expression of xylella fastidiosa fimbrial and afimbrial proteins

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 2010, p. 4250–4259 Vol. 76, No. 13 0099-2240/10/$12.00 doi:10.1128/AEM.02114-09 Copyright © 2010, American Society for Microbiology. All Rights Reserved. Expression of Xylella fastidiosa Fimbrial and Afimbrial Proteins during Biofilm Formation R. Caserta, 1,2 M. A. Takita, 1 M. L. Targon, 1 L. K. Rosselli-Murai, 2 A. P. de Souza, 2 L. Peroni, 3 D. R. Stach-Machado, 3 A. Andrade, 4 C. A. Labate, 4 E. W. Kitajima, 5 M. A. Machado, 1 and A. A. de Souza 1 * Centro APTA Citros Sylvio Moreira/IAC, Rodovia Anhanguera Km 158, Cordeiro ´polis SP, Brazil 13490-970 1 ; Universidade Estadual de Campinas/UNICAMP, Centro de Biologia Molecular e Engenharia Gene ´tica, Departamento de Gene ´tica e Evoluc ¸a ˜o, Instituto de Biologia, P.O. Box 6010, Campinas SP, Brazil 13083-970 2 ; Universidade Estadual de Campinas/UNICAMP, Laborato ´rio de Imunologia Aplicada, Departamento de Microbiologia e Imunologia, Rua Monteiro Lobato s/n, Campinas SP, Brazil 13083-970 3 ; Escola Superior de Agricultura “Luiz de Queiroz”/USP, Laborato ´rio Max Feffer de Gene ´tica de Plantas, Departamento de Gene ´tica, P.O. Box 83, Piracicaba SP, Brazil 13400-970 4 ; and Escola Superior de Agricultura “Luiz de Queiroz”/USP, Nu ´cleo de Apoio a ` Pesquisa em Microscopia Eletro ˆnica Aplicada a ` Pesquisa Agropecua ´ria (NAP/MEPA), Piracicaba SP, Brazil 13418-900 5 Received 9 September 2009/Accepted 4 May 2010 Complete sequencing of the Xylella fastidiosa genome revealed characteristics that have not been described previously for a phytopathogen. One characteristic of this genome was the abundance of genes encoding proteins with adhesion functions related to biofilm formation, an essential step for colonization of a plant host or an insect vector. We examined four of the proteins belonging to this class encoded by genes in the genome of X. fastidiosa: the PilA2 and PilC fimbrial proteins, which are components of the type IV pili, and XadA1 and XadA2, which are afimbrial adhesins. Polyclonal antibodies were raised against these four proteins, and their behavior during biofilm development was assessed by Western blotting and immunofluorescence assays. In addition, immunogold electron microscopy was used to detect these proteins in bacteria present in xylem vessels of three different hosts (citrus, periwinkle, and hibiscus). We verified that these proteins are present in X. fastidiosa biofilms but have differential regulation since the amounts varied temporally during biofilm formation, as well as spatially within the biofilms. The proteins were also detected in bacteria colonizing the xylem vessels of infected plants. Aggregative growth is a common feature in the microbial world, and its discovery radically changed our concept of mi- crobial growth dynamics. A cellular aggregate adhering to a surface is known as a biofilm. It has important characteristics, such as greater resistance to antimicrobial compounds (34, 54), increased capacity of the cells to take up nutrients from the environment (59), and higher detoxification efficiency resulting from an increase in expression of genes encoding efflux pumps (43). These characteristics give the biofilm cells a great adap- tive advantage. Biofilm growth also confers advantages to plant pathogens by promoting virulence and protection against plant defense responses. Bacteria can colonize different niches in the plant, from aerial surfaces to roots and the vascular system, and biofilm formation can play a role at all of these sites of colo- nization. In the vessels, biofilms are very important since the cells need to survive in a competitive habitat where plant de- fense compounds are produced in response to infection (7). Biofilm development is divided into at least the following five phases: (i) reversible attachment, (ii) irreversible attach- ment, (iii) beginning of maturation, (iv) mature biofilm, and (v) dispersion (13, 50). In Xylella fastidiosa strain 9a5c, the maturation phase occurs between days 15 and 20 in vitro, while dispersion occurs between days 25 and 30, as observed by our analysis of biofilm formation using different methods, includ- ing scanning electron microscopy and quantification of exopo- lysaccharides, biomass, and the total protein (unpublished data). The establishment and development of biofilms of plant- colonizing bacteria share several features with the establish- ment and development of biofilms of human bacterial patho- gens, such as regulation by quorum sensing, nutrient starvation regulation, and phase variation. Motility is also an important factor not only for the initiation and development of the bio- film but also for dispersion (50). Attachment is mediated by surface-associated structures, which include both polysaccha- rides and proteins classified as fimbrial and afimbrial adhesins, depending on the structure to which they contribute. Fimbrial adhesins form filamentous structures, while afimbrial adhesins produce projections on the outer membrane (23). X. fastidiosa, a Gram-negative phytopathogen that grows as a biofilms in both plant xylem vessels and the cybarium of insect vectors, is a major threat to plant production around the world. In Brazil, it has a major economic impact on citriculture since it causes citrus variegated chlorosis disease (CVC) (35, 39, 42). The biofilm formed by X. fastidiosa blocks the xylem vessels of susceptible citrus plants, impairing water flow. This blockage leads to a drastic reduction in fruit size (32) and, * Corresponding author. Mailing address: Centro APTA Citros Syl- vio Moreira/IAC, Rodovia Anhanguera Km 158, Cordeiro ´polis SP, Brazil 13490-970. Phone and fax: 55-19-35461399. E-mail: alessandra @centrodecitricultura.br. Published ahead of print on 14 May 2010. 4250 on March 15, 2018 by guest http://aem.asm.org/ Downloaded from

