This study was supported by United States Department of Agriculture, Agricultural Research Service. Trade names or commercial products in this publication are mentioned solely for the purpose of providing specific information and does not imply recommendation or endorsement by the United States Department of Agriculture. USDA is an equal opportunity provider and employer.

1. The Peripheral Fringe of Bacterial Colony
<ol>
	<li>Grow X. fastidiosa (Xf) Temecula wild type22, pilG deletion mutant Xf&#916;pliG (using previously described deletion strategy23), and its complementary Xf&#916;pliG-C (using previously described chromosome-based genetic complementation strategy24) on PD2 medium agar plates25 at 28 &#176;C for 5-7 days.</li>
	<li>Autoclave cellophane (1 x 1 cm2) in water at 121 &#176;C (249 &#176;F) for 15 min. Pick up one piece of cellophane, drain the water by touching one corner of cellophane on an empty Petri dish, carefully lay the cellophane over 15% of the agar surface and air dry.</li>
	<li>Pick up individual X. fastidiosa colonies with sterile rounded toothpicks and spot cells aseptically onto a sterilized sheet of cellophane overlaid on the 15% of agar surface in the agar plates. Incubate the plates at 28 &#176;C for 2-3 days.</li>
	<li>Examine the edge morphology of the colonies using a dissecting microscope with a 2X objective lens and a 10X ocular lens. Photograph the peripheral fringe around colonies.</li>
</ol>
2. Microscopy and Microfluidic Flow Chambers
<ol>
	<li>Fabricate microfluidic devices using photo-lithographic procedures similar to those previously described5,18. Design four parallel channels with computer-assisted design software on master silicon wafer using standard lithographic methods26.</li>
	<li>Create the microfluidic chambers from silicon wafer master with polydimethylsiloxane (PDMS). Pour unpolymerized PDMS over the silicon wafer master and cure it at 60 &#176;C for 1 hr. Peel off the PDMS replica from the wafer master and trim the PDMS replica with a blade into a 22 mm x 40 mm as the same size of a glass coverslip.</li>
	<li>Expose the PDMS replica, a glass coverslip (22 x 40 mm2), and a microscope slide (51 x 76 mm2) to air plasma at 30 W for 2 min27. Sandwich the PDMS body between the glass coverslip and the glass microscope to build the micro&#64258;uidic chamber.</li>
	<li>Drill a hole (5.5 mm diameter) through the PDMS at each end of the patterned-channel. Cut the silicone rubber tubing into 12-20 cm long. Insert one end of the silicone rubber tubing (5.1 mm outside diameter, 2.1 mm inside diameter, 0.8 mm wall) into each opening end of the channels of the PDMS replica, and seal it with unpolymerized PDMS at 60 &#176;C for 1 hr.</li>
	<li>Connect another end of the tubing to the barbed end of plastic luer connectors. Wrap the assembled microfluidic chambers with the aluminum foil and autoclave them for 20 min.</li>
	<li>Collect bacteria cells of X. fastidiosa wild type, mutant Xf&#916;pliG, and complementary Xf&#916;pliG-C via scraping, using disposable inoculating loops from PD2 medium plates. Adjust cell density to an optical density of 0.05 at 600 nm in PD2 broth as described previously23. Collect the bacterial cell solution into a 1 ml Gastight syringe.</li>
	<li>Mount the microfluidic device on an inverted microscope stage. Connect an inlet tube to the 5 ml Gastight syringe containing PD2 broth. Fit the 5 ml Gastight syringe with the syringe pumps.</li>
	<li>Connect the outlet tube to a waste reservoir. Maintain a medium flow rate of 0.2 &#181;l min-1 for 30 min to stabilize the system.</li>
	<li>Connect side-inlet tubes to a 1 ml Gastight syringe containing the bacterial cell solution. Flush the bacterial cell solution through the rubber tubing until the channel is reached. Maintain a medium flow rate of 0.2 &#181;l min-1 for another 30 min in order to flush unbound cells from the chamber prior to image capture.</li>
	<li>Mount the microscope shutter under the field-adjusted part of the microscope to control the light. Connect the shutter to the shutter control system and connect the shutter control system to the computer.</li>
	<li>Mount a digital camera to the video port of the microscope and connect it to the computer. Run the time-lapse recording software, select the &#34;shutter&#34; function from the menu, and recognize automatically the installed shutter as default in the software to establish connections to the shutter with the software.</li>
	<li>Select the &#34;digital camera&#34; function from the menu of the time-lapse recording software to automatically recognize the digital camera as the default capture device in the software and establish communication with the digital camera with the software.</li>
	<li>Locate the bacterial cells in one of the channels using 20X phase-contrast optics, then switch to the 40X objective lens prior to image capture.</li>
	<li>Run the time-lapse recording software, select the &#34;image acquisition&#34; function using default parameters from the menu to acquire the images from the microscope. Next, open the &#34;Acquire time-lapse&#34; function and set the time interval to 30 sec5,18,28 for duration of 6-24 hr depending on experiment needed to observe twitching motility of X. fastidiosa5,18,28. Click &#34;OK&#34; to start the time-lapse recording. Click &#34;Stack function&#34; from menu, select &#34;save as&#34; to stack the images in the destination folder on the computer after finishing the recording.</li>
	<li>For multiple channels, capture time-lapse images from the first channel every 30 sec for 6 hr. Move the objective lens of the microscope to the next channel to locate the target cells. Repeat the time-lapse function as described above to capture images in each of four channels sequentially if experiment is set up to utilize four channels. Continue the time-lapse image capture for as long as three consecutive days. All experiments were conducted at room temperature (23 &#177; 2 &#176;C).</li>
	<li>Compile the time-lapse images into a video file using the time-lapse recording imaging software. Run time-lapse recording software, click &#34;Stack function&#34; from the menu, and select &#34;open stack function&#34; to open the stacked files from the computer.</li>
	<li>Start the &#34;Make movie&#34; function from the stack module, selecting all images and choosing the &#34;AVI&#34; output format. Click &#34;save as&#34; to store the video file in the destination folder on the computer.</li>
	<li>Select the compiled movies from the destination folder on the computer and play them. Then, observe the motility of the single cells through the resulting visualization of the twitching motility activity of bacteria cells in the generated video files.</li>
</ol>

