越境"检测是最新的，游泳细菌可以准备过渡到集体移动成群。综合起来，这些协议代表了一种系统而有力的方法，用于识别运动机械的部件，并描述它们在游泳和蜂拥而至的不同方面的作用。它们可以很容易地适应研究其他细菌物种的动能。

我们实验室34的最新研究详细介绍了大肠杆菌株中伪还原体的分离，其活动性因信号分子c-di-GMP的高水平而受损。这种菌株（JP1442）有两种突变:yhjH和+ycgR。YhjH是大肠杆菌中降解c-di-GMP的最活性磷二酯酶。缺乏 Yhjh 会导致 c - di - gmp 水平升高和抑制动能。Ycgr 是 c - di - gmp 效应器。在与 c-di-GMP 的复合体中，YcgR 与旗杆转子结合，首先诱导 CCW 电机旋转，然后...

Watch this Scientific Journal Video about Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series at JoVE.com

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

许多细菌使用旗菌驱动的活力来导航他们的环境，并单独和集体殖民有利的环境。这里展示的是使用三种既定的方法，利用动感作为选择工具，以确定有助于游泳和蜂拥而至的活力的组件/路径。

通过在系列中应用三种既定技术，调查大 肠杆菌 的弗拉盖拉驱动的动性

Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, ...

investigating-flagella-driven-motility-escherichia-coli-applying

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

7.5K 次观看.  The University of Texas at Austin. 许多细菌使用旗菌驱动的活力来导航他们的环境，并单独和集体殖民有利的环境。这里展示的是使用三种既定的方法，利用动感作为选择工具，以确定有助于游泳和蜂拥而至的活力的组件/路径。

视频: Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

可视化单分子络合物在体内使用高级荧光显微

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

科研

生物学

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the concentration of specific chemoeffectors by comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement. Although multiple, competing gradients often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to concentration gradients of attractants and repellents. Here, we describe the development of a microfluidic chemotaxis model for presenting precise and stable concentration gradients of chemoeffectors to bacteria and quantitatively investigating their response to the applied gradient. The device is versatile in that concentration gradients of any desired absolute concentration and gradient strength can be easily generated by diffusive mixing. The device is demonstrated using the response of Escherichia coli RP437 to gradients of amino acids and nickel ions.

This protocol describes the development of a microfluidic device for investigating bacterial chemotaxis in stable concentration gradients of chemoeffectors.

一个微流体装置量化细菌趋稳定的浓度梯度

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the ...

Quantifying Bacterial Surface Swarming Motility on Inducer Gradient Plates

Bacterial swarming motility is a common microbiological phenotype that bacterial communities use to migrate over semisolid surfaces. In investigations of induced swarming motility, specific concentration&#160;of an inducer may not be able to report events occurring within the optimal concentration range to elicit the desired responses from a species. Semisolid plates containing multiple concentrations are commonly used to investigate the response within an inducer concentration range. However, separate semisolid plates increase variations in medium viscosity and moisture content within each plate due to nonuniform solidification time.
This paper describes a one-step method to simultaneously test surface swarming motility on a single gradient plate, where the isometrically arranged test wells allow the simultaneous acquisition of multiconcentration responses. In the present work, the surface swarming of Escherichia coli K12 and Pseudomonas aeruginosa PAO1 were evaluated in response to a concentration gradient of inducers such as resveratrol and arabinose. Periodically, the swarm morphologies were imaged using an imaging system to capture the entire surface swarming process.
The quantitative measurement of the swarm morphologies was acquired using ImageJ software, providing analyzable information of the swarm area. This paper presents a simple gradient swarm plate method that provides qualitative and quantitative information about the inducers&#39; effects on surface swarming, which can be extended to study the effects of other inducers on a broader range of motile bacterial species.

Here, we describe the use of inducer gradient plates to evaluate bacterial swarming motility while simultaneously obtaining multiple concentration responses.

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 ...

生物化学

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

Xylella fastidiosa is a Gram-negative non-flagellated bacterium that causes a number of economically important diseases of plants. The twitching motility provides X. fastidiosa a means for long-distance intra-plant movement and colonization, contributing toward pathogenicity in X. fastidiosa. The twitching motility of X. fastidiosa is operated by type IV pili. Type IV pili of Xylella fastidiosa are regulated by pilG, a chemotaxis regulator in Pil-Chp operon encoding proteins that are involved with signal transduction pathways. To elucidate the roles of pilG in the twitching motility of X. fastidiosa, a pilG-deficient mutant Xf&#916;pilG and its complementary strain Xf&#916;pilG-C containing native pilG were developed. A microfluidic chambers integrated with a time-lapse image recording system was used to observe twitching motility in Xf&#916;pilG, Xf&#916;pilG-C and its wild type strain. Using this recording system, it permits long-term spatial and temporal observations of aggregation, migration of individual cells and populations of bacteria via twitching motility. X. fastidiosa wild type and complementary Xf&#916;pilG-C strain showed typical twitching motility characteristics directly observed in the microfluidic flow chambers, whereas mutant Xf&#916;pliG exhibited the twitching deficient phenotype. This study demonstrates that pilG contributes to the twitching motility of X. fastidiosa. The microfluidic flow chamber is used as a means for observing twitching motility.

