这项研究是由美国农业部农业研究服务支持。本出版物中的商品名称或商业产品只是提到提供具体信息之目的，并不由农业部美国农业部暗示推荐或认可。美国农业部是一个平等机会提供者和雇主。

1.细菌菌落外周边缘<ol><li> 十，成长fastidiosa（Xf为 ）蒂梅丘拉野生型22，pilG缺失突变Xf为 ΔpliG（使用先前描述删除策略23），以及其互补XfΔpliG-C（使用先前描述基于染色体遗传互补策略24）上的 PD2培养基琼脂板25在28℃下5-7天。 </li><li>高压釜玻璃纸（1×1 cm 2）的水在121℃（249°F）的15分钟。拿起一块玻璃纸，通过在一个空的培养皿接触玻璃纸的一个角落里沥干水，仔细地打好玻璃纸在琼脂表面和空气干燥的15％。 </li><li>拿起个别X. fastidiosa菌落用无菌牙签圆润和现货细胞无菌到在琼脂平板覆盖在琼脂表面的15％的玻璃纸杀菌片。孵育所述板■在28°C 2-3天。 </li><li>检查使用具有2X物镜解剖显微镜和10X目镜殖民地的边缘形态。照片周围的殖民地外围边缘。 </li></ol> 2.显微镜和微流控流动室<ol><li>先前描述5,18使用类似于光刻过程制造微流体装置。使用标准光刻方法26设计与电脑辅助设计软件的主硅片上四个并行通道。 </li><li>与聚二甲基硅氧烷（PDMS）创建一个从硅片主微流控室。倒未聚合的PDMS在硅片主，并在60℃下固化它为1小时。剥离从晶片主与PDMS复制品并修剪PDMS复制品用刀片成22毫米×40mm的作为玻璃盖玻片的大小相同。 </li><li>露出PDMS复制品，在玻璃盖玻片（22×40mm的2），以及一个microscOPE幻灯片（为50 x 75 平方毫米）空气等离子体在30W 2分钟27。夹层玻璃盖玻片，玻璃显微镜打造微FL uidic室之间的PDMS身体。 </li><li>在图案化的通道的每一端钻透PDMS中的孔（5.5毫米直径）。切硅橡胶管插入长12-20厘米。插入硅橡皮管的一端（外径5.1毫米，内径2.1毫米，0.8毫米壁）插入PDMS复制品的信道的各开口端部，并在60℃下用未聚合的PDMS密封它为1小时。 </li><li>管子的另一端连接到塑料鲁尔连接器的倒刺末端。包组装微流体腔室与铝箔和高压灭菌他们20分钟。 </li><li> 十，收集细菌细胞fastidiosa野生型，突变体Xf为 ΔpliG，互补XfΔpliG-C通过刮，用一次性接种从PD2中板循环。调整细胞密度到0.05在PD2肉汤600纳米的光密度如前面23所描述。收集细菌细胞溶液到1毫升气密注射器。 </li><li>安装在倒置显微镜阶段微流体装置。入口管连接到包含PD2肉汤中的5毫升注射器气密性。适应中的5毫升注射器气密用注射器泵。 </li><li>出口管连接到废物储存器。保持0.2微升min的介质流速-1 30分钟以稳定该系统。 </li><li>侧入口管连接到含有细菌细胞溶液1毫升气密注射器。冲洗通过橡胶管的细菌细胞溶液，直到达到信道。以冲洗从室图像捕获之前未结合的细胞保持0.2微升min的介质流速-1另外的30分钟。 </li><li>安装在显微镜来控制光的场调整部分在显微镜下快门。活门连接到快门共ntrol系统和闸门控制系统连接到计算机。 </li><li>安装一台数码相机到显微镜的视频端口，并将其连接到电脑。运行时间推移录音软件，从菜单中选择了"快门"功能，并自动识别软件安装快门为默认建立与软件的快门连接。 </li><li>从时间推移刻录软件的菜单中选择"数码相机"功能可以自动识别数码相机作为软件默认的捕获设备，并建立与数码相机相关的软件通信。 </li><li>定位在使用20X相衬光学通道之一的细菌细胞，然后切换到之前的图像捕获的40X物镜。 </li><li>运行时间推移录音软件，使用从菜单中默认参数，以获得从显微镜图像选择"图像采集"功能。接下来，打开"采集延时"功能，时间间隔设置为30秒5,18,28为根据，以观察X的运动性抽动实验需要6-24小时的时间fastidiosa 5,18,28。点击"确定"，启动延时录制。点击"堆栈功能"，从菜单中选择"另存为"，以完成录音后，堆在目标文件夹中的图像在计算机上。 </li><li>对于多个通道，从第一通道捕获时间推移的图像，每隔30秒6小时。移动显微镜的物镜到下一个通道来定位目标细胞。如上所述，如果实验设置为利用四个通道捕捉图像中的每一个顺序地四个通道重复时间推移功能。继续只要连续三天的时间推移图像采集。所有实验均在室温（23±2℃）下进行。 </li><li>编译时间推移图像成使用时间推移录制的图像处理软件的视频文件。运行时间推移录音软件，单击"堆栈功能"，从菜单，并选择"打开堆叠功能"，从计算机打开堆放的文件。 </li><li>从堆栈模块启动"制作电影"功能，选择所有图像并选择"AVI"格式输出。点击"另存为"的视频文件存储在计算机上的目标文件夹。 </li><li>选择在计算机上从目标文件夹编译电影并播放它们。然后，通过细菌细胞的抽搐运动活动中所生成的视频文件生成的可视化观察单个细胞的运动。 </li></ol>