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Page 1: Expression of Xylella fastidiosa Fimbrial and Afimbrial Proteins

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 2010, p. 4250–4259 Vol. 76, No. 130099-2240/10/$12.00 doi:10.1128/AEM.02114-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Expression of Xylella fastidiosa Fimbrial and AfimbrialProteins during Biofilm Formation�

R. Caserta,1,2 M. A. Takita,1 M. L. Targon,1 L. K. Rosselli-Murai,2 A. P. de Souza,2 L. Peroni,3D. R. Stach-Machado,3 A. Andrade,4 C. A. Labate,4 E. W. Kitajima,5

M. A. Machado,1 and A. A. de Souza1*Centro APTA Citros Sylvio Moreira/IAC, Rodovia Anhanguera Km 158, Cordeiropolis SP, Brazil 13490-9701; Universidade Estadual de

Campinas/UNICAMP, Centro de Biologia Molecular e Engenharia Genetica, Departamento de Genetica e Evolucao,Instituto de Biologia, P.O. Box 6010, Campinas SP, Brazil 13083-9702; Universidade Estadual de Campinas/UNICAMP,

Laboratorio de Imunologia Aplicada, Departamento de Microbiologia e Imunologia, Rua Monteiro Lobato s/n, Campinas SP,Brazil 13083-9703; Escola Superior de Agricultura “Luiz de Queiroz”/USP, Laboratorio Max Feffer de Genetica de Plantas,

Departamento de Genetica, P.O. Box 83, Piracicaba SP, Brazil 13400-9704; and Escola Superior deAgricultura “Luiz de Queiroz”/USP, Nucleo de Apoio a Pesquisa em

Microscopia Eletronica Aplicada a Pesquisa Agropecuaria (NAP/MEPA),Piracicaba SP, Brazil 13418-9005

