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>

<ol>
	<li>Purcell, A. H., Hopkins, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fastidious+xylem-limited+bacterial+plant+pathogens.">Fastidious xylem-limited bacterial plant pathogens.</a> Annu. Rev. Phytopathol. 34, 131-151 (1996).</li><li>Purcell, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xylella+fastidiosa,+a+regional+problem+or+global+threat.">Xylella fastidiosa, a regional problem or global threat.</a> J. Plant Pathology. 79, 99-105 (1997).</li><li>Hopkins, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xylella+fastidiosa:+Xylem-limited+bacterial+pathogen+of+plants.">Xylella fastidiosa: Xylem-limited bacterial pathogen of plants.</a> Annu. Rev. Phytopathol. 27, 271-290 (1989).</li><li>Mattick, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+IV+pili+and+twitching+motility.">Type IV pili and twitching motility.</a> Annu. Rev. Microbiol. 56, 289-314 (2002).</li><li>Meng, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upstream+migration+of+Xylella+fastidiosa+via+pilus-driven+twitching+motility.">Upstream migration of Xylella fastidiosa via pilus-driven twitching motility.</a> J. Bacteriol. 187, 5560-5567 (2005).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+I+and+type+IV+pili+of+Xylella+fastidiosa+affect+twitching+motility,+biofilm+formation+and+cell-cell+aggregation.">Type I and type IV pili of Xylella fastidiosa affect twitching motility, biofilm formation and cell-cell aggregation.</a> Microbiology. 153, 719-726 (2007).</li><li>Simpson, A. J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genome+sequence+of+the+plant+pathogen+Xylella+fastidiosa.">The genome sequence of the plant pathogen Xylella fastidiosa.</a> Nature. 406, 151-157 (2000).</li><li>Maier, B., Potter, L., So, M., Long, C. D., Seifert, H. S., Sheetz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+pilus+motor+forces+exceed+100+pN.">Single pilus motor forces exceed 100 pN.</a> Proc. Natl. Acad. Sci. USA. 99, 16012-16017 (2002).</li><li>Touhami, A., Jericho, M. H., Boyd, J. M., Beveridge, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+characterization+and+determination+of+adhesion+forces+of+Pseudomonas+aeruginosa+pili+by+using+atomic+force+microscopy.">Nanoscale characterization and determination of adhesion forces of Pseudomonas aeruginosa pili by using atomic force microscopy.</a> J. Bacteriol. 188, 370-377 (2006).</li><li>Skerker, J. M., Berg, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+observation+of+extension+and+retraction+of+type+IV+pili.">Direct observation of extension and retraction of type IV pili.</a> Proc. Natl. Acad. Sci. USA. 98, 6901-6904 (2001).</li><li>Brown, D. C., Larson, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvements+to+parallel+plate+flow+chambers+to+reduce+reagent+and+cellular+requirements.">Improvements to parallel plate flow chambers to reduce reagent and cellular requirements.</a> BMC Immunol. 2, 9 (2001).</li><li>Thomas, W. E., Nilsson, L. M., Forero, M., Sokurenko, E. V., Vogel, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shear-dependent+'stick-and-roll'+adhesion+of+type+1+fimbriated+Escherichia+coli.">Shear-dependent 'stick-and-roll' adhesion of type 1 fimbriated Escherichia coli.</a> Mol. Microbiol. 53, 1545-1557 (2004).</li><li>Thomas, W. E., Trintchina, E., Forero, M., Vogel, V., Sokurenko, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+adhesion+to+target+cells+enhanced+by+shear+force.">Bacterial adhesion to target cells enhanced by shear force.</a> Cell. 109, 913-923 (2002).</li><li>Bahar, O., Fuente, D. L., Burdman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+adhesion,+biofilm+formation+and+motility+of+Acidovorax+citrulli+using+microfluidic+flow+chambers.">Assessing adhesion, biofilm formation and motility of Acidovorax citrulli using microfluidic flow chambers.</a> FEMS Microbiol. Lett. 312, 33-39 (2010).</li><li>Thomas, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+a+laminar+flow+system+to+explain+shear-enhanced+bacterial+adhesion.">Using a laminar flow system to explain shear-enhanced bacterial adhesion.</a> Proceedings of ICMM2005, Third International Conference on Microchannels and Mini-channels. , 751-759 (2005).</li><li>Fuente, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+adhesion+forces+of+type+I+and+type+IV+pili+of+Xylella+fastidiosa+bacteria+by+use+of+a+microfluidic+flow+chamber.