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作者宣称，他们没有竞争的经济利益。

在这项研究中，我们的特点X的运动行为fastidiosa PilG突变Xf为 ΔpilG和新设计的多个并行的纳米通道微FL uidic室互补XfΔpilG-Ç株。新设计的微FL uidic腔室可以具有100微米的纳米通道多达四个平行腔室宽度相比仅与单个50微米宽的通道18早期的设计。改进的更宽纳米通道便于引入细菌细胞与媒体的流动。此外，该微流体室1）简单的构建和组装; 2）相对便宜;?...

叶缘焦枯病菌是革兰氏阴性非鞭 ​​毛，病原菌引起许多经济上重要的作物疾病，包括皮尔斯在葡萄病（ 葡萄属）1,2，3条。这种细菌是有限的导水木质部船只。葡萄感染引起木质部导管和水分胁迫的结果和营养不足3堵塞。成功定植取决于细菌的从感染的初始位点移动到植物3的其余部分的能力。抽搐蠕动是鞭毛无关细菌移动的装置通过扩展，附件，和极性IV型菌毛4已特征在于X的缩回fastidiosa 5,6,7。 抽搐运动已由激光镊子和原子力显微镜（AFM）8,9,10观察。使用这些技术，叔由N型菌毛IV产生的魔力motilities 淋球菌和P.由FL uorescently标签菌毛和显微镜捕捉他们的动作绿脓杆菌进行了表征。虽然这两种方法有详细的个体细菌的粘合力，该程序是复杂和耗时9,10。微FL uidic室，观察单个细胞的长途迁徙，以及细菌细胞5,6小聚集。这些室被设计成与隔时图像记录系统11,12,13,14一体的板一个微制造纳米通道。微FL uidic室装置用于研究细菌的运动行为和细胞 - 细胞相互作用提供几个优点:（i）其提供了具有多个信道功能的集成平台; （ii）其可以检查运动和单细胞的细菌的纳米级特征的聚合; （三）它允许直接米细菌细胞和时间推移分析icroscopic图像记录，（ⅳ）它提供了一种在微环境的细菌的个体和/或群体的长期的空间和时间的观测; （五）培养基中的信道的流量可以精确控制和培养基（ⅵ）仅有一个非常小的体积（1ml）中，需要对每个实验。 最近，微FL uidic溢流系统已被用于研究在各种微环境14,15,16细菌细胞的行为。的密合性和E的表面附着大肠杆菌 15，X。 fastidiosa 16和敏捷食酸citrulli 14到玻璃表面用微FL uidic室评估。由敏捷食citrulli的IV型菌毛介导的聚集和生物膜的形成进行了分析14。另外，A的运动citrulli下溢流Ç观察onditions表明，IV型菌毛可能在殖民化中发挥重要作用，传播的A. citrulli在木质部导管在边溢流条件。 铜绿假单胞菌和X的抽搐motilities fastidiosa细胞在微加工流动室5,6,17针对流体当前成功观察。 IV型菌毛短少X的pilB和pilQ突变fastidiosa被发现深刻地改变微FL uidic装置5,6,18的溢流条件下抽搐运动的速度。在微FL uidic器件细菌粘附和运动进行的研究表明，微FL uidic室是特别适合于在体外分析菌毛介导的细菌的抽搐运动和迁移。这些结果可以解释这有利于细胞间的连接，聚集和殖民内的抽动介导的迁移机制主机，最终导致全身感染。 X的弼热电联产操纵子fastidiosa包含pilG，霹雳，pilJ，丸剂，chpB和CHPC其中编码信号转导通路20。跨膜化学感受器结合化学刺激在周质结构域和在其细胞质部分以最终控制细菌抽搐蠕动激活信号级联。在X的弼CHP操纵子fastidiosa，一个磷酸化蛋白班车是PilG同源到崔泰源。在E.大肠杆菌和P.假单胞菌，崔泰源是在与该鞭毛马达蛋白19，21交互趋化系统的响应调节。虽然弼CHP操纵子在X的贡献对毒力fastidiosa最近检查20，pilG在趋操纵子的作用，响应于环境信号及X的调节/马达IV型菌毛fastidiosa是UNC李尔。为了阐明X的抽动运动的活动趋调节pilG的洞察力fastidiosa，微FL uidic室被用来评估X的抽搐蠕动fastidiosa。 十fastidiosa的pilG的特征在于，通过比较一个缺失突变体 Xf为 ΔpliG，互补应变XfΔpliG-C及其野生型在体外 的表型 。结果突出pilG在X的抽动运动中的作用fastidiosa。