Received 9 September 2009/Accepted 4 May 2010

Complete sequencing of the Xylella fastidiosa genome revealed characteristics that have not been describedpreviously for a phytopathogen. One characteristic of this genome was the abundance of genes encodingproteins with adhesion functions related to biofilm formation, an essential step for colonization of a plant hostor an insect vector. We examined four of the proteins belonging to this class encoded by genes in the genomeof X. fastidiosa: the PilA2 and PilC fimbrial proteins, which are components of the type IV pili, and XadA1 andXadA2, which are afimbrial adhesins. Polyclonal antibodies were raised against these four proteins, and theirbehavior during biofilm development was assessed by Western blotting and immunofluorescence assays. Inaddition, immunogold electron microscopy was used to detect these proteins in bacteria present in xylemvessels of three different hosts (citrus, periwinkle, and hibiscus). We verified that these proteins are present inX. fastidiosa biofilms but have differential regulation since the amounts varied temporally during biofilmformation, as well as spatially within the biofilms. The proteins were also detected in bacteria colonizing thexylem vessels of infected plants.

Aggregative growth is a common feature in the microbialworld, and its discovery radically changed our concept of mi-crobial growth dynamics. A cellular aggregate adhering to asurface is known as a biofilm. It has important characteristics,such as greater resistance to antimicrobial compounds (34, 54),increased capacity of the cells to take up nutrients from theenvironment (59), and higher detoxification efficiency resultingfrom an increase in expression of genes encoding efflux pumps(43). These characteristics give the biofilm cells a great adap-tive advantage.

Biofilm growth also confers advantages to plant pathogensby promoting virulence and protection against plant defenseresponses. Bacteria can colonize different niches in the plant,from aerial surfaces to roots and the vascular system, andbiofilm formation can play a role at all of these sites of colo-nization. In the vessels, biofilms are very important since thecells need to survive in a competitive habitat where plant de-fense compounds are produced in response to infection (7).

Biofilm development is divided into at least the followingfive phases: (i) reversible attachment, (ii) irreversible attach-ment, (iii) beginning of maturation, (iv) mature biofilm, and

(v) dispersion (13, 50). In Xylella fastidiosa strain 9a5c, thematuration phase occurs between days 15 and 20 in vitro, whiledispersion occurs between days 25 and 30, as observed by ouranalysis of biofilm formation using different methods, includ-ing scanning electron microscopy and quantification of exopo-lysaccharides, biomass, and the total protein (unpublisheddata). The establishment and development of biofilms of plant-colonizing bacteria share several features with the establish-ment and development of biofilms of human bacterial patho-gens, such as regulation by quorum sensing, nutrient starvationregulation, and phase variation. Motility is also an importantfactor not only for the initiation and development of the bio-film but also for dispersion (50). Attachment is mediated bysurface-associated structures, which include both polysaccha-rides and proteins classified as fimbrial and afimbrial adhesins,depending on the structure to which they contribute. Fimbrialadhesins form filamentous structures, while afimbrial adhesinsproduce projections on the outer membrane (23).

X. fastidiosa, a Gram-negative phytopathogen that grows asa biofilms in both plant xylem vessels and the cybarium ofinsect vectors, is a major threat to plant production around theworld. In Brazil, it has a major economic impact on citriculturesince it causes citrus variegated chlorosis disease (CVC) (35,39, 42). The biofilm formed by X. fastidiosa blocks the xylemvessels of susceptible citrus plants, impairing water flow. Thisblockage leads to a drastic reduction in fruit size (32) and,

* Corresponding author. Mailing address: Centro APTA Citros Syl-vio Moreira/IAC, Rodovia Anhanguera Km 158, Cordeiropolis SP,Brazil 13490-970. Phone and fax: 55-19-35461399. E-mail: [email protected].

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consequently, severe economic losses resulting from reducedplant productivity (4).