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PilJ+localizes+to+cell+poles+and+is+required+for+type+IV+pilus+extension+in+Pseudomonas+aeruginosa.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+type+I+and+type+IV+pilus+biosynthetic+genes+affect+twitching+motility+rates+in+Xylella+fastidiosa.">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><li>Ferandez, A., Hawkins, A. C., Summerfield, D. T., Harwood, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cluster+II+che+genes+from+Pseudomonas+aeruginosa+are+required+for+an+optimal+chemotactic+response.">Cluster II che genes from Pseudomonas aeruginosa are required for an optimal chemotactic response.</a> J. Bacteriol. 184, 4374-4383 (2002).</li><li>Cursino, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+an+Operon,+Pil-Chp,+That+Controls+Twitching+Motility+and+Virulence+in+Xylella+fastidiosa.">Identification of an Operon, Pil-Chp, That Controls Twitching Motility and Virulence in Xylella fastidiosa.</a> Mol. Plant Microbe Interact. 10, 1198-1206 (2011).</li><li>Hazelbauer, G. L., Falke, J. J., Parkinson, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+chemoreceptors:+High-performance+signaling+in+networked+arrays.">Bacterial chemoreceptors: High-performance signaling in networked arrays.</a> Trends Biochem. Sci. 33, 9-19 (2008).</li><li>Costa, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+hosts+of+Xylella+fastidiosa+in+and+near+southern+California+vineyards.">Plant hosts of Xylella fastidiosa in and near southern California vineyards.</a> Plant Dis. 88, 1255-1261 (2004).</li><li>Shi, X. Y., Dumenyo, C. K., Hernandez-Martinez, R., Azad, H., Cooksey, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+regulatory+pathways+in+Xylella+fastidiosa:+genes+and+phenotypes+controlled+by+algU.">Characterization of regulatory pathways in Xylella fastidiosa: genes and phenotypes controlled by algU.</a> Appl. Environ. Microbiol. 73, 6748-6756 (2007).</li><li>Matsumoto, A., Young, G. M., Igo, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome-Based+Genetic+Complementation+System+for+Xylella+fastidiosa.">Chromosome-Based Genetic Complementation System for Xylella fastidiosa.</a> Appl. Environ. Microbiol. 75, 1679-1687 (2009).</li><li>Davis, M. J., Purcell, A. H., Thomson, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+Media+for+the+Pierce's+Disease+Bacterium.">Isolation Media for the Pierce's Disease Bacterium.</a> Phytopathology. 70, 425-429 (1980).</li><li>Xia, Y. N., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+lithography.">Soft lithography.</a> Annu. Rev. Mater. Sci. 28, 153-184 (1998).</li><li>Chaudhury, M. K., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+interfacial+interactions+between+semispherical+lenses+and+flat+sheets+of+poly-(dimethylsiloxane)+and+their+chemical+derivatives.">Direct measurement of interfacial interactions between semispherical lenses and flat sheets of poly-(dimethylsiloxane) and their chemical derivatives.</a> Langmuir. 7, 1013-1025 (1991).</li><li>Cruz, L. F., Parker, J. K., Cobine, P. A., De La Fuente, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-enhanced+twitching+motility+in+Xylella+fastidiosa+is+linked+to+a+single+PilY1+homolog.">Calcium-enhanced twitching motility in Xylella fastidiosa is linked to a single PilY1 homolog.</a> Appl. Environ. Microbiol. 80, 7176-7196 (2014).</li></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>Biology materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>X. fastidiosa (Xf) Temecula wild type</td>
			<td></td>
			<td></td>
			<td>Costa, H. S., et al., 2004 22</td>
		</tr>
		<tr>
			<td>pilG deletion mutant Xf&#916;pliG</td>
			<td></td>
			<td></td>
			<td>Shi, X. Y., et al., 2007 26</td>
		</tr>
		<tr>
			<td>pilG complementary strain Xf&#916;pliG-C&#160;</td>
			<td></td>
			<td></td>
			<td>Davis, M. J., wt al. 1998 23</td>
		</tr>
		<tr>
			<td>Physical materials and equipment</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&#160;</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 UNIBLITZ, US</td>
			<td>#LS2T2</td>
			<td>Control camera&#8217;s exposure time</td>
		</tr>
		<tr>
			<td>Microscope Shutter Control system</td>
			<td>The UNIBLITZ, US</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&#160;</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 o...