<table><tbody><tr><td>&lt;strong&gt;Biology materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;X. fastidiosa (Xf)&lt;/em&gt; Temecula wild type</td><td></td><td></td><td>Costa, H. S., &lt;em&gt;et al.&lt;/em&gt;, 2004 &lt;sup&gt;22&lt;/sup&gt;</td></tr><tr><td>&lt;em&gt;pilG&lt;/em&gt; deletion mutant &lt;em&gt;Xf&amp;Delta;pliG&lt;/em&gt;</td><td></td><td></td><td>Shi, X. Y., &lt;em&gt;et al.&lt;/em&gt;, 2007 &lt;sup&gt;26&lt;/sup&gt;</td></tr><tr><td>&lt;em&gt;pilG&lt;/em&gt; complementary strain &lt;em&gt;Xf&amp;Delta;pliG&lt;/em&gt;-C&amp;nbsp;</td><td></td><td></td><td>Davis, M. J., &lt;em&gt;et al.&lt;/em&gt; 1998 &lt;sup&gt;23&lt;/sup&gt;</td></tr><tr><td>&lt;strong&gt;Physical materials and equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Disposable inoculating loops</td><td>VWR international, Radnor, PA</td><td>#22-363-607</td><td>quantitative procedures such as bacterial collection</td></tr><tr><td>Polydimethylsiloxane (PDMS)</td><td>Dow Corning Corporation</td><td>#0002709226</td><td>Sylgard 184 silicone Elastomeric Kits</td></tr><tr><td>AmScope MD2000 digital camera</td><td>AmScope, Irvine, CA</td><td>SE305R-AZ-E</td><td>Image, video recording and measurement&amp;nbsp;</td></tr><tr><td>Tubes line</td><td>Edgewood, NY</td><td>#T4300</td><td>Connected to the syringe and microfluidic chamber</td></tr><tr><td>Plastic luer connectors</td><td>Edgewood, NY</td><td></td><td>Connected to the syringe and microfluidic chamber</td></tr><tr><td>Syringe pumps</td><td>Pico Plus, Harvard Apparatus, MA</td><td>#702209</td><td>The flow rate can be adjusted while the pump is running.</td></tr><tr><td>Syringes</td><td>Gastight, Hemilton Company, Reno, NV</td><td>#1005</td><td>Provide the flowing broth</td></tr><tr><td>Inverted Olympus IMT-2 microscope</td><td>Olympus</td><td>IMT-2 FLuoro PHase</td><td>Image observation and recording</td></tr><tr><td>SPOT-RT digital camera</td><td>Diagnostic Instruments, Inc., MI</td><td>RT230</td><td>Image, video recording and measurement</td></tr><tr><td>Microscope Shutter</td><td>The &lt;em&gt;UNIBLITZ, US&lt;/em&gt;</td><td>#LS2T2</td><td>Control camera&amp;rsquo;s exposure time</td></tr><tr><td>Microscope Shutter Control system</td><td>The &lt;em&gt;UNIBLITZ, US&lt;/em&gt;</td><td>VCM-D1</td><td>VCM-D1 Single Channel CE/UL/CSA Approved Shutter Driver</td></tr><tr><td>MetaMorph Image software</td><td>Universal Imaging Corp., PA</td><td></td><td>Real-time super-resolution image processing&amp;nbsp;</td></tr></tbody></table>

