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.
iosa EPS production could be regulated by a quorum-sensing
mechanism (32, 36). The goal of this study was to determine if
X. fastidiosa produces an EPS similar to xanthan gum and to
investigate when and where X. fastidiosa EPS is present during
biofilm formation in vitro and in planta.
MATERIALS AND METHODS
Bacterial strains and growth conditions. X. fastidiosa Fetzer (18) and X.
fastidiosa Temecula green fluorescent protein (GFP) (31) were grown at 28°C in
PD3 liquid and solid media (6).
Isolation of X. campestris pv. campestris gumI mutant EPS and preparation of
X. campestris pv. campestris EPS antiserum. X. campestris pv. campestris EPS
was purified according to methods described previously by Ielpi et al. (22). This
EPS was generously provided by Luis Ielpi (University of Buenos Aires, Buenos
Aires, Argentina). Approximately 1 mg gumI mutant X. campestris pv. campestris
gum was mixed with Freund’s complete adjuvant and injected into New Zealand
White rabbits. Subsequent booster injections were made every 2 weeks with the
same amount of immunogen mixed with Freund’s incomplete adjuvant. Sera
were collected at 2-week intervals and tested by indirect enzyme-linked immu-
nosorbent assay (ELISA) against the gumI mutant X. campestris pv. campestris
EPS. The serum with the highest titer was chosen for use in ELISA and immu-
nolocalization. Preimmune serum used as a negative control in the ELISA never
cross-reacted with gumI mutant X. campestris pv. campestris EPS (data not
shown). For convenience, these antibodies will be referred to as X. fastidiosa EPS
Specificity of anti-EPS antiserum. The anti-EPS antiserum was tested for
cross-reactivity with X. fastidiosa lipopolysaccharide (LPS). X. fastidiosa cells
were harvested from PD3 plates, adjusted to an optical density at 600 nm
(OD600) of 0.6. in 1.5 ml sterile distilled H2O, and pelleted by centrifugation. X.
fastidiosa LPS was extracted with hot phenol as described previously by DeLoney
et al. (7) and resuspended in 20 ?l of sample buffer. Five-microliter aliquots of
LPS were subjected to sodium deoxycholate polyacrylamide gel electrophoresis
at 30 mA until the loading dye reached the bottom of the gel. The gel was silver
stained according to methods described previously by Tsai and Frasch (44). X.
fastidiosa LPS electrophoresed on a second sodium deoxycholate polyacrylamide
gel was transferred onto a nitrocellulose membrane at 100 V for 1 h, and the
membrane was cut into three pieces. After blocking with 1% nonfat milk in
phosphate-buffered saline (PBS), the membranes were probed with a 1:400
dilution of either polyclonal anti-EPS antiserum, preimmune rabbit serum, or
polyclonal anti-X. fastidiosa antiserum raised against whole X. fastidiosa cells in
PBS for 1 h at 37°C with gentle shaking. The membranes were washed three
times for 10 min each with PBS–0.05% Tween 20 (PBST). The membranes were
incubated with a 1:1,500 dilution of goat anti-rabbit alkaline phosphatase con-
jugate (Bio-Rad Laboratories, Hercules, CA) for 1 h at 37°C and washed four
times for 10 min each with PBST. Bound conjugate was detected using nitroblue
tetrazolium–5-bromo-4-chloro-3-indolyl phosphate (Bio-Rad Laboratories, Her-
Preparation of F(ab)2fragments. Immunoglobulin G (IgG) from the poly-
clonal anti-EPS antiserum was purified by protein A affinity column chromatog-
raphy using low-salt conditions and eluting with 0.1 M glycine (pH 3.0) (17).
F(ab)2fragments were prepared by digesting purified anti-EPS IgG with 0.1 ?g
pepsin/mg IgG in 0.1 M sodium acetate buffer (pH 4.5) at 37°C. F(ab)2fragments
were purified by passing the digestion reaction mixture through a protein A
affinity column to remove any undigested IgG.