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 ...

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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Research

JoVE Journal

Immunology and Infection

10.6K Views.  University of California, Davis. 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 ...

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

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. The NALT are two parallel bell-shaped structures located in the nasal passages above the hard palate, and are usually considered to be secondary components of the mucosal-associated lymphoid system4-6. Located within the NALT are discrete compartments of B and T lymphocytes interspersed with antigen-presenting dendritic cells4,7,8. These cells are surrounded by an epithelial cell layer intercalated with M-cells that are responsible for antigen retrieval from the mucosal surfaces of the air passages9,10. Naive lymphocytes circulating through the NALT are poised to respond to first encounters with respiratory pathogens7. While NALT disappear in humans by the age of two years, the Waldeyer's Ring and similarly structured lymphatic organs continue to persist throughout life6. In contrast to humans, mice retain NALT throughout life, thus providing a convenient animal model for the study of immune responses originating within the nasal sinuses11.
Cultures of single-cell suspensions of NALT are not practical due to low yields of mononuclear cells. However, NALT biology can be examined by ex vivo culturing of the intact organ, and this method has the additional advantage of maintaining the natural tissue structure. For in vivo studies, genetic knockout models presenting defects limited to NALT are not currently available due to a poor understanding of the developmental pathway. For example, while lymphotoxin-&alpha; knockout mice have atrophied NALT, the Peyer's patches, peripheral lymph nodes, follicular dendritic cells and other lymphoid tissues are also altered in these genetically manipulated mice12,13. As an alternative to gene knockout mice, surgical ablation permanently eliminates NALT from the nasal passage without affecting other tissues. The resulting mouse model has been used to establish relationships between NALT and immune responses to vaccines1,3. Serial collection of serum, saliva, nasal washes and vaginal secretions is necessary for establishing the basis of host responses to vaccination, while immune responses originating directly from NALT can be confirmed by tissue culture. The following procedures outline the surgeries, tissue culture and sample collection necessary to examine local and systemic humoral immune responses to intranasal (IN) vaccination.

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue NALT in Mouse Responses to Vaccines

Methods to examine contributions of the nasopharyngeal-associated lymphoreticular tissues (NALT) to nasal and systemic immune responses of mice to intranasal vaccines are described. We demonstrate a surgical procedure to establish a NALT-dependent mouse model and ex vivo cultures of extracted NALT.

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. ...

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin acquisition at the bacterial cell surface plays in resistance to the host immune system. Flow cytometry experiments identified human plasma vitronectin as a ligand for the bacterial receptor outer membrane protein H of Haemophilus influenzae type f. An enzyme-linked immunosorbent assay was employed to characterize the protein-protein interactions between purified recombinant protein H and vitronectin, and binding affinity was assessed using bio-layer interferometry. The biological importance of the binding of vitronectin to protein H at the bacterial cell surface in evasion of the host immune response was confirmed using a serum resistance assay with normal and vitronectin-depleted human serum. The importance of vitronectin in bacterial adherence was analyzed using glass slides with and without vitronectin coating, followed by Gram staining. Finally, bacterial adhesion to human alveolar epithelial cell monolayers was investigated. The protocols described here can be easily adapted to the study of any bacterial species of interest.

