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Applied and Environmental Microbiology, November 2007, p. 7252-7258, Vol. 73, No. 22
0099-2240/07/$08.00+0     doi:10.1128/AEM.00895-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Detection and Visualization of an Exopolysaccharide Produced by Xylella fastidiosa In Vitro and In Planta

M. Caroline Roper,¹ L. Carl Greve,² John M. Labavitch,² and Bruce C. Kirkpatrick^1*

Department of Plant Pathology,¹ Department of Plant Sciences, University of California, Davis, Davis, California 95616²

Received 20 April 2007/ Accepted 29 August 2007

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Many phytopathogenic bacteria, such as Ralstonia solanacearum, Pantoea stewartii, and Xanthomonas campestris, 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.

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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 biofilms (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). Bacterial 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). Secretion 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 products resulting from enzymatic action so the bacteria can utilize these products as a carbon source (20).

Grapevines infected with X. fastidiosa have extensive vascular 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 development, delayed shoot growth, and, ultimately, vine death. Extensive 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 because other phytopathogenic bacteria produce EPSs that are involved in virulence and contribute to vascular blockage (9, 26).

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 producing 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 producing 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 analysis 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 expressed (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. fastidiosa 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.

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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 immunosorbent assay (ELISA) against the gumI mutant X. campestris pv. campestris EPS. The serum with the highest titer was chosen for use in ELISA and immunolocalization. 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 antibodies.

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 µl 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 conjugate (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, Hercules, CA).

Preparation of F(ab)₂ fragments.
Immunoglobulin G (IgG) from the polyclonal anti-EPS antiserum was purified by protein A affinity column chromatography using low-salt conditions and eluting with 0.1 M glycine (pH 3.0) (17). F(ab)₂ fragments were prepared by digesting purified anti-EPS IgG with 0.1 µg pepsin/mg IgG in 0.1 M sodium acetate buffer (pH 4.5) at 37°C. F(ab)₂ fragments 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 sandwich 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, Rochester, NY) were coated with 5 µg/ml anti-X. fastidiosa EPS F(ab)₂ fragments in 0.015 M Na₂CO₃-0.035 M NaHCO₃ (pH 9.6). Wells were blocked with 1% nonfat milk in 1x PBS (pH 7.2) for 1 h at room temperature and then washed three times with 1x 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 (OD₆₀₀ of 0.25), and serially diluted to10⁷, 10⁶, 10⁵, and 10⁴ CFU/ml in PBS. The cell-free supernatant was prepared by filtering the supernatant of a 9-day-old X. fastidiosa liquid culture through a 0.2-µm 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 different 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 µg/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 Aldrich, 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 (Molecular Devices, Sunnyvale, CA). Each X. fastidiosa cell concentration was measured 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 10⁸ CFU/ml (OD₆₀₀, 0.25), and the cells were serially diluted to 10⁷, 10⁶, 10⁵, and 10⁴ CFU/ml in PBS. One-milliliter aliquots of each of the cell concentrations were centrifuged at 16,000 x 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 vortexing 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 µl 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 1x 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-µl drop of the bacterial suspension. Control plants were mock inoculated with distilled H₂O in the same manner. Fourteen weeks following inoculation, symptomatic leaves were harvested 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 incubated 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 (Zeiss, Germany). Observations of petiole sections were made with either a Nikon 40x PlanAPO oil immersion lens (numerical aperture, 1.4) or a Nikon 100x 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 sequentially. For each image, 12 images were Kalman averaged to eliminate noise. Displayed images were processed with LaserSharp 2000 software (Zeiss, Germany).

Images of in vitro-formed X. fastidiosa biofilms were captured using an Olympus FV1000 FluoView confocal laser scanning biological microscope mounted on an Olympus IX81 microscope. An Olympus 60x 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 µm. Displayed images were processed with Olympus FV software.

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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. fastidiosa 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. fastidiosa 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 10⁶ CFU/ml but not below 10⁶ CFU/ml (Fig. 1). A 10⁸-CFU/ml concentration of X. fastidiosa cells harvested from solid medium contained approximately 6 µg of EPS/ml, and a 10⁸-CFU/ml concentration of cells harvested from biofilm rings formed in shaken flasks contained approximately 10 µg/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 µg/ml.

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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 standard deviations.

Additionally, washing once with 1x 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 attached 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 µg/ml) (data not shown). PD3 liquid medium used as a negative control in the ELISA never cross-reacted with the anti-EPS antiserum (data not shown).

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FIG. 2. Protein A double-antibody sandwich ELISA of X. fastidiosa cells harvested from solid PD3 medium and either unwashed, washed once with 1x 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 honest significance.

In vitro visualization of X. fastidiosa EPS.
Confocal microscopy 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 counterstained 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 µm). 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 component 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).

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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, counterstained with Syto9, and observed with an Olympus 60x PlanAPO oil immersion lens (numerical aperture, 1.42). X. fastidiosa cells are depicted 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 µm. Scale bars, 15 µm (except in A, where the scale bar was 10 µm).

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). Interestingly, in some instances, X. fastidiosa EPS was seen attached to the walls of the vessel lumen and not associated with X. fastidiosa 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 fluorescent conjugate had no red fluorescence associated with them (data not shown).

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FIG. 4. CLSM images of leaf petioles from grapevines infected with GFP-labeled X. fastidiosa cells and observed with either a Nikon 100x PlanAPO oil immersion lens (numerical aperture, 1.2) (A to C) or a Nikon 40x 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 µm (A to C) and 50 µm (D to I).

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Protein A double-antibody sandwich ELISA using antibodies raised against a modified xanthan gum polymer structurally similar to the X. fastidiosa EPS (5, 22) allowed a reliable quantification 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, suggesting that EPS production is induced when X. fastidiosa colonizes 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. fastidiosa 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 microcolonies then merged and formed confluent biofilms that became thicker over time. Confocal images of 8-day-old mature biofilms indicate that X. fastidiosa EPS is found throughout the biofilm matrix.

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 polysaccharide 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 biofilms. 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, attempts in our laboratory to construct X. fastidiosa EPS-negative 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 biofilm maturation rather than initial attachment (39). However, those authors reported that these mutants showed no measurable 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 precipitation 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 include 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 endoglucanases that could potentially degrade the β-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 produces 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 control measures for PD by mediating biofilm formation in plant and insect vectors.

 
This research was supported by grants from the University of California 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 manuscript.

 
* Corresponding author. Mailing address: Department of Plant Pathology, University of California, Davis, One Shields Avenue, Davis, CA 95616. Phone: (530) 752-2831. Fax: (530) 752-5674. E-mail: bckirkpatrick{at}ucdavis.edu

Published ahead of print on 7 September 2007.

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Applied and Environmental Microbiology, November 2007, p. 7252-7258, Vol. 73, No. 22
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