Quantification of X. fastidiosa EPS in vitro. Protein A double-antibody sand-
wich ELISA was used to detect X. fastidiosa EPS associated with X. fastidiosa
Fetzer cells grown for 9 days on solid PD3 medium, in biofilms that formed at the
air-liquid interface in liquid PD3 medium, and in the cell-free supernatant of that
liquid culture. The wells of a microtiter plate (Maxisorp; Nunc Products, Roch-
ester, NY) were coated with 5 ?g/ml anti-X. fastidiosa EPS F(ab)2fragments in
0.015 M Na2CO3–0.035 M NaHCO3(pH 9.6). Wells were blocked with 1%
nonfat milk in 1? PBS (pH 7.2) for 1 h at room temperature and then washed
three times with 1? PBST (pH 7.2). X. fastidiosa cells grown on solid medium,
biofilms harvested from liquid medium, or cell-free supernatants from cells
grown in liquid medium were used as ELISA samples. For both liquid- and
solid-medium cultures, the cells were grown for 9 days, harvested, adjusted to a
concentration of 108(OD600of 0.25), and serially diluted to107, 106, 105, and 104
CFU/ml in PBS. The cell-free supernatant was prepared by filtering the super-
natant of a 9-day-old X. fastidiosa liquid culture through a 0.2-?m filter (Nalgene,
Rochester, NY). PD3 medium with no X. fastidiosa was used as a negative
control in the ELISA. The modified xanthan gum, which was used as an antigen
to produce the anti-EPS antiserum, was dissolved in PBS and adjusted to dif-
ferent concentrations to form a standard curve. The samples were added to the
F(ab)2-coated microtiter wells and incubated for 1.5 h at 37°C followed by
washing three times with PBST. Whole anti-X. fastidiosa EPS IgG was added to
the wells at a concentration of 5 ?g/ml. Plates were incubated for 1.5 h at 37°C
followed by three washes with PBST. Protein A-alkaline phosphatase conjugate
(Sigma Aldrich, St. Louis, MO) was diluted 1:1,500 in PBS, added to the wells,
and incubated for 1 h at 37°C. Sigmafast p-nitrophenyl phosphate (Sigma Al-
drich, St. Louis, MO) was prepared according to the manufacturer’s instructions
and used as a substrate. The substrate color was allowed to develop for 1 h, and
the absorbance was measured at 405 nm in an Emax microplate reader (Molec-
ular Devices, Sunnyvale, CA). Each X. fastidiosa cell concentration was mea-
sured in duplicate on each microtiter plate, and each experiment was repeated
five times to give 10 repetitions per treatment.
Cellular association of X. fastidiosa EPS. To determine if the X. fastidiosa EPS
was loosely associated with the cells as an extracellular matrix or tightly bound as
a capsular polysaccharide, we washed the cells that were grown on solid medium
either once with PBS or once with 0.5 M NaCl (pH 7.2). X. fastidiosa Fetzer cells
were grown on PD3 solid medium and resuspended in PBS, the concentration
was adjusted to 108CFU/ml (OD600, 0.25), and the cells were serially diluted to
107, 106, 105, and 104CFU/ml in PBS. One-milliliter aliquots of each of the cell
concentrations were centrifuged at 16,000 ? g for 10 min, and the cell pellet was
resuspended by vigorously vortexing in either PBS or 0.5 M NaCl (pH 7.2). The
cells were pelleted again by centrifugation and resuspended by vigorous vortex-
ing in PBS. These washed cells were used as antigen samples in the above-
described protein A double-antibody sandwich ELISA test. X. fastidiosa Fetzer
cells that were suspended in PBS and not subjected to either washing regimen
were used as unwashed controls. Each X. fastidiosa cell concentration for each
treatment was measured in duplicate on a microtiter plate, and each experiment
was repeated five times to give 10 repetitions per treatment.
Statistical analysis. The ELISA data were analyzed using analysis of variance
in blocks, with blocks as a random factor. Group differences were assessed using
Tukey’s honest significant difference.