This report describes protocols for characterizing interactions between bacterial outer membrane proteins and the human complement regulator vitronectin. The protocols can be used to study the binding reactions and biological function of vitronectin in any bacterial species.

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) containing immunologically active molecules. Knowledge regarding the molecular mechanisms of vesicle biogenesis, the content of the vesicles, and their functions at the pathogen-host interface is very limited. Addressing these questions requires rigorous procedures for isolation, purification, and validation of EVs. Previously, vesicle production was found to be enhanced when M. tuberculosis was exposed to iron restriction, a condition encountered by Mtb in the host environment. Presented here is a complete and detailed protocol to isolate and purify EVs from iron-deficient mycobacteria. Quantitative and qualitative methods are applied to validate purified EVs.

Mycobacterium tuberculosis shows increased production and release of extracellular vesicles in response to low iron conditions. This work details a protocol for generating low iron conditions and methods for the purification and characterization of mycobacterial extracellular vesicles released in response to iron deficiency.

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) ...

Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density and the location of immune cells in the TME have important diagnostic and prognostic values. Multiomic profiling of the TME has exponentially increased our understanding of the numerous cellular and molecular networks regulating tumor initiation and progression. However, these techniques do not provide information about the spatial organization of cells or cell-cell interactions. Affordable, accessible, and easy to execute multiplexing techniques that allow spatial resolution of immune cells in tissue sections are needed to complement single cell-based high-throughput technologies. Here, we describe a strategy that integrates serial imaging, sequential labeling, and image alignment to generate virtual multiparameter slides of whole tissue sections. Virtual slides are subsequently analyzed in an automated fashion using user-defined protocols that enable identification, quantification, and mapping of cell populations of interest. The image analysis is done, in this case using the analysis modules Tissuealign, Author, and HISTOmap. We present an example where we applied this strategy successfully to one clinical specimen, maximizing the information that can be obtained from limited tissue samples and providing an unbiased view of the TME in the entire tissue section.

Here, we describe a simple and accessible strategy for visualizing, quantifying, and mapping immune cells in formalin-fixed paraffin-embedded tumor tissue sections. This methodology combines existing imaging and digital analysis techniques with the purpose of expanding the multiplexing capability and multiparameter analysis of imaging assays.

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

Characterization of Immune Cells and Proinflammatory Mediators in the Pulmonary Environment

Immune cell expansion, activation, and trafficking to the lungs, which are controlled by the expression of multiple cytokines and chemokines, may be altered by severe brain injury. This is evidenced by the fact that pneumonia is a major cause of mortality in patients who have suffered from ischemic stroke. The goal of this protocol is to describe the use of multicolor flow cytometric analysis to identify 13 types of immune cells in the lungs of mice, including alveolar macrophages, interstitial macrophages, CD103+ or CD11b+ dendritic cells (DCs), plasmacytoid DCs, eosinophils, monocytes/monocyte-derived cells, neutrophils, lymphoid-derived&#160; T and B cells, NK cells, and NKT cells, following ischemic stroke induction by transient middle cerebral artery occlusion. Moreover, we describe the preparation of lung homogenates using a bead homogenization method, to determine the expression levels of 13 different cytokines or chemokines simultaneously by multiplex bead arrays coupled with flow cytometric analysis. This protocol can also be used to investigate the pulmonary immune response in other disease settings, such as infectious lung disease or allergic disease.

This protocol describes the use of flow cytometry to identify the changes in immune cell composition, cytokine profile, and chemokine profile in the pulmonary environment following transient middle cerebral artery occlusion, a murine model of ischemic stroke.

Immune cell expansion, activation, and trafficking to the lungs, which are controlled by the expression of multiple cytokines and chemokines, may be ...