Name: Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa (Video) | JoVE Journal
Uploaded: 2016-04-08T14:00:00.000+00:00
Duration: 8 min 44 s
Description: Learn twitching motility visualization in Xylella fastidiosa using nano-microfluidic flow chambers. This method reveals bacterial behavior mechanisms underlying Pierce's disease in grapevines.

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5 minutes

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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<li>Thomas, W. E., Nilsson, L. M., Forero, M., Sokurenko, E. V., Vogel, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Shear-dependent+%27stick-and-roll%27+adhesion+of+type+1+fimbriated+Escherichia+coli%5BTitle%5D)+AND+%22Mol.+Microbiol%22%5BJournal%5D)">Shear-dependent 'stick-and-roll' adhesion of type 1 fimbriated Escherichia coli.</a> Mol. Microbiol. 53, 1545-1557 (2004).</li>
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<li>Thomas, W. E. Using a laminar flow system to explain shear-enhanced bacterial adhesion. Proceedings of ICMM2005, Third International Conference on Microchannels and Mini-channels. Toronto, Ontario, Canada, , 751-759 (2005).</li>
<li>Fuente, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessing+adhesion+forces+of+type+I+and+type+IV+pili+of+Xylella+fastidiosa+bacteria+by+use+of+a+microfluidic+flow+chamber%5BTitle%5D)+AND+%22Appl.+Environ.+Microbiol%22%5BJournal%5D)">Assessing adhesion forces of type I and type IV pili of Xylella fastidiosa bacteria by use of a microfluidic flow chamber.</a> Appl. Environ. Microbiol. 73, 2690-2696 (2007).</li>
<li>DeLange, P. A., Collins, T. L., Pierce, G. E., Robinson, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PilJ+localizes+to+cell+poles+and+is+required+for+type+IV+pilus+extension+in+Pseudomonas+aeruginosa%5BTitle%5D)+AND+%22Curr+Microbiol%22%5BJournal%5D)">PilJ localizes to cell poles and is required for type IV pilus extension in Pseudomonas aeruginosa.</a> Curr Microbiol. 55, 389-395 (2007).</li>
<li>Fuente, D. L., Burr, T. J., Hoch, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mutations+in+type+I+and+type+IV+pilus+biosynthetic+genes+affect+twitching+motility+rates+in+Xylella+fastidiosa%5BTitle%5D)+AND+%22J.+Bacteriol%22%5BJournal%5D)">Mutations in type I and type IV pilus biosynthetic genes affect twitching motility rates in Xylella fastidiosa.</a> J. Bacteriol. 189, 7507-7510 (2007).</li>
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</ol>

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 introdu...

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

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 XfΔpliG-C strain (Figure 1). Mutant XfΔpliG, 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...

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.

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

The over goal of this protocol is to functionally characterize the genes responsbile for the twitching motility of bacteria using a nano microfluidic flow chamber integrated with a time-lapse image recording system.This method can help answer key questions about a bacteria phenotype and associated gene function, such as the twitching motility of bacteria.The main advantage of this technique is that it provides a special and a tempered observation for aggregation migration of individual cells and populations.Demonstrating this procedure will be Dr.Xiangyang Shi, a post-doc from my laboratory.Using a standardized deletion strategy, grow Temecula wild-type Xylella fastidiosa, grow pilG deletion mutants, and grow the complimentary strain all on PD2 medium agar plates.Incubate the bacteria at 28

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Learn twitching motility visualization in Xylella fastidiosa using nano-microfluidic flow chambers. This method reveals bacterial behavior mechanisms underlying Pierce's disease in grapevines.

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

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