Immunolocalization of X. fastidiosa EPS in in vitro biofilms. Fifty-milliliter
Falcon tubes containing 20 ml of PD3 liquid medium were each inoculated with
200 ?l of a 108-CFU/ml suspension of X. fastidiosa cells harvested from a PD3
plate containing 7-day-old X. fastidiosa colonies. An autoclaved glass microscope
slide was placed vertically in each tube, and the tubes were shaken in an upright
position for either 1, 2, 4, or 8 days to allow the formation of an X. fastidiosa
biofilm at the air-liquid interface on the microscope slide. At each time point,
slides were removed from the tube and gently heat fixed. There were three
replicates for each time point, and the experiment was repeated three times. The
X. fastidiosa biofilm was probed with a 1:400 dilution of anti-EPS antiserum or
preimmune serum and 1% bovine serum albumin (BSA) in PBS, incubated at
37°C for 1 h, and washed gently three times with 0.2% BSA in 1? PBS (BSA-
PBS). Slides were incubated for 1 h at 37°C with a 1:1,000 dilution of Alexa Fluor
546 goat anti-rabbit fluorescent conjugate in BSA-PBS (Invitrogen, Carlsbad,
CA) and washed gently four times with BSA-PBS. In order to visualize X.
fastidiosa cells, slides were counterstained with the nucleic acid stain Syto 9
(Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions and
immediately mounted in Slowfade mounting fluid (Invitrogen, Carlsbad, CA).
Immunolocalization of X. fastidiosa EPS in PD-infected petioles. Greenhouse-
grown Vitis vinifera cv. Thompson seedless plants were pinprick inoculated (19)
with a 108-CFU/ml suspension of X. fastidiosa Temecula GFP cells (31). Each
plant was inoculated twice on the stem with a 20-?l drop of the bacterial
suspension. Control plants were mock inoculated with distilled H2O in the same
manner. Fourteen weeks following inoculation, symptomatic leaves were har-
vested from PD-infected grapevines, and healthy leaves were harvested from
mock-inoculated grapevines. The petioles were removed from these leaves, cut
into 2-cm pieces, and fixed in 1.5% glutaraldehyde–0.3% paraformaldehyde–
0.025 M PIPES [piperazine-N,N?-bis(2-ethanesulfonic acid)] buffer (pH 7.2) for
48 h with gentle shaking. Following fixation, the petioles were rinsed in 0.025 M
PIPES (pH 7.2) and cross sectioned by hand with a double-edged razor blade.
For immunolocalization, the petiole cross sections were incubated with a 1:400
dilution of X. fastidiosa EPS antiserum or preimmune serum and 1% BSA at
37°C for 1 h. Sections were gently washed once with BSA-PBS and then incu-
bated with an Alexa Fluor 568 goat anti-rabbit conjugate in BSA-PBS at 37°C for
1 h (Invitrogen, Carlsbad, CA). Sections were washed gently four times with
BSA-PBS (pH 7.2) and immediately mounted in Slowfade antifade mounting
medium (Invitrogen, Carlsbad, CA).
Confocal laser scanning microscopy (CLSM). Images of grapevine petiole
cross sections were captured with a Bio-Rad MRC 1024 confocal laser scanning
microscope mounted on a Nikon (Melville, NY) Microphot-SA microscope
VOL. 73, 2007EXOPOLYSACCHARIDE PRODUCED BY XYLELLA FASTIDIOSA7253
(Zeiss, Germany). Observations of petiole sections were made with either a
Nikon 40? PlanAPO oil immersion lens (numerical aperture, 1.4) or a Nikon
100? PlanAPO oil immersion lens (numerical aperture, 1.2). GFP was excited
using the 488-nm laser, and a 522 nm/32 nm barrier filter was placed in front of
the detector. Alexa Fluor 568 was excited with the 568-nm laser, and a 598 nm/
40 nm band pass filter was placed in front of the detector. To avoid cross talk
between the green and red detection channels, the images were collected se-
quentially. For each image, 12 images were Kalman averaged to eliminate noise.
