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Harvey C. Hoch

Professor and Department Chair
Co-Director, Nanobiotechnology Center

Department of Plant Pathology

Cornell University – NYSAES

Tel: (315) 787-2332

E-Mail: hch1@cornell.edu

Webpage:  
http://www.cals.cornell.edu/cals/plpath/directory/geneva.cfm?netId=hch1
http://www.nbtc.cornell.edu/

Thomas J. Burr

Professor, Associate Dean of the College of Agriculture and Life Sciences, and Goichman Family Director of Cornell's New York State Agricultural Experiment Station at Geneva

Department of Plant Pathology

Cornell University – NYSAES

Tel: (315) 787-2312

E-Mail: tjb1@cornell.edu

Webpage:
http://www.cals.cornell.edu/cals/plpath/directory/geneva.cfm?netId=tjb1

   

Contributions made from:
Yizhi Meng, former Postdoctoral Associate (email address:  ymeng@sunysb.edu)
Leonardo De La Fuente, Postdoctoral Associate (email address:  ld82@cornell.edu)
http://www.nysaes.cornell.edu/pp/postra/delafuente/
Luciana Cursino Parent, Postdoctoral Associate (email address:  lcs33@cornell.edu)
http://www.nysaes.cornell.edu/pp/postra/cursino/
Paulo Zaini, Postdoctoral Associate (email address:  paz22@cornell.edu)
 

Xylella fastidiosa is a Gram-negative xylem limited bacterium that causes a number of plant diseases including Pierce's Disease of grape. A particularly puzzling and important question is how the bacteria migrate within the xylem vascular system ‘upstream’ against the direction of xylem sap flow. Using microfluidic chambers, constructed of polydimethylsiloxane (PDMS) through which nutrient media flowed at a constant rate, it was observed that the non-flagellated bacteria migrate on the chamber surface with ‘twitching’ movements characteristic of type IV pili.
Wild-type X. fastidiosa cells possess both short type I (0.4-1.0 μm in length) and longer type IV pili (1.0-5.8 μm in length) at the same pole (Meng at al., J. Bacteriology 187: 5560-5567, 2005).  A number of mutants deficient for expression of either pilus type have been created and used to examine motility, colonization, and biofilm development in this pathogen.
 

  Xylella. fastidiosa Movies 1-14   Pseudomonas aeruginosa movies


Xylella fastidiosa colony morphology. Twitching motility of wild-type X. fastidiosa within the peripheral fringe of a 24-hour-old colony on the surface of nutrient agar. Scale bars, 20 μm; time, h:min (similar to Movie1a, J. Bacteriol. 187: 5560-5567, 2005).

Xylella fastidiosa colony morphology.  Colony expansion through cell division of mutant 1A2 (pilB).  Scale bars, 20 μm; time, h:min (similar to Movie1b, J. Bacteriol. 187: 5560-5567, 2005).

Xylella fastidiosa wild-type cells in microfluidic chamber with flow.  Many cells of X. fastidiosa wild-type strain exhibit twitching motility (R to L) against a continuous stream of medium flowing through a microfluidic chamber.  Selected cells are encircled in red.  Within the larger red circle are several bacteria attached in a short chain end-to-end; at 3:13 (h:min) the distal bacteria detach.  Several cells, either intermittently attached or not attached at all, and are carried with the flowing medium.  Medium flows from left to right at 20,000 μm/min.  Scale bar, 10 μm; time, h:min (similar to Movie2, J. Bacteriol. 187: 5560-5567, 2005).

Xylella fastidiosa wild-type cells in microfluidic chamber without flow.  Xylella fastidiosa wild-type strain exhibits twitching motility in a microfluidic chamber with stagnant non-flowing culture medium.  Selected cells are noted with colored circles.  The green encircled bacterium migrates in a nearly complete circle.  Scale bar, 10 μm; time, h:min (similar to Movie3, J. Bacteriol. 187: 5560-5567, 2005).

Xylella fastidiosa mutant 1A2 in microfluidic chamber.  Cells of X. fastidiosa mutant 1A2 (deficient for the pilB gene; cells possess only short type I pili) do not exhibit twitching motility in the microfluidic chamber and remain firmly attached, often at one pole with the other pole wafting in the direction of the flow of the medium.  Medium flows from left to right at 20,000 μm/min.  Scale bar, 10 μm; time, h:min (similar to Movie 4, J. Bacteriol. 187: 5560-5567, 2005).