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The authors declare that they have no competing financial interests.

In this study, we characterized the motion behavior of X. fastidiosa PilG mutant Xf&#916;pilG and its complementary Xf&#916;pilG-C strains in newly designed multiple parallel-nano-channel micro&#64258;uidic chambers. The newly designed micro&#64258;uidic chambers can have up to four parallel chambers with 100 &#181;m nano-channel in width compared to earlier designs with only a single 50 &#181;m wide channel18. The improved wider nano-channel facilitates the introduction of bacterial cells with the flowing of the media. In addition, this microfluidics chamber is 1) simple to build and assemble; 2) relatively inexpensive; and 3) easily applicable to varying experimental requirements. As a result, this chamber design permits the long-term observation of the motions of the bacterial cells under varieties experimental microenvironment.
Stabilizing the &#64258;ow of the currents through the micro&#64258;uidic channel is the critical step to creating an intact flowing-micro&#64258;uidic microenvironment for the observation of the motility of the bacterial cells under a variety of experimental environments. The flushing of micro&#64258;uidic chambers and connecting tubing with the media prior to the introduction of bacterial cells is also an important step to stabilize the flow in the chamber. However, the high speed of the flow will flush the bacterial cells out of the chamber without retaining cells in the course of the chamber. The proper speed of the media flowing through the micro&#64258;uidic channels needs to be adjusted using a syringe pump. During the experiments in this study, the flowing was set and stabilized by the pump at 0.2 to 1 &#181;l min-1 for at least 30 min prior to introducing the bacterial cells into the channel. Once the cells were introduced into the micro&#64258;uidic channels, the medium &#64258;ow was maintained at 0.2 &#181;l min-1 for 30 to 60 min to stabilize the system and remove nonattached cells. It is very important to stabilize the flow and to keep the background clear in order to observe the motion of the bacterial cells. Images were captured every 30 sec to con&#64257;rm the twitching motility activity of the introduced cells maintaining a constant flow speed at 0.2 &#181;l min-1. If the experiments require the collection of the bacterial cells in the chamber, the cells can be flushed out by gradually increasing the &#64258;ow rates of media from 0.2 to 110 &#181;l min-1 by adjusting the syringe pump speed.
The bacterial cell activities in the micro&#64258;uidic chambers were assessed through the inverted microscope using 40X phase-contrast optics and time-lapse images recorded with a digital camera, which was controlled by the imaging software. The flow speed and the interval time for image capture can be adjusted accordingly with experimental requirements. However, in most cases, flow speed for a medium flow rate is set to 0.2 &#181;l min-1 with time-lapse images recorded every 30 sec for the pilus-mediated bacterial cells. In other instances, if the medium &#64258;ow rate is increased to 0.1 &#181;l min-1 during the course of experimentation, the behavior of the bacterial cells will be recorded by capturing images every 10 to 15 sec accordingly. The time-lapse images were taken every 30 sec for duration of 6-24 hr and saved as a source images files for each tested strain. The videos were then complied from the images taken from 6-8 hr from each strain, which shown contrast phenotypes in twitching motilities between mutant and wild type/complement strains (Supplemental V1, V2, V3).
The significance of the nano-micro&#64258;uidic chamber devices over macroscale parallel-plate &#64258;ow chambers includes the direct examination of the motions and the aggregations of single cells of bacteria. In addition to low-cost5 and low reagent and sample volume requirements, the advantages of micro&#64258;uidic chambers are the ease of construction of a &#64258;ow microenvironment for bacterial culture and accurate control over &#64258;uid &#64258;ow rate. The multiple parallel channels permit the observation of differential bacterial strains in a single experiment setup which provides compatible data for analysis. The flagellar-independent twitching motility of the bacteria with the polar pili is particularly suitable for analysis in this nano-micro&#64258;uidic chamber. However, this micro&#64258;uidic chamber is less suitable for flagellar-dependent bacterial movement, in which the movement of bacteria is usually too quick and exhibits random directions. This limitation sometimes could be compromised by adjusting the medium flow rate to 0.05 &#181;l min-1 and changing the time-lapse image capture rate at every 1 to 2 sec for 1 hr to analyze the motions of the flagellar-mediated bacteria in the nano-scale environment.
Microfluidic chamber devices used herein provide direct visual evidence for functional evaluation of the PilG responsible for motion behavior in vitro. Additionally, this study also reveals that cell to cell aggregation through the twitching motility is essential for biofilm formation, signaling and pathogenicity in X. fastidiosa. Visualization of bacteria twitching motility by microfluidic devices provides a new approach to study gene function involving behavior in bacteria which is not easily measured by other assay methods. This approach can be applied to other bacterial systems. The microfluidic chamber devices provide a flow system for characterizing the physiological behavior of bacterial cells associated with cell-cell attachments, cell aggregations, motion patterns and biofilm formation.

