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 1...

<ol>
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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. 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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>

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...

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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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10.6K visualizações.  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 ...