APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2007, p. 7252–7258
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 73, No. 22
Detection and Visualization of an Exopolysaccharide Produced by
Xylella fastidiosa In Vitro and In Planta?
M. Caroline Roper,1L. Carl Greve,2John M. Labavitch,2and Bruce C. Kirkpatrick1*
Department of Plant Pathology,1and Department of Plant Sciences,2University of California, Davis, Davis, California 95616
Received 20 April 2007/Accepted 29 August 2007
Many phytopathogenic bacteria, such as Ralstonia solanacearum, Pantoea stewartii, and Xanthomonas campes-
tris, produce exopolysaccharides (EPSs) that aid in virulence, colonization, and survival. EPS can also
contribute to host xylem vessel blockage. The genome of Xylella fastidiosa, the causal agent of Pierce’s disease
(PD) of grapevine, contains an operon that is strikingly similar to the X. campestris gum operon, which is
responsible for the production of xanthan gum. Based on this information, it has been hypothesized that X.
fastidiosa is capable of producing an EPS similar in structure and composition to xanthan gum but lacking the
terminal mannose residue. In this study, we raised polyclonal antibodies against a modified xanthan gum
polymer similar to the predicted X. fastidiosa EPS polymer. We used enzyme-linked immunosorbent assay to
quantify production of EPS from X. fastidiosa cells grown in vitro and immunolocalization microscopy to
examine the distribution of X. fastidiosa EPS in biofilms formed in vitro and in planta and assessed the
contribution of X. fastidiosa EPS to the vascular occlusions seen in PD-infected grapevines.
Xylella fastidiosa is the causal agent of Pierce’s disease (PD)
of grapevine and many other economically important diseases
(21). This gram-negative bacterium lives in plant xylem vessels
as well as the foregut and mouthparts of its xylem-feeding
insect vectors. In both environments, X. fastidiosa forms bio-
films (3, 10, 15, 29, 33). Biofilms protect microbial communities
from antibiotics, dehydration, host defenses, and other stresses
while contributing to adhesion and virulence by allowing the
coordinated expression of pathogenicity genes via quorum
sensing (16, 41, 48). The biofilm matrix includes nucleic acids,
proteins, humic substances, and exopolysaccharide (EPS). Bac-
terial EPS is an important structural component of this matrix
and aids in the adhesion of bacteria to surfaces and to each
other as well as imparting stability and structure to the mature
biofilm (2, 42, 48).
In addition to aiding in adhesion and stability, it is theorized
that the viscous nature of EPS also helps localize and stabilize
hydrolytic enzymes produced by the bacteria. X. fastidiosa uses
plant cell wall-degrading enzymes to digest the pit membrane
barriers separating xylem vessels from one another in order to
facilitate systemic movement throughout grapevines (35). Se-
cretion and trapping enzymes in close proximity to the pit
membrane would be particularly adaptive in the xylem sap
environment. Besides localizing the enzymes, X. fastidiosa EPS
could also serve to concentrate and entrap the hydrolytic prod-
ucts resulting from enzymatic action so the bacteria can utilize
these products as a carbon source (20).
Grapevines infected with X. fastidiosa have extensive vascu-
lar occlusions and exhibit symptoms similar but not identical to
water stress (43). Symptoms associated with PD of Vitis vinifera
grapevines include leaf scorching (necrosis and chlorosis),
berry desiccation, leaf abscission, irregular periderm develop-
ment, delayed shoot growth, and, ultimately, vine death. Ex-
tensive vascular blockage is the generally accepted cause for
the symptoms (13, 14). Pectic gels, tyloses, and X. fastidiosa
biofilms contribute to these vascular occlusions (24, 40). We
hypothesize that X. fastidiosa produces an EPS that contributes
to the vascular occlusion seen in PD-infected grapevines be-
cause other phytopathogenic bacteria produce EPSs that are
involved in virulence and contribute to vascular blockage
Electron micrographs indicate that X. fastidiosa cells in
planta are embedded in an amorphous extracellular matrix
hypothesized to be bacterial EPS (3, 29, 40). In addition to
microscopic evidence, in silico analysis of the X. fastidiosa
genome strongly suggests that X. fastidiosa is capable of pro-
ducing an EPS that is similar to xanthan gum (5). The X.
fastidiosa genome contains homologs to 9 of the 12 genes
found in the well-characterized gum operon of X. campestris
pv. campestris, but it is missing the X. campestris pv. campestris
gumI, gumG, and gumL homologs (1, 37, 46). The nine X.
fastidiosa gum genes are also arranged in an order identical to
that of their X. campestris pv. campestris homologs. Thus, da
Silva et al. (5) proposed that X. fastidiosa is capable of pro-
ducing an EPS similar to xanthan gum, but X. fastidiosa EPS is
likely missing the terminal mannosyl residue found on the
repeating side chains based on the absence of the X. campestris
pv. campestris gumI, gumG, and gumL homologs. These genes
are involved in the addition and decoration of the terminal
mannosyl residue in X. campestris pv. campestris (23).
Furthermore, Fourier transform infrared spectroscopy anal-
ysis detected carbohydrates associated with X. fastidiosa cells
(10), and computer analysis of codon usage predicted that the
X. fastidiosa gum genes have the potential to be highly ex-
pressed (12). Microarray studies showed that the gum genes
are expressed in both planktonic and biofilm states (10), but
expression levels of the X. fastidiosa gum genes gumC, gumD,
and gumJ are affected by cell density, suggesting that X. fastid-
* Corresponding author. Mailing address: Department of Plant Pathol-
ogy, University of California, Davis, One Shields Avenue, Davis, CA
95616. Phone: (530) 752-2831. Fax: (530) 752-5674. E-mail: bckirkpatrick
?Published ahead of print on 7 September 2007.
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