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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

In this study, a nano-microfluidic flow chamber was employed to visualize and functionally characterize the twitching motility of Xylella fastidiosa, a bacterium that causes Pierce's disease in grapevine.

Abstract

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 XfΔpilG and its complementary strain XfΔpilG-C containing native pilG were developed. A microfluidic chambers integrated with a time-lapse image recording system was used to observe twitching motility in XfΔpilG, XfΔpilG-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 XfΔpilG-C strain showed typical twitching motility characteristics directly observed in the microfluidic flow chambers, whereas mutant XfΔpliG 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.

Introduction

Xylella fastidiosa is a Gram-negative non-flagellated, pathogenic bacterium that causes a number of economically important crop diseases, including Pierce'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 fluorescently 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 microfluidic 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. Microfluidic 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 microfluidic flow 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 microfluidic chambers. The aggregation and biofilm formation mediated by type IV pili of Acidovorax citrulli were analyzed14. Furthermore, the motion of A. citrulli observed under flow conditions demonstrated that the type IV pili may play important roles in the colonization and spread of A. citrulli in xylem vessels under sap flow 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 flow conditions in microfluidic devices5,6,18. The studies conducted on bacterial adhesion and motility in microfluidic devices indicated that the microfluidic 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 microfluidic 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 XfΔpliG, complementary strain XfΔpliG -C and its wild type in vitro. The results highlight the role of pilG in the twitching motility of X. fastidiosa.

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Protocol

1. The Peripheral Fringe of Bacterial Colony

  1. Grow X. fastidiosa (Xf) Temecula wild type22, pilG deletion mutant XfΔpliG (using previously described deletion strategy23), and its complementary XfΔpliG-C (using previously described chromosome-based genetic complementation strategy24) on PD2 medium agar plates25 at 28 °C for 5-7 days.
  2. Autoclave cellophane (1 x 1 cm2) in water at 121 °C (249 °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.
  3. 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 °C for 2-3 days.
  4. 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.

2. Microscopy and Microfluidic Flow Chambers

  1. 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.
  2. Create the microfluidic chambers from silicon wafer master with polydimethylsiloxane (PDMS). Pour unpolymerized PDMS over the silicon wafer master and cure it at 60 °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.
  3. 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 microfluidic chamber.
  4. 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 °C for 1 hr.
  5. 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.
  6. Collect bacteria cells of X. fastidiosa wild type, mutant XfΔpliG, and complementary XfΔpliG-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.
  7. 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.
  8. Connect the outlet tube to a waste reservoir. Maintain a medium flow rate of 0.2 µl min-1 for 30 min to stabilize the system.
  9. 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 µl min-1 for another 30 min in order to flush unbound cells from the chamber prior to image capture.
  10. 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.
  11. 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 "shutter" function from the menu, and recognize automatically the installed shutter as default in the software to establish connections to the shutter with the software.
  12. Select the "digital camera" 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.
  13. 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.
  14. Run the time-lapse recording software, select the "image acquisition" function using default parameters from the menu to acquire the images from the microscope. Next, open the "Acquire time-lapse" 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 "OK" to start the time-lapse recording. Click "Stack function" from menu, select "save as" to stack the images in the destination folder on the computer after finishing the recording.
  15. 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 ± 2 °C).
  16. Compile the time-lapse images into a video file using the time-lapse recording imaging software. Run time-lapse recording software, click "Stack function" from the menu, and select "open stack function" to open the stacked files from the computer.
  17. Start the "Make movie" function from the stack module, selecting all images and choosing the "AVI" output format. Click "save as" to store the video file in the destination folder on the computer.
  18. 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.

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Results

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 o...

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Discussion

In this study, we characterized the motion behavior of X. fastidiosa PilG mutant XfΔpilG and its complementary XfΔpilG-C strains in newly designed multiple parallel-nano-channel microfluidic chambers. The newly designed microfluidic chambers can have up to four parallel chambers with 100 µm nano-channel in width compared to earlier designs with only a single 50 µm wide channel18. The improved wider nano-channel facilitates the introdu...

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Disclosures

The authors declare that they have no competing financial interests.

Acknowledgements

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.

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Materials

NameCompanyCatalog NumberComments
Biology materials
X. fastidiosa (Xf) Temecula wild typeCosta, H. S., et al., 2004 22
pilG deletion mutant XfΔpliGShi, X. Y., et al., 2007 26
pilG complementary strain XfΔpliG-C Davis, M. J., et al. 1998 23
Physical materials and equipment
Disposable inoculating loopsVWR international, Radnor, PA#22-363-607quantitative procedures such as bacterial collection
Polydimethylsiloxane (PDMS)Dow Corning Corporation#0002709226Sylgard 184 silicone Elastomeric Kits
AmScope MD2000 digital cameraAmScope, Irvine, CASE305R-AZ-EImage, video recording and measurement 
Tubes lineEdgewood, NY#T4300Connected to the syringe and microfluidic chamber
Plastic luer connectorsEdgewood, NYConnected to the syringe and microfluidic chamber
Syringe pumpsPico Plus, Harvard Apparatus, MA#702209The flow rate can be adjusted while the pump is running.
SyringesGastight, Hemilton Company, Reno, NV#1005Provide the flowing broth
Inverted Olympus IMT-2 microscopeOlympusIMT-2 FLuoro PHaseImage observation and recording
SPOT-RT digital cameraDiagnostic Instruments, Inc., MIRT230Image, video recording and measurement
Microscope ShutterThe UNIBLITZ, US#LS2T2Control camera’s exposure time
Microscope Shutter Control systemThe UNIBLITZ, USVCM-D1VCM-D1 Single Channel CE/UL/CSA Approved Shutter Driver
MetaMorph Image softwareUniversal Imaging Corp., PAReal-time super-resolution image processing 

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