visualization of twitching motility and characterization of the role of the pilg in xylella fastidiosa

叶缘焦枯病菌是革兰氏阴性非鞭 ​​毛的细菌引起的若干植物的经济上重要的疾病。抽搐蠕动提供X. fastidiosa长距离内厂运行和殖民化，手段在十朝贡献致病fastidiosa，X的运动性抽动fastidiosa是由IV型菌毛操作。 叶缘焦枯病菌的IV型菌毛由pilG，在弼CHP操纵子编码的蛋白质趋化性调节器所涉及的信号转导通路调节。为了阐明pilG的角色X的抽动运动fastidiosa，一个pilG缺陷型突变体Xf为 ΔpilG及其互补株XfΔpilG-含有C本地pilG被开发。与时间推移图像记录系统集成的微流控室，观察抽搐蠕动在XfΔpILG，XfΔpilG-C及其野生型菌株。 利用这种记录系统中，其允许聚合的长期的空间和时间的观察，个体细胞，并通过抽动蠕动细菌种群的迁移。X. fastidiosa野生型和互补XfΔpilG-C株显示，微流体流室直接观察到典型的抽动运动特征，而突变XfΔpliG展出抽搐缺陷表型。这项研究表明，pilG有助于X的运动性抽动fastidiosa。微流体流动室被用作用于观察抽动运动的装置。

表示IV型菌毛介导的抽搐运动的周菌落边缘的情况下，在X的菌落观察fastidiosa野生型和互补Xf为 ΔpliG-C菌株（ 图1）。突变XfΔpliG，然而，并没有表现出在菌落周围的周边的边缘（ 图1）。在纳米微流体流动腔室的细菌细胞的延时成像显示，在野生型X的观察抽搐蠕动fastidiosa和互补XfΔpliG-C（补充V1?...

Watch this Scientific Journal Video about Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa at JoVE.com

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

在这项研究中，纳米微流体流动室被用于可视化和功能上表征叶缘焦枯病菌，导致皮尔斯病在葡萄细菌的抽搐蠕动。

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 ...

visualization-twitching-motility-characterization-role-pilg-xylella

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

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

10.7K 次观看.  University of California, Davis. 在这项研究中，纳米微流体流动室被用于可视化和功能上表征叶缘焦枯病菌，导致皮尔斯病在葡萄细菌的抽搐蠕动。 

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

Fluorescence Live-cell Imaging of the Complete Vegetative Cell Cycle of the Slow-growing Social Bacterium Myxococcus xanthus

Fluorescence live-cell imaging of bacterial cells is a key method in the analysis of the spatial and temporal dynamics of proteins and chromosomes underlying central cell cycle events. However, imaging of these molecules in slow-growing bacteria represents a challenge due to photobleaching of fluorophores and phototoxicity during image acquisition. Here, we describe a simple protocol to circumvent these limitations in the case of Myxococcus xanthus (which has a generation time of 4 - 6 h). To this end, M. xanthus cells are grown on a thick nutrient-containing agar pad in a temperature-controlled humid environment. Under these conditions, we determine the doubling time of individual cells by following the growth of single cells. Moreover, key cellular processes such as chromosome segregation and cell division can be imaged by fluorescence live-cell imaging of cells containing relevant fluorescently labeled marker proteins such as ParB-YFP, FtsZ-GFP, and mCherry-PomX over multiple cell cycles. Subsequently, the acquired images are processed to generate montages and/or movies.