Due to the economic damage caused by CVC, there hasbeen a major effort to generate more information about itsbiology. This led to sequencing of the genome of the pathogen.The X. fastidiosa genome harbors a wide variety of genes en-coding adhesins (53). Bacterial cell surface adhesins are im-portant in the initial phases of adherence to surfaces, as well asin bacterium-bacterium interactions and microcolony develop-ment (15). Insight into X. fastidiosa has also come from ge-nome analysis of a strain of X. fastidiosa which causes Pierce’sdisease of grape (58). Studies of this strain showed that thecellular aggregation process involves type I and type IV fim-brial adhesins. The two types of fimbriae present differentadhesion forces that help bacteria adhere to a substrate (10,30). Adhesion proteins have also been demonstrated to medi-ate adherence to carbohydrates of leafhopper foregut surfaces(27). In addition, both fimbrial and afimbrial adhesins areimportant for plant pathogenicity (38, 41). However, the ex-pression of these proteins during X. fastidiosa biofilm forma-tion either in vitro or in planta is still poorly understood. For X.fastidiosa strains causing CVC, nothing is known about the roleof these proteins in pathogenicity or biofilm formation, al-though some adhesion-encoding genes, such as pilA2, pilC,xadA1, and xadA2, were found to be upregulated either invirulent strains of X. fastidiosa or during biofilm formation (12,14). These results suggest that the biofilm mode of growth isimportant for successful colonization of the citrus host by X.fastidiosa strains that cause CVC. In this work we focused onthe temporal expression of the PilA2 and PilC fimbrial proteinsand XadA1 and XadA2, which are afimbrial adhesins, duringin vitro development of X. fastidiosa CVC biofilms. We dem-onstrated that the temporal and spatial patterns of expressionof the fimbrial and afimbrial adhesins are very different duringbiofilm development in vitro. Moreover, we also verified thatthese adhesins are present in X. fastidiosa cells in symptomaticplants of three different hosts (citrus, periwinkle, and hibiscus).

MATERIALS AND METHODS

X. fastidiosa strain and in vitro growth conditions. X. fastidiosa subsp. paucastrain 9a5c (51), for which a genome sequence is available and which wasobtained from INRA (Institut National de La Recherche Agronomique, Bor-deaux, France), was used in all studies. Bacteria were extracted from petioles ofsymptomatic plants that had been ground in phosphate-buffered saline (PBS),and the suspensions were spread on periwinkle wilt (PW) medium (8). The firstcolonies were observed between days 10 and 15 after inoculation, and cells wereinoculated into 50 ml of PW medium and grown at 130 rpm and 28°C. Thecultures were transferred weekly to fresh medium (1 week corresponded to onepassage) and used to obtain both biofilm and planktonic cells. Biofilms wererecovered from passages 1 (7 days, 14 generations) to 8 (56 days, 112 genera-tions). Planktonic cells which did not form a biofilm were collected after thepassage 18 (126 days, 252 generations) in PW medium and were used after 10days of growth. For X. fastidiosa strain 9a5c, the doubling time was 12 h.

Construction of expression vectors. In order to amplify highly antigenic re-gions of the target proteins for antibody production, primers were designed forflanking coding sequences having hydrophilic and antigenic regions (Lasergene99; DNASTAR). More than one pair of primers were designed for differentantigenic regions of the fimbrial proteins PilA2 (Tfp pilus assembly protein,major pilin FimA/PilA [type IV fimbrillin]) (Bioinformatics Laboratory openreading frame identification number [LBI ORF ID] XF2539) and PilC (type IVfimbrial assembly protein PilC) (LBI ORF ID XF2538) and the afimbrial pro-teins XadA2 (surface protein adhesin YadA-like) (LBI ORF ID XF1529) andXadA1 (surface protein adhesin YadA-like) (LBI ORF ID XF1516). Theseproteins were originally annotated FimA, PilC, Hsf, and UspA1 but later were

reannotated as indicated above; the current accepted annotation is available atwww.xylella.lncc.br. EcoRI and HindIII sites were placed in the 5� and 3� regionsof each primer for directional cloning in pET28a (Novagen). The Primer Selectsoftware from Lasergene99 (DNASTAR) was used to design the primers. Theprimer sequences are shown in Table 1.