Displayed images were processed with LaserSharp 2000 software (Zeiss,
Images of in vitro-formed X. fastidiosa biofilms were captured using an Olym-
pus FV1000 FluoView confocal laser scanning biological microscope mounted
on an Olympus IX81 microscope. An Olympus 60? PlanAPO oil immersion lens
(numerical aperture, 1.42) was used to observe the X. fastidiosa biofilms. Syto 9
was excited with the 488-nm laser, and a 505 nm/530 nm barrier filter was placed
in front of the detector. Alexa Fluor 546 was excited with the 543-nm laser, and
a 545 nm/555 nm barrier filter was placed in front of the detector. Cross talk was
avoided as described above. X. fastidiosa biofilms were imaged through the z axis,
with each z section equaling 0.4 ?m. Displayed images were processed with
Olympus FV software.
Specificity of anti-EPS antiserum. The X. fastidiosa anti-
EPS antiserum did not cross-react with X. fastidiosa LPS that
was transferred onto a nitrocellulose membrane, indicating
that the anti-EPS antiserum is EPS specific. Polyclonal X. fas-
tidiosa antiserum did cross-react with X. fastidiosa LPS bound
to nitrocellulose, demonstrating that the X. fastidiosa LPS was
successfully transferred onto the nitrocellulose membranes
(data not shown).
In vitro quantification and cellular association of X. fastid-
iosa EPS. We used protein A double-antibody sandwich
ELISA to detect EPS from X. fastidiosa cells grown on plates,
in biofilms harvested from liquid flask cultures, and in the
cell-free culture supernatant. We detected X. fastidiosa EPS
from both growth conditions at concentrations of 108, 107, and
106CFU/ml but not below 106CFU/ml (Fig. 1). A 108-CFU/ml
concentration of X. fastidiosa cells harvested from solid me-
dium contained approximately 6 ?g of EPS/ml, and a 108-
CFU/ml concentration of cells harvested from biofilm rings
formed in shaken flasks contained approximately 10 ?g/ml
EPS compared to a standard curve of known concentrations of
the modified xanthan gum used as an antigen in the ELISA
(Fig. 1). The smallest detectable amount of modified xanthan
gum in the ELISA was 1 ?g/ml.
Additionally, washing once with 1? PBS or 0.5 M NaCl
removed a portion of the X. fastidiosa EPS, demonstrating that
some of the X. fastidiosa EPS is loosely associated with the cells
(Fig. 2). No washing scheme completely washed off all of the
EPS, indicating that a significant portion of the EPS associated
with X. fastidiosa cells grown on solid medium remained at-
tached to the cells, presumably as a tightly bound capsular
polysaccharide. We hypothesized that the interaction of the X.
fastidiosa EPS with the cell is ionic in nature and reasoned that
increasing the ionic strength of the wash buffer would liberate
more EPS. However, the same amount of EPS was removed
when cells were washed with a solution of low (PBS) or high
(0.5 M NaCl) ionic strength. It is possible that the PBS is
sufficient to wash off all of the ionically bound EPS and could
explain why washing with 0.5 M NaCl did not liberate more
EPS than the PBS wash. In contrast, when grown in liquid
medium, a significant portion of the EPS was found in the
cell-free supernatant as an extracellular fraction rather than
tightly bound to the cells (approximately 5 ?g/ml) (data not
shown). PD3 liquid medium used as a negative control in the
ELISA never cross-reacted with the anti-EPS antiserum (data
FIG. 1. Protein A double-antibody sandwich ELISA using serial
dilutions of X. fastidiosa cells harvested from solid PD3 medium or
from biofilms formed in liquid PD3 medium as an antigen. F(ab)2
fragments prepared from anti-EPS IgG were used to trap the antigen,
and purified anti-EPS IgG was used as the probe. Error bars indicate
FIG. 2. Protein A double-antibody sandwich ELISA of X. fastidiosa
cells harvested from solid PD3 medium and either unwashed, washed
once with 1? PBS (pH 7.2), or washed once with 0.5 M NaCl. F(ab)2
fragments prepared from anti-EPS IgG were used to trap the antigen,
and purified anti-EPS IgG was used as the probe. Error bars indicate
standard deviations, and letters indicate groups assigned by Tukey’s
7254ROPER ET AL.APPL. ENVIRON. MICROBIOL.
In vitro visualization of X. fastidiosa EPS. Confocal micros-
copy was performed on in vitro X. fastidiosa biofilms that
developed after 1, 2, 4, and 8 days of growth in PD3 medium.