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

In this study, a nano-microfluidic flow chamber was employed to visualize and functionally characterize the twitching motility of Xylella fastidiosa, a bacterium that causes Pierce&#39;s disease in grapevine.

抽动运动功能的可视化和表征的角色的<em&gt; PilG</em&gt;在<em&gt;叶缘焦枯病菌</em

Xylella fastidiosa is a Gram-negative non-flagellated bacterium that causes a number of economically important diseases of plants. The twitching motility ...

免疫与感染

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for&#160;in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and&#160;inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.

This study details purification of KIF1A(1-393LZ), a member of kinesin-3 family, using Sf9-baculovirus expression system. In vitro single-molecule and multi-motor gliding analysis of these purified motors exhibited robust motility properties comparable to motors from mammalian cell lysate. Thus, Sf9-baculovirus system is amenable to express and purify motor protein of interest.

Sf9纯化超加工驱动蛋白-3系列电机的单分子分析

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved ...

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process starts by screening for colonies that survive on minimal media, assuming that only Escherichia coli harboring the correct plasmid will be able to thrive in the specific conditions. Since the process of building large genetic circuits, requiring the assembly of many DNA parts, is challenging, circuit components are often distributed between multiple plasmids at different copy numbers requiring the use of several antibiotics. Mutations in the plasmid can destroy transcription of the antibiotic resistance genes and interject with resources management in the cell leading to necrosis. The selected colony is set on a glass-bottom Petri dish and a few focus planes are selected for microscopy tracking in both bright field and fluorescent domains. The protocol maintains the image focus for more than 12 hours under initial conditions that cannot be regulated, creating a few difficulties. For example, dead cells start to accumulate in the lenses' field of focus after a few hours of imaging, which causes toxins to buildup and the signal to blur and decay. Depletion of nutrients introduces new metabolic processes and hinder the desired response of the circuit. The experiment's temperature lowers the effectivity of inducers and antibiotics, which can further damage the reliability of the signal. The minimal media gel shrinks and dries, and as a result the optical focus changes over time. We developed this method to overcome these challenges in Escherichia coli, similar to previous works developing analogous methods for other micro-organisms. In addition, this method offers an algorithm to quantify the total stochastic noise in unaltered and altered cells, finding that the results are consistent with flow analyzer predictions as shown by a similar coefficient of variation (CV).

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

This work presents a microscopy method that allows live imaging of a single cell of Escherichia coli for analysis and quantification of the stochastic behavior of synthetic gene circuits.

使用延时显微镜连续测量 大肠杆菌 中的生物噪声

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process ...

生物工程

Generation of Null Mutants to Elucidate the Role of Bacterial Glycosyltransferases in Bacterial Motility

The study of glycosylation in prokaryotes is a rapidly growing area. Bacteria harbor different glycosylated structures on their surface whose glycans constitute a strain-specific barcode. The associated glycans show higher diversity in sugar composition and structure than those of eukaryotes and are important in bacterial-host recognition processes and interaction with the environment. In pathogenic bacteria, glycoproteins have been involved in different stages of the infectious process, and glycan modifications can interfere with specific functions of glycoproteins. However, despite the advances made in the understanding of glycan composition, structure, and biosynthesis pathways, understanding of the role of glycoproteins in pathogenicity or interaction with the environment remains very limited. Furthermore, in some bacteria, the enzymes required for protein glycosylation are shared with other polysaccharide biosynthetic pathways, such as lipopolysaccharide and capsule biosynthetic pathways. The functional importance of glycosylation has been elucidated in several bacteria through mutation of specific genes thought to be involved in the glycosylation process and the study of its impact on the expression of the target glycoprotein and the modifying glycan. Mesophilic Aeromonas have a single and O-glycosylated polar flagellum. Flagellar glycans show diversity in carbohydrate composition and chain length between Aeromonas strains. However, all strains analyzed to date show a pseudaminic acid derivative as the linking sugar that modifies serine or threonine residues. The pseudaminic acid derivative is required for polar flagella assembly, and its loss has an impact on adhesion, biofilm formation, and colonization. The protocol detailed in this article describes how the construction of null mutants can be used to understand the involvement of genes or genome regions containing putative glycosyltransferases in the biosynthesis of a flagellar glycan. This includes the potential to understand the function of the glycosyltransferases involved and the role of the glycan. This will be achieved by comparing the glycan deficient mutant to the wild-type strain.

