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

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, a....

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, car....

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 observed in.......

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Biology materials
X. fastidiosa (Xf) Temecula wild type			Costa, H. S., et al., 2004 22
pilG deletion mutant XfΔpliG			Shi, X. Y., et al., 2007 26
pilG complementary strain XfΔpliG-C			Davis, M. J., et al. 1998 23
Physical materials and equipment
Disposable inoculating loops	VWR international, Radnor, PA	#22-363-607	quantitative procedures such as bacterial collection
Polydimethylsiloxane (PDMS)	Dow Corning Corporation	#0002709226	Sylgard 184 silicone Elastomeric Kits
AmScope MD2000 digital camera	AmScope, Irvine, CA	SE305R-AZ-E	Image, video recording and measurement
Tubes line	Edgewood, NY	#T4300	Connected to the syringe and microfluidic chamber
Plastic luer connectors	Edgewood, NY		Connected to the syringe and microfluidic chamber
Syringe pumps	Pico Plus, Harvard Apparatus, MA	#702209	The flow rate can be adjusted while the pump is running.
Syringes	Gastight, Hemilton Company, Reno, NV	#1005	Provide the flowing broth
Inverted Olympus IMT-2 microscope	Olympus	IMT-2 FLuoro PHase	Image observation and recording
SPOT-RT digital camera	Diagnostic Instruments, Inc., MI	RT230	Image, video recording and measurement
Microscope Shutter	The UNIBLITZ, US	#LS2T2	Control camera’s exposure time
Microscope Shutter Control system	The UNIBLITZ, US	VCM-D1	VCM-D1 Single Channel CE/UL/CSA Approved Shutter Driver
MetaMorph Image software	Universal Imaging Corp., PA		Real-time super-resolution image processing