Biofilms were labeled with anti-EPS antibodies, followed by an
anti-rabbit Alexa Fluor 546 fluorescent conjugate, and coun-
terstained with the nucleic acid stain Syto 9. X. fastidiosa cells
stained with Syto 9 are depicted in green and X. fastidiosa EPS
is depicted in red in Fig. 3. The overhead and sagittal images
presented in Fig. 3 are composite images of sections through
the z axis of the X. fastidiosa biofilm (z section, 0.4 ?m). After
24 h of growth, microcolonies attached to the glass slide were
seen, and small amounts of EPS were associated with them
(Fig. 3A). The microcolonies began to merge after 48 h of
growth, and more EPS was visible (Fig. 3B). After 4 days, a
confluent biofilm on the glass slide was formed and became
thicker over time. An 8-day-old biofilm was considered to be
mature because the thickness and appearance of an 8-day-old
biofilm were similar to those of the 10-day-old biofilm (data
not shown). The composite overhead and sagittal images of the
sequential z series clearly show that EPS is a significant com-
ponent of the biofilm matrix and is distributed throughout the
biofilm. Controls labeled with preimmune serum followed by
the Alexa Fluor 546 fluorescent conjugate did not have any red
fluorescence associated with them (data not shown).
In planta visualization of X. fastidiosa EPS. We used CLSM
to determine if X. fastidiosa produces an EPS in planta and if
this EPS is a component of the vascular occlusions found in
PD-infected grapevines. X. fastidiosa EPS was visualized in X.
fastidiosa GFP-infected petiole cross sections labeled with anti-
EPS antiserum followed by an anti-rabbit Alexa Fluor 546
fluorescent conjugate. X. fastidiosa GFP cells are depicted in
green and the X. fastidiosa EPS is depicted in red in Fig. 4. The
confocal images show that X. fastidiosa produces an EPS in
planta that is usually colocalized with X. fastidiosa bacterial
aggregates found in the xylem vessels (Fig. 4A to F). Interest-
ingly, in some instances, X. fastidiosa EPS was seen attached to
the walls of the vessel lumen and not associated with X. fas-
tidiosa cells (Fig. 4G to I). We found no bacterial cells or EPS
in the mock-inoculated controls (data not shown). In addition,
the numerous tyloses and gels observed in PD-infected plants
never fluoresced. X. fastidiosa GFP-infected petioles incubated
with preimmune serum followed by the Alexa Fluor 546 fluo-
FIG. 3. Accumulation, over time, of X. fastidiosa EPS in biofilms
formed on glass slides placed in liquid cultures in PD3 medium. Shown
are CLSM images of X. fastidiosa in vitro biofilms labeled with anti-
EPS antibodies followed by anti-rabbit IgG Alexa Flour 546, counter-
stained with Syto9, and observed with an Olympus 60? PlanAPO oil
immersion lens (numerical aperture, 1.42). X. fastidiosa cells are de-
picted in green, and EPS is depicted in red. Green and red channels
have been merged in all panes. Images presented are overhead (xy)
and sagittal (xz) images. The step size for each z section was 0.4 ?m.
Scale bars, 15 ?m (except in A, where the scale bar was 10 ?m).
FIG. 4. CLSM images of leaf petioles from grapevines infected
with GFP-labeled X. fastidiosa cells and observed with either a Nikon
100? PlanAPO oil immersion lens (numerical aperture, 1.2) (A to C)
or a Nikon 40? PlanAPO oil immersion lens (numerical aperture, 1.4)
(D to I). Petiole cross sections were labeled with anti-EPS antibodies
followed by anti-rabbit IgG Alexa Fluor 568. (A, D, and G) Green
channel, with X. fastidiosa cells depicted in green. (B, E, and H) Red
channel, with X. fastidiosa EPS depicted in red. (C, F, and I) Green and
red channels merged. A to F depict X. fastidiosa EPS colocalized with
EPS. G to I depict X. fastidiosa EPS not associated with X. fastidiosa
cell masses. Arrows indicate developing tyloses. Note that there is no
fluorescence associated with the tyloses. Scale bars, 15 ?m (A to C)
and 50 ?m (D to I).