Xylella fastidiosa is a Gram-negative non-flagellated, pathogenic bacterium that causes a number of economically important crop diseases, including Pierce&#39;s disease in grapevine (Vitis vinifera L.)1,2, 3. This bacterium is limited to the water-conducting xylem vessels. Infection of grapevine causes the blockage of xylem vessels and results in water stress and nutritional deficiencies3. Successful colonization depends on the ability of the bacterium to move from the initial site of infection to the rest of the plant3. Twitching motility is a means of flagellar-independent bacterial movement through the extension, attachment, and retraction of the polar type IV pili4 that has been characterized in X. fastidiosa5,6,7.
The twitching motility has been observed by laser tweezers and atomic force microscopy (AFM) 8,9,10. Using these techniques, twitching motilities generated by type IV pilus of N. gonorrhoeae and P. aeruginosa were characterized by &#64258;uorescently labeling pili and capturing their movements microscopically. Although both methods have detailed the adhesive force of individual bacteria, the procedures are complicated and time consuming9,10. The micro&#64258;uidic chambers were used to observe long-distance migration of individual cells as well as small aggregates of bacterial cells5,6. These chambers were designed as a microfabricated-nano-channel in a plate integrated with a time-lapse image recording system11,12,13,14. Micro&#64258;uidic chamber devices offer several advantages for studying the movement behavior and cell-cell interactions of bacteria: (i) it provides an integrated platform with multiple channel capabilities; (ii) it can examine the motions and aggregations of single cells in the nano-scale features of bacteria; (iii) it allows for direct microscopic image recording of bacterial cells and time-lapse analysis, (iv) it provides long-term spatial and temporal observations of individual and/or populations of bacteria in a micro-environment; (v) the flow rate of culture medium in a channel can be precisely controlled and (vi) only a very small volume (1 ml) of culture medium is required for each experiment.
Recently, the micro&#64258;uidic &#64258;ow system has been employed to investigate the behaviors of bacterial cells under various microenvironments 14,15,16. The adhesiveness and the surface attachment of E. coli15, X. fastidiosa16, and Acidovorax citrulli14 to glass surfaces were assessed using micro&#64258;uidic chambers. The aggregation and bio&#64257;lm formation mediated by type IV pili of Acidovorax citrulli were analyzed14. Furthermore, the motion of A. citrulli observed under &#64258;ow conditions demonstrated that the type IV pili may play important roles in the colonization and spread of A. citrulli in xylem vessels under sap &#64258;ow conditions. The twitching motilities of Pseudomonas aeruginosa and X. fastidiosa cells were successfully observed against a fluid current in a microfabricated flow chamber5,6,17. Type IV pilus deficient pilB and pilQ mutants of X. fastidiosa were found to profoundly alter the speed of twitching motility under the &#64258;ow conditions in micro&#64258;uidic devices5,6,18. The studies conducted on bacterial adhesion and motility in micro&#64258;uidic devices indicated that the micro&#64258;uidic chambers are particularly suitable for analyzing the twitching motility and migration of pili-mediated bacteria in vitro. These results explain the twitching-mediated migration mechanism which facilitates cell-cell attachment, aggregation and colonization within the host, eventually lead to systemic infection.
Pil-Chp operon of X. fastidiosa contains pilG, pilI, pilJ, pilL, chpB and chpC which encode signal transduction pathways20. The transmembrane chemoreceptors bind chemical stimuli in the periplasmic domain and activate a signaling cascade in their cytoplasmic portion to ultimately control bacterial twitching motility. In the Pil-Chp operon of X. fastidiosa, a phospho-shuttle protein PilG is a homologue to CheY. In E. coli and P. aeruginosa, CheY is the response regulator in chemotaxis systems that interact with the flagella motor proteins19,21. Although the contributions of the Pil-Chp operon toward virulence in X. fastidiosa were examined recently20, the role of pilG in chemotaxis operon in response to the environmental signals and to regulated/motor type IV pili of X. fastidiosa is unclear. To elucidate the insight of chemotaxis regulator pilG in the activity of twitching motility of X. fastidiosa, a micro&#64258;uidic chamber is used to assess the twitching motility of X. fastidiosa. The pilG of X. fastidiosa is characterizedby comparing the phenotypes of a deletion mutant Xf&#916;pliG, complementary strain Xf&#916;pliG -C and its wild type in vitro. The results highlight the role of pilG in the twitching motility of X. 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