Fluorescence Live-cell Imaging of the Complete Vegetative Cell Cycle of the Slow-growing Social Bacterium Myxococcus xanthus

Bacterial cells are spatially highly organized. To follow this organization over time in slow growing Myxococcus xanthus cells, a set-up for fluorescence live-cell imaging with high spatiotemporal resolution over several generations was developed. Using this method, spatiotemporal dynamics of important proteins for chromosome segregation and cell division could be determined.

生长缓慢的社会细菌Myxococcus xanthus的全营养细胞周期的荧光活细胞成像

Fluorescence live-cell imaging of bacterial cells is a key method in the analysis of the spatial and temporal dynamics of proteins and chromosomes ...

科研

生物化学

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique&#39;s relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

In this protocol, fluorescently labeled T. cruzi&#160;were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy.

斑马鱼幼虫动物模型的建立研究锥虫运动性体内

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

发育生物学

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

生成和多表型高内涵筛选<em&gt;贝氏柯克斯体</em&gt;转座子突变体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

免疫与感染

Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography

A swarm of the &delta;-proteobacterium Myxococcus xanthus contains millions of cells that act as a collective, coordinating movement through a series of signals to create complex, dynamic patterns as a response to environmental cues. These patterns are self-organizing and emergent; they cannot be predicted by observing the behavior of the individual cells. Using a time-lapse microcinematography tracking assay, we identified a distinct emergent pattern in M. xanthus called chemotaxis, defined as the directed movement of a swarm up a nutrient gradient toward its source 1.
In order to efficiently characterize chemotaxis via time-lapse microcinematography, we developed a highly modifiable plate complex (Figure 1) and constructed a cluster of 8 microscopes (Figure 2), each capable of capturing time-lapse videos. The assay is rigorous enough to allow consistent replication of quantifiable data, and the resulting videos allow us to observe and track subtle changes in swarm behavior. Once captured, the videos are transferred to an analysis/storage computer with enough memory to process and store thousands of videos. The flexibility of this setup has proven useful to several members of the M. xanthus community.

Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography

To study Myxococcus xanthus swarm behavior, we have designed a time-lapse microcinematography protocol that can be modified for different assays. It employs standard growth conditions adapted for microscopy, and yields reproducible results by the use of inexpensive, reusable silicone gaskets. We have used this method to quantify multicellular chemotaxis.

在录制多细胞行为黏细菌生物膜，使用定时Microcinematography

A swarm of the &delta;-proteobacterium Myxococcus xanthus contains millions of cells that act as a collective, coordinating movement through a series of ...

生物学

A Semi-quantitative Approach to Assess Biofilm Formation Using Wrinkled Colony Development

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a biofilm, bacterial cells often exhibit altered physiology, including enhanced resistance to antibiotics and other environmental stresses 1. Additionally, biofilms can play important roles in host-microbe interactions. Biofilms develop when bacteria transition from individual, planktonic cells to form complex, multi-cellular communities 2. In the laboratory, biofilms are studied by assessing the development of specific biofilm phenotypes. A common biofilm phenotype involves the formation of wrinkled or rugose bacterial colonies on solid agar media 3. Wrinkled colony formation provides a particularly simple and useful means to identify and characterize bacterial strains exhibiting altered biofilm phenotypes, and to investigate environmental conditions that impact biofilm formation. Wrinkled colony formation serves as an indicator of biofilm formation in a variety of bacteria, including both Gram-positive bacteria, such as Bacillus subtilis 4, and Gram-negative bacteria, such as Vibrio cholerae 5, Vibrio parahaemolyticus 6, Pseudomonas aeruginosa 7, and Vibrio fischeri 8.
The marine bacterium V. fischeri has become a model for biofilm formation due to the critical role of biofilms during host colonization: biofilms produced by V. fischeri promote its colonization of the Hawaiian bobtail squid Euprymna scolopes 8-10. Importantly, biofilm phenotypes observed in vitro correlate with the ability of V. fischeri cells to effectively colonize host animals: strains impaired for biofilm formation in vitro possess a colonization defect 9,11, while strains exhibiting increased biofilm phenotypes are enhanced for colonization 8,12. V. fischeri therefore provides a simple model system to assess the mechanisms by which bacteria regulate biofilm formation and how biofilms impact host colonization.
In this report, we describe a semi-quantitative method to assess biofilm formation using V. fischeri as a model system. This method involves the careful spotting of bacterial cultures at defined concentrations and volumes onto solid agar media; a spotted culture is synonymous to a single bacterial colony. This 'spotted culture' technique can be utilized to compare gross biofilm phenotypes at single, specified time-points (end-point assays), or to identify and characterize subtle biofilm phenotypes through time-course assays of biofilm development and measurements of the colony diameter, which is influenced by biofilm formation. Thus, this technique provides a semi-quantitative analysis of biofilm formation, permitting evaluation of the timing and patterning of wrinkled colony development and the relative size of the developing structure, characteristics that extend beyond the simple overall morphology.