VOL. 73, 2007 EXOPOLYSACCHARIDE PRODUCED BY XYLELLA FASTIDIOSA7255
rescent conjugate had no red fluorescence associated with
them (data not shown).
Protein A double-antibody sandwich ELISA using antibod-
ies raised against a modified xanthan gum polymer structurally
similar to the X. fastidiosa EPS (5, 22) allowed a reliable quan-
tification of X. fastidiosa EPS associated with cells grown on
solid medium or harvested from biofilm rings that formed at
the air-liquid interface in shaken liquid cultures. Under both
growth regimens, X. fastidiosa makes a relatively small amount
of EPS (Fig. 1) compared to X. campestris pv. campestris,
which produces copious amounts of EPS in culture. Unlike X.
campestris pv. campestris, X. fastidiosa does not form mucoid
colonies on solid media, so it was expected that X. fastidiosa
would produce a small amount of EPS in vitro. Likewise,
Fourier transform infrared spectroscopy analysis showed that
only a small amount of carbohydrate is associated with X.
fastidiosa biofilms attached to glass (33). Interestingly, when
grown in shaken flasks, X. fastidiosa produced more EPS than
when grown on solid medium (Fig. 1), and a significant portion
of the EPS was released into the supernatant, whereas on solid
medium, the majority of the EPS was tightly bound to the cells
as capsular polysaccharide (Fig. 2). These data suggest that
different growth conditions (solid versus liquid medium) affect
the amount of EPS being produced.
The relatively small amount of EPS production could be due
in part to the fastidious nature of X. fastidiosa when grown in
vitro, coupled with the fact that PD3 medium is complex and
not representative of the nutrient-poor xylem sap habitat
where X. fastidiosa is found in nature. Indeed, the expression of
several X. fastidiosa gum genes is upregulated in planta, sug-
gesting that EPS production is induced when X. fastidiosa col-
onizes grapevines (28). Picomolar amounts of EPS polymers
can cause the clogging of pit pore membranes and limit water
flow in other plant species (45). Thus, it is feasible that X.
fastidiosa EPS could contribute to the vascular clogging that is
associated with PD symptom development, especially if the
majority of EPS is extracellular, as suggested by the data that
we collected from X. fastidiosa cells grown in liquid medium.
Others studies proposed that EPS is not involved in the
initial attachment of X. fastidiosa cells to surfaces but rather
contributes to the three-dimensional architecture and stability
of the mature biofilm, as observed for other bacterial species
(4, 15, 30, 33, 47). Scanning electron micrographs of X. fastid-
iosa cells grown in culture demonstrate that there is no EPS
associated with the initial X. fastidiosa microcolonies that form,
but large aggregates of X. fastidiosa cells are embedded in an
extracellular matrix presumed to be EPS (27, 29). In an effort
to investigate if X. fastidiosa EPS was present during early
biofilm formation, we monitored the presence of EPS in X.
fastidiosa biofilms over time by immunolocalization followed
by CLSM. After 24 h, small aggregates of X. fastidiosa cells had
attached to glass slides, and these aggregates had small
amounts of EPS associated with them (Fig. 3A). These micro-
colonies then merged and formed confluent biofilms that be-
came thicker over time. Confocal images of 8-day-old mature
biofilms indicate that X. fastidiosa EPS is found throughout the
As quantified by ELISA, X. fastidiosa biofilms formed in
vitro do not have large amounts of EPS associated with them,
but visualization of the EPS by CLSM clearly indicates that the
EPS is a substantial component of the mature biofilm matrix in
vitro. The patchy distribution of EPS in biofilms that formed in
vitro could be due to the possibility that not all cells in the
biofilm are producing EPS or to the inability of the anti-EPS
antibodies to fully penetrate the biofilm matrix. Additionally,
the ELISA data suggest that more of the EPS is present as an
extracellular EPS rather than as tightly bound capsular poly-
saccharide when X. fastidiosa is grown in liquid culture versus
on solid medium. Therefore, it is possible that the EPS is
sloughed off the cells in the biofilms seen in Fig. 3, which might
explain why EPS is not seen coating all of the cells. In other
bacterial species, mutants that are deficient in EPS production
can still attach to surfaces but are unable to form mature
biofilms with the same three-dimensional architecture as wild-
type cells (25, 47). We hypothesize that this may also be the
case for X. fastidiosa based on the observation that there is very
little EPS present during initial microcolony formation but that
substantial amounts of EPS are associated with mature bio-
films. The use of X. fastidiosa mutants compromised in EPS
production would enable us to confirm the role of EPS in
initial attachment and overall biofilm formation. However, at-
tempts in our laboratory to construct X. fastidiosa EPS-nega-
tive mutants have been unsuccessful using a PD strain of X.