Each 25-�l PCR mixture used to obtain amplicons contained 100 ng of total9a5c DNA, 50 ng of each primer (forward and reverse), 2.5 mM deoxynucleosidetriphosphates (dNTP), 1.25 �l of 50 mM MgSO4, 2.5 �l of 10� buffer (Invitro-gen), 1 U of Taq Platinum high-fidelity polymerase (Invitrogen), and Milli-QH2O. The amplification program was one cycle at 94°C for 3 min, followed by 35cycles of 55°C for 1 min, 72°C for 1.5 min, and 94°C for 1 min and then 10 minat 72°C for further extension. The amplified fragments were analyzed in a 1%agarose gel stained with ethidium bromide and were visualized under UV light.The fragments were excised from the gel and purified using a GFX PCR DNAand gel band purification kit (Amersham Biosciences). The amplicons werecloned in the pGEM-T vector (Promega). Escherichia coli DH5� competent cellswere transformed with the constructs, and the transformants were grown in LBcontaining 100 �g/ml of ampicillin.

The recombinant plasmids were sequenced with the two primers used foramplification of the target genes and primers for the pGEM-T vector (T7 andSP6 promoter primers). For sequencing the xadA1-1 and xadA1-2 fragments (1.5and 2.0 kb, respectively), internal primers were also used in order to obtain thecomplete nucleotide sequences. The reaction mixtures were prepared by follow-ing the Applied Biosystems instructions for a BigDye Terminator cycle sequenc-ing Ready Reaction DNA sequencing kit (v 3.0), and the resulting DNAs wererun in an ABI 3730 automatic sequencer (Applied Biosystems). The quality of acloned DNA fragment was confirmed by performing similarity searches againstthe X. fastidiosa database using BlastN and BlastX (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi) tools. Plasmids containing sequences that exhibited100% identity with the expected nucleotide sequences were cleaved with EcoRIand HindIII (Invitrogen) for directional cloning in pET28a using T4 DNA ligase(Invitrogen). pET28a was designed for expression of the recombinant proteinfused to a six-His tag in both the N- and C-terminal regions of the protein. E. coliRosetta competent cells were transformed with the recombinant plasmids, andselection was performed in LB plates containing 100 �g/ml of kanamycin. PCRcolony screening was used to exclude false positives.

Expression of the target proteins and purification. The target proteins wereinduced using 1 mM isopropyl-�-D-thiogalactopyranoside (IPTG) in a 100-mlculture incubated for 2 h at 37°C. For the proteins that appeared in the insolublephase, a lower temperature (25°C) was used for induction. The cells were col-lected by centrifugation at 4,000 � g for 20 min at 8°C, suspended in 1 ml ofbuffer 1 (50 mM Tris, 300 mM NaCl; pH 7.5) containing 1 mg/ml lysozyme and1 mM phenylmethylsulfonyl fluoride (PMSF), and lysed by sonication on ice withan ultrasonic cell disruptor (Unique). The cell debris and supernatant wereseparated by centrifugation at 12,000 rpm for 15 min at 8°C, and the expressionpattern for each sample was monitored by SDS-PAGE performed using a 12%or 15% separation gel under reducing conditions as described by Laemmli (29);the gel was stained with Coomassie brilliant blue R-250 and destained withmethanol-acetic acid.

Soluble proteins were purified using an immobilized metal affinity chromatog-raphy (IMAC) column packed with 1.0 ml of nickel-nitrilotriacetic acid (Ni-NTA) resin. Bound proteins were eluted with 4 ml of 200 mM imidazole in 50mM Tris (pH 7.5), 300 mM NaCl. Fractions were used to estimate the totalprotein concentration (Bradford assay) and were analyzed by SDS-PAGE.

Even at low induction temperatures PilC was found only in the insolublefraction of the extract. Therefore, this protein was cut out of the gel for immu-nization.