We provide a simple, semi-quantitative method to investigate biofilm formation in vitro. This method takes advantage of the Zeiss stemi 2000-C Dissecting Microscope (with camera attachment) to monitor both the timing and pattern of biofilm formation, as assessed by the development of wrinkled colonies.

一个半定量的方法来评估生物膜形成皱殖民地的发展

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a ...

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method has been developed for observing interspecies interactions between two bacterial pathogens. The use of this method to interrogate interactions between a motile Gram-negative pathogen, Pseudomonas aeruginosa and a non-motile Gram-positive pathogen, Staphylococcus aureus is demonstrated here. This protocol consists of co-inoculating each species between a coverslip and an agarose pad, which maintains the cells in a single plane and allows for visualization of bacterial behaviors in both space and time.
Furthermore, the time-lapse microscopy demonstrated here is ideal for visualizing the early interactions that take place between two or more bacterial species, including changes in bacterial species motility in monoculture and in coculture with other species. Due to the nature of the limited sample space in the microscopy setup, this protocol is less applicable for studying later interactions between bacterial species once cell populations are too high. However, there are several different applications of the protocol which include the use of staining for imaging live and dead bacterial cells, quantification of gene or protein expression through fluorescent reporters, and tracking bacterial cell movement in both single species and multispecies experiments.

This live-bacterial cell imaging protocol allows for visualization of interactions between multiple bacterial species at the single-cell level over time. Time-lapse imaging allows for observation of each bacterial species in monoculture or coculture to interrogate interspecies interactions in multispecies bacterial communities, including individual cell motility and viability.

单细胞物种间细菌相互作用的动力学可视化

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method ...

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 ...

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

Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, to elucidate the function and assembly of flagellar parts, and to examine and understand patterns of movement. Here we demonstrate a combination of three of these methodologies. Motility in soft agar is the oldest, offering a strong selection for isolating gain-of-function suppressor mutations in motility-impaired strains, where motility is restored through a second mutation. The cell-tethering technique, first employed to demonstrate the rotary nature of the flagellar motor, can be used to assess the impact of signaling effectors on the motor speed and its ability to switch rotational direction. The &#8220;border-crossing&#8221; assay is more recent, where swimming bacteria can be primed to transition into moving collectively as a swarm. In combination, these protocols represent a systematic and powerful approach to identifying components of the motility machinery, and to characterizing their role in different facets of swimming and swarming. They can be easily adapted to study motility in other bacterial species.

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

Many bacteria use flagella-driven motility to navigate their environment and colonize favorable surroundings both individually and as a collective. Demonstrated here is the use of three established methods that exploit motility as a selection tool to identify components/pathways contributing to swimming and swarming motility.

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, ...

抽动运动功能的可视化和表征的角色的

摘要

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此视频中的章节

生长缓慢的社会细菌Myxococcus xanthus的全营养细胞周期的荧光活细胞成像

斑马鱼幼虫动物模型的建立研究锥虫运动性体内

生成和多表型高内涵筛选

在录制多细胞行为黏细菌生物膜，使用定时Microcinematography

一个半定量的方法来评估生物膜形成皱殖民地的发展

单细胞物种间细菌相互作用的动力学可视化

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

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

抽动运动功能的可视化和表征的角色的

摘要

探索更多视频

此视频中的章节

生长缓慢的社会细菌Myxococcus xanthus的全营养细胞周期的荧光活细胞成像

斑马鱼幼虫动物模型的建立研究锥虫运动性体内

生成和多表型高内涵筛选

在录制多细胞行为黏细菌生物膜，使用定时Microcinematography

一个半定量的方法来评估生物膜形成皱殖民地的发展

单细胞物种间细菌相互作用的动力学可视化

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

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

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