Xylella fastidiosa mutant 6E11 colony morphology.  Twitching motility of X. fastidiosa mutant 6E11 (deficient for the fimA gene; cells possess only type IV pili) on a cellophane surface overlaid on nutrient agar. Twitching motility occurs as individual cells and as groupings of cells that ‘explore’ at the colony periphery.  Time, min:sec.

Xylella bacteria migrate against a rapid stream of media flow.  Flow (L to R) is 20,000 µm/min for both top and bottom frames. Top frame is ‘near’-real time with time marked as sec:tenths sec.  Numerous non-attached bacteria are carried with the flow.  Bottom frame is time lapsed with time indicated as h:min.  Many bacteria show twitching movement, both with and against direction of flow.

To view the top and bottom frames as individual movies of smaller file sizes, select (Movie7a.AVI) and (Movie7b.AVI) (File size: 18.5 MB each)

 
Time-lapse movie depicting Xylella fastidiosa wild-type (WT),  pilB- (mutant 1A2, type I pili only), and fimA- (mutant 6E11, type IV pili only) cells on microfluidic channel surfaces.  Media flow rate within the channels was increased sequentially at one minute intervals from 2 to 220 µl/min.  At higher flow rates WT and fimA- cells detached from the glass surface, while pilB- cells were dragged downstream, and in general remained attached.  Flow direction is left to right. (Applied and Environmental Microbiology, 73: 2690-2696)

Xylella fastidiosa mutant TM14 (pilY1) exhibits type IV pili twitching-motility (right to left) against a stream of medium flowing 50,000 µm/min (L to R).  Cells observed traveling from left to right are either floating free with the medium or they are dragged on the chamber surface with the flow of medium (Movie for Microbiology, 153: 719-726)

 
Comparative paths of type IV pilus-mediated twitching movement of Xylella fastidiosa wild type (WT) cells, and pilY1- (mutant TM14, deficient in pilus adhesion protein) and fimA- (mutant 6E11, deficient in shorter type I pili) cells on microfluidic channel surfaces.  Traces correspond to migratory paths of the cells (right to left) against medium flow (left to right) of three representative cells for each cell type over the same period of time.  Note that fimA mutants moved greater distances than the length of the field of view during the time interval shown. (J. Bacteriol., 189: 7507–7510)

 
Individual cells of chpY mutant, grown on cellophane overlaid on PW agar, are shown moving via type IV pili twitching within the confines of a colony margin.  Individual cells and rafts of cell aggregates similar to those observed in the complemented chpY mutant and wild-type isolates are not observed in this mutant. It appears that motile cells are not able to break away from the colony margin to form a colony fringe.

 
Individual cells of the complemented chpY mutant grown on cellophane overlaid on PW agar, are shown moving via type IV pili twitching within the peripherial fringe of the colony. Occasionally individual cells and, more often, rafts of cell aggregates similar to those previously shown for the wild-type isolate migrate to and fro from the peripheral fringe, often on apparent ‘slime’ trails. 

 
   
Autoaggregation of Xylella fastidiosa cells a microfabricated fluidic chambers after 9-days growth.  Widely dispersed cells aggregated over a period of 10 hours.  Individual cells can be seen moving via type IV pilus twitching-motility toward the developing aggregate.

 
Autoaggregation of Xylella fastidiosa cells in a microfabricated fluidic chamber.  By 11-days, much of the chamber was colonized with densely packed cells and cell aggregates, the later which merged with other aggregates to form large masses of X. fastidiosa.  As the large aggregates were transported with the flow of media they dispersed the cells within the smaller aggregates.  Dispersed cells re-aggregated within 10-20 minutes.  The small aggregates re-merged with other aggregates and individual cells. 

 
Autoaggregation of wild-type Xylella fastidiosa cells in a microfabricated fluidic chamber.  Individual Xf cells aggregate into small masses which merge and form larger aggregates.  This is under no flow conditions.  Time is hours: minutes.

 
Pseudomonas aeruginosa strain PAKfliC::gmR (courtesy of Prof. Howard Berg, Harvard), a flagella-minus mutant was previously shown to exhibit type IV pili twitching motility (Skerker, J. M. & Berg, H. C. (2001) Proc. Natl. Acad. Sci. USA 98, 6901-6904) also exhibited directed ‘upstream’ migration in the microfluidic chamber.  All cells, influenced by the medium current, were prostrate to the substratum.  Flow is left to right. Scale bar, 10 μm; time, min:sec.

 
Under no-flow conditions Pseudomonas aeruginosa strain PAKfliC::gmR exhibited type IV pili twitching motility; however, the direction of migration was random.  Also, many cells, attached at one pole to the substratum, remained upright as they migrated. Scale bar, 10 μm; time, min:sec.
   
   
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