Here, we describe the construction of null mutants of Aeromonas in specific glycosyltransferases or regions containing glycosyltransferases, the motility assays, and flagella purification performed to establish the involvement and function of their encoded enzymes in the biosynthesis of a glycan, as well as the role of this glycan in bacterial pathogenesis.

产生空突变体以阐明细菌糖基转移酶在细菌运动中的作用

The study of glycosylation in prokaryotes is a rapidly growing area. Bacteria harbor different glycosylated structures on their surface whose glycans ...

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more &#8220;temperate swarmers&#8221; that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. &#8220;Wettability&#8221;, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.

Swarming motility is influenced by physical and environmental factors. We describe a two-phase protocol and guidelines to circumvent the challenges commonly associated with swarm assay preparation and data collection. A macroscopic imaging technique is employed to obtain detailed information on swarm behavior that is not provided by current analysis techniques.

准备，成像和定量的细菌表面运动性测定

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar ...

Biophysical Characterization of Flagellar Motor Functions

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel in response to a variety of environmental stimuli and are among the triggers for surface colonization and infections. The precise mechanisms by which motors remodel and promote cellular adaptation likely depend on key motor attributes. The photomultiplier-based bead-tracking technique presented here enables accurate biophysical characterization of motor functions, including adaptations in motor speeds and switch-dynamics. This approach offers the advantage of real-time tracking and the ability to probe motor behavior over extended durations. The protocols discussed can be readily extended to study flagellar motors in a variety of bacterial species.

Recent findings suggest that bacterial flagellar motors sense a variety of environmental signals and remodel in response. The bead-assays discussed here are expected to help explain the role of remodeling in cellular adaptation to environmental stressors.

鞭毛马达功能的生物物理表征

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel ...

Visualizing Bacterial Motility Based on a Color Reaction

Bacterial motility is crucial for bacterial pathogenicity, biofilm formation, and drug resistance. Bacterial motility is crucial for the invasion and/or dissemination of many pathogenic species. Therefore, it is important to detect bacterial motility. Bacterial growth conditions, such as oxygen, pH, and temperature, can affect bacterial growth and the expression of bacterial flagella. This can lead to reduced motility or even loss of motility, resulting in the inaccurate evaluation of bacterial motility. Based on the color reaction of 2,3,5-triphenyl tetrazolium chloride (TTC) by intracellular dehydrogenases of living bacteria, TTC was added to traditional semisolid agar for bacterial motility detection. The results showed that this TTC semisolid agar method for the detection of bacterial motility is simple, easy to operate, and does not involve large and expensive instruments. The results also showed that the highest motility was observed in semisolid medium prepared with 0.3% agar. Compared with the traditional semisolid medium, the results are easier to evaluate and more accurate.

Here, we present a protocol to detect bacterial motility based on a color reaction. Key advantages of this method are that it is easy to evaluate and more accurate, and does not require specialized equipment.

基于颜色反应可视化细菌运动

Bacterial motility is crucial for bacterial pathogenicity, biofilm formation, and drug resistance. Bacterial motility is crucial for the invasion and/or ...

通过在系列中应用三种既定技术，调查大肠杆菌的弗拉盖拉驱动的动性

摘要

探索更多视频

此视频中的章节

可视化单分子络合物在体内使用高级荧光显微

一个微流体装置量化细菌趋稳定的浓度梯度

量化诱导剂梯度板上细菌表面聚集运动

抽动运动功能的可视化和表征的角色的

Sf9纯化超加工驱动蛋白-3系列电机的单分子分析

使用延时显微镜连续测量大肠杆菌中的生物噪声

产生空突变体以阐明细菌糖基转移酶在细菌运动中的作用

准备，成像和定量的细菌表面运动性测定

鞭毛马达功能的生物物理表征

基于颜色反应可视化细菌运动

通过在系列中应用三种既定技术，调查大 肠杆菌 的弗拉盖拉驱动的动性

摘要

探索更多视频

此视频中的章节

可视化单分子络合物在体内使用高级荧光显微

一个微流体装置量化细菌趋稳定的浓度梯度

量化诱导剂梯度板上细菌表面聚集运动

抽动运动功能的可视化和表征的角色的

Sf9纯化超加工驱动蛋白-3系列电机的单分子分析

使用延时显微镜连续测量 大肠杆菌 中的生物噪声

产生空突变体以阐明细菌糖基转移酶在细菌运动中的作用

准备，成像和定量的细菌表面运动性测定

鞭毛马达功能的生物物理表征

基于颜色反应可视化细菌运动

通过在系列中应用三种既定技术，调查大肠杆菌的弗拉盖拉驱动的动性

使用延时显微镜连续测量大肠杆菌中的生物噪声