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.

The presence of a peripheral colony fringe indicative of type IV pilus-mediated twitching motility, was observed in the colonies of X. fastidiosa wild type and complementary Xf&#916;pliG-C strain (Figure 1). Mutant Xf&#916;pliG, however, did not exhibit a fringe around the periphery of the colonies (Figure 1). Time-lapse imaging of bacterial cells in nano-microfluidic flow chambers revealed that twitching motility was observed in both wild type X. fastidiosa and the complementary Xf&#916;pliG-C (Supplemental V1, V3), whereas Xf&#916;pilG mutant cells did not exhibit twitching motility throughout the experiment (Supplemental V2). Cells of mutant Xf&#916;pilG formed relatively small loose aggregates in PD2 broth (Supplemental V2). In contrast, cells of X. fastidiosa wild type and complementary Xf&#916;pilG-C developed larger aggregates in PD2 broth (Figure 2, (Supplemental V1, V3).
<img alt="Figure 1" src="/files/ftp_upload/53816/53816fig1.jpg" /> 
Figure 1: The peripheral fringe of bacterial colony. Colony margin characteristics of X. fastidiosa from wild type, mutant Xf&#916;pilG, and complementary Xf&#916;pilG-C grown on PD2 agar covered with a sterilized sheet of cellophane. With the exception of mutant Xf&#916;pilG, all colonies exhibited a peripheral fringe, indicating type IV pilus-mediated twitching motility. Pictures were taken after 5 days of growth on culture medium. Magnification bar, 0.5 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53816/53816fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53816/53816fig2.jpg" /> 
Figure 2: The twitching motility of X. fastidiosa cells in nano-microfluidic flow chamber. The twitching motility of all tested strain cells was recorded during 6 days of observation. The assessments were conducted from three independent video segments. Magnification bar, 20 &#181;m. 
Note: The twitching motility of X. fastidiosa cells is characterized by single cell movement across glass surfaces through the extension, attachment, and retraction of the polar type IV pili. The single cell was observed in the migration preferentially against a fluid current in a microfabricated flow chamber. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53816/53816fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplemental Figure 1" src="/files/ftp_upload/53816/53816supfig1.jpg" /> 
Supplemental Figure 1: A four-channel microfluidic flow chamber. Each channel with media in and media out connectors at each end. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53816/53816supfig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplemental Movie 1" src="/files/ftp_upload/53816/WT-xylella_fastidiosa.jpg" /> 
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53816/WT-xylella_fastidiosa.avi" target="_blank">Supplemental Movie 1: Twitching motility. (Right click to download).</a> Twitching of wild-type X. fastidiosa in a microfluidic flow chamber.
<img alt="Supplemental Movie 2" src="/files/ftp_upload/53816/pilG-mutant.jpg" /> 
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53816/pilG-mutant.avi" target="_blank">Supplemental Movie 2: Impaired twitching motility. (Right click to download).</a> Motility of Xf mutant. Xf&#916;pilG observed in amicrofluidic flow chamber.
<img alt="Supplemental Movie 3" src="/files/ftp_upload/53816/pilG-complement.jpg" /> 
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53816/pilG-complement.avi" target="_blank">Supplemental Movie 3: Restored twitching motility. (Right click to download).</a> Twitching motility of Xf complementary strain. Xf&#916;pilG-C observed in a microfluidic flow chamber.

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

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.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.7K Views.  University of California, Davis. 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's disease in grapevine.

Video: 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.

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

Biochemistry

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.

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

Developmental Biology

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.

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

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.

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

Biology

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.

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.

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.

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.

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.

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.

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

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

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Rozdziały w tym wideo

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

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

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

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

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

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

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

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Biophysical Characterization of Flagellar Motor Functions

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