Identification of purified proteins by LC-MS/MS. After obtaining the targetproteins, we confirmed their identity by mass spectrometry analysis. For this,samples were excised from an SDS-PAGE gel and digested as described byFiorani Celedon et al. (16), using trypsin (Invitrogen), before sequence analysis.The peptide mixtures were identified by online liquid chromatography (LC)using a Cap-LC coupled to a quantitative time of flight (Q-TOF) Ultima APImass spectrometer (Waters). Five microliters of sample was loaded onto ananoease trapping column (0.18 by 23.5 mm; Waters) for preconcentration,which was followed by separation of peptides in a nanoease LC column (Sym-metry 300 C18 3.5 �m; 75 by 100 mm; Waters). Peptides were eluted using a60-min linear gradient of solvent B (95% [vol/vol] acetonitrile [ACN] and 0.1%[vol/vol] formic acid in water) at a flow rate of 250 nl min�1. Solvent A consistedof 5% (vol/vol) ACN and 0.1% (vol/vol) formic acid in water. The whole analysiswas performed using the positive-ion mode with a needle voltage of 3 kV. Themass range used was m/z 300 to 2,000, and tandem mass spectrometry (MS/MS)spectra were acquired for the most intense peaks (�15 counts). Multiple charged

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precursor ions were selected for fragmentation and peptide sequencing usingautomated data-dependent acquisition (DDA) MassLynx software (Waters),switching from the MS mode to the MS/MS mode and then returning to the MSmode. The resulting fragmented spectra were processed using the ProteinLynxv4.0 software (Waters). MASCOT MS/MS Ion Search (Matrix Science) was usedto blast the sequences against the Swiss-Prot and NCBI nr databases. CombinedMS-MS/MS searches were conducted with a parent ion mass tolerance of 50ppm, an MS/MS mass tolerance of 0.1 Da, carbamidomethylation of cysteine(fixed modification), and methionine oxidation (variable modification). Accord-ing to MASCOT probability analysis, only significant (P � 0.05) hits wereaccepted.

Antibody production. Antibodies against the target proteins were obtainedusing New Zealand White rabbits. The purified proteins, as well as PilC (excisedfrom an SDS-PAGE gel), were individually mixed with Freund’s complete ad-juvant (Sigma) and injected into individual rabbits. Proteins mixed with Freund’sincomplete adjuvant were injected two more times, at 10 and 20 days after thefirst injection. The concentration of inoculated proteins was 150 �g. The qualityof the antibodies was tested by performing a direct enzyme-linked immunosor-bent assay (ELISA), using 3 �g of the target proteins as antigens and PBS as thenegative control. The antibody dilutions varied from 1:10,000 to 1:256,000. Theantibodies produced positive results even at a dilution of 1:250,000, and nocross-reaction was observed with any of the other proteins used for antibodyproduction.

Extraction of total protein for X. fastidiosa biofilm growth phases. Threereplicate experiments were carried out for extraction of biofilm proteins. Cellsgrown in PW medium until the passage 8 were transferred to flasks containing 50ml of medium and grown under the conditions described above. The biofilms thatattached to the glass at the medium-air interface were collected after 3, 5, 10, 15,20, and 30 days of growth, which corresponded to the different biofilm develop-mental phases (13), and total protein was extracted. To do this, the medium wasdiscarded, and 1 ml of wash buffer (50 mM Tris [pH 8.0], 25 mM NaCl, 5 mMEDTA [pH 8.0], 2% Triton X-100) was used to collect biofilm cells. Lysozymeand PMSF (1 mM each) were added to the extract, and the samples were kept

on ice for 20 min. Cells were lysed by sonication, which was followed by centrif-ugation at 10,000 � g for 10 min. The supernatant and the pellet were used forprotein analysis. For planktonic conditions, the cells were obtained after passage18, when no biofilm was observed at the medium-air interface, and were collectedby centrifugation at 6,000 � g for 5 min. Total protein was extracted by treatingthe cells as described above for the biofilms. For all of the experiments, theamount of total protein obtained from each biofilm phase was normalized beforethe gel was loaded, using a bovine serum albumin (BSA) standard curve forcomparison, for which R2 was 0.956.

Western blot assays. X. fastidiosa proteins from different biofilm developmen-tal phases and planktonic growth were quantified using the procedure describedby Lowry et al. (31). For Western blot assays, for each of the growth conditions6 �g of proteins was separated by 9% or 12% SDS-PAGE, depending on theprotein size. Individual Western blot experiments were performed for each of theantibodies tested, and preimmune rabbit antiserum was used to verify that therewas a possible nonspecific cross-reaction. After electrophoresis, gels were incu-bated in Tris-glycine buffer (48 mM Tris, 39 mM glycine, 0.04% SDS, 20%methanol) for 1 h, and the proteins were transferred to a nitrocellulose mem-brane (Hybond-C Ultra; Amersham) according to the specifications of the man-ufacturer (Pharmacia) for the Multiphor II Novablot electrophoretic transferunit. Membranes were treated by following Amersham’s Hybond-C nitrocellu-lose membrane protocol, using 1� TBS (150 mM NaCl, 10 mM Tris-HCl; pH8.0), a 16-h blocking period, and incubation with antibodies at a 1:10,000 dilutionfor 6 h. The membranes were incubated with a 1:10,000 dilution of goat anti-rabbit alkaline phosphatase conjugate (Sigma) for 1 h at 37°C and washed threetimes with 1� TBS, 0.1% Tween 20. For detection of the proteins, the mem-branes were incubated in a freshly prepared substrate solution (0.1 M Tris-HCl[pH 9.5], 0.1 M NaCl, 5 mM MgCl2, 2 mg 5-bromo-4-chloro-3-indolylphosphate,4 mg Nitro Blue Tetrazolium). When the desired staining was observed, thereaction was stopped by washing the membranes in distilled water. At least threemembranes were tested in each biological experiment.

Signal intensity was quantified using Image J 1.42q (http://rsb.info.nih.gov/ij)software. The values were used for statistical analyses (P � 0.05, t test).

TABLE 1. Sequences of primers used to amplify target genes

Fragment Primera Primer sequence (5�–3�) Fragment size (bp)

pilC-1 eco_for (F) GAATTCTGGGGCACGAGCTTTTTAGAT 568rev_hind4 (R) AAGCTTCAGCCCTGACCAGATTGCGATAA

pilC-2 eco_for (F) GAATTCTGGGGCACGAGCTTTTTAGAT 646hind_rev1 (R) AAGCTTTTCCCTTCAGAGCCTCAATGTTC

pilC-3 eco_for5 (F) GAATTCGAAGAGCGCTTATGGGTGTGG 586hind_rev5 (R) AAGCTTCAGCCCTGACCAGATTGCGATAAAG

xadA1-1 f_eco2 (F) GAATTCCAAGGCGTCGACTCGGTTGCTCTA 1.564 � 103

r_hind1 (R) AAGCTTTCGCCGAAATGCTGACACCACTT

xadA1-2 feco_long5 (F) GAATTCGTGGCGGCATCGGTGAAG 2.056 � 103

rhind_in4 (R) AAGCTTTAGCCGCCGCCATACTGTTA

pilA2-1 f_eco (F) CGAATTCAAAGTTAAGGGCAACTCTCAAGT 148rev_hind (R) TAAGCTTTTATTTAGAGGCAATGCATCCAGAAGGT

pilA2-2 f2_eco (F) GGAATTCAATTATGTCGCCAGGTCCCAACT 450rev_hind (R) TAAGCTTTTATTTAGAGGCAATGCATCCAGAAGGT

pilA2-3 f_eco_int (F) CGAATTCATGAAGAAGCAACAAGGTTTTA 520rev_hind (R) TAAGCTTTTATTTAGAGGCAATGCATCCAGAAGGT

xadA2-7 f1_eco (F) TGAATTCGCGGGCAGCAAGGTGATTAGC 882R_hind (R) CAAGCTTAGCGTTCACCCCTTATC

xadA2-8 f_ecolong (F) GAATTCGCGGGTGCAGTGTCA 1.6 � 103

r1_hind (R) CAAGCTTAATTCCCACCTCAATACATCC

xadA2-9 f1_eco (F) TGAATTCGCGGGCAGCAAGGTGATTAGC 1 � 103

r1_hind (R) CAAGCTTAATTCCCACCTCAATACATCC

a F