fastidiosa, but gumB and gumF (two of the genes in the nine-
gene X. fastidiosa gum operon) mutants have been constructed
in the citrus variegated chlorosis strain of X. fastidiosa. These
mutants still attached to surfaces but have a reduced capacity
to form biofilms, indicating that EPS is likely involved in bio-
film maturation rather than initial attachment (39). However,
those authors reported that these mutants showed no measur-
able differences in EPS production compared to the wild type.
These findings were based on the wet weight of precipitable
material from a cell-free supernatant obtained after growth in
the defined medium XDM2(8). Based on our findings that X.
fastidiosa does not produce large amounts of EPS in PD3
medium and preliminary data for EPS production in XDM2
medium, we believe that EPS quantification based on precip-
itation followed by weighing of the collected precipitate is not
sufficiently sensitive to detect differences in EPS production. It
is likely that a portion of the collected precipitate would in-
clude proteins and other materials secreted by X. fastidiosa as
well as medium components (38).
In almost all cases, we saw X. fastidiosa EPS colocalizing
with X. fastidiosa cell aggregates found in the xylem vessels of
plants. These biofilms either partially or completely filled the
cross section of the xylem vessel, where they contribute to
major blockages of the xylem network. However, in some
cases, we found X. fastidiosa EPS in xylem vessels but not
associated with bacterial cells. We speculate that this is likely a
result of EPS being carried away from the biofilm by the xylem
sap flow. EPS can also be shed from microbial aggregates and
be adsorbed other places as bacteria are liberating themselves
from the biofilm matrix in order to colonize new niches (11).
This sloughing off of cells from the biofilm can occur as a result
of enzymatic degradation of the EPS polymer (48). Hydrolytic
enzymes are present in bacterial biofilms, and X. fastidiosa
possesses several open reading frames encoding putative en-
7256 ROPER ET AL.APPL. ENVIRON. MICROBIOL.
doglucanases that could potentially degrade the ?-1,4-glucan
backbone of the X. fastidiosa EPS. At least one recombinant X.
fastidiosa endoglucanase, when expressed in Escherichia coli, is
capable of cleaving carbohydrate polymers with a 1,4-linked
glucan backbone (34, 49); thus, this enzyme has the potential
to digest X. fastidiosa EPS.
In conclusion, we have demonstrated that X. fastidiosa pro-
duces an EPS similar in structure to that predicted by in silico
analysis and that this EPS is part of the mature biofilm matrix.
We also show that X. fastidiosa EPS contributes to the vascular
occlusions seen in PD-infected grapevine petioles; however, its
precise role in virulence remains unclear. Because biofilms are
often associated with persistent infections, understanding the
properties and components of the X. fastidiosa biofilm and the
factors regulating its development could lead to possible con-
trol measures for PD by mediating biofilm formation in plant
and insect vectors.
This research was supported by grants from the University of Cali-
fornia and the California Department of Food and Agriculture
Pierce’s Disease/Glassy-Winged Sharpshooter Research programs.
We thank Luis Ielpi (University of Buenos Aires, Buenos Aires,
Argentina) for the generous gift of the modified xanthan gum and
Steve Lindow (University of California, Berkeley, CA) for GFP-X.
fastidiosa. We also thank Hera Vlamakis (Harvard Medical School,
Boston, MA) for helpful suggestions and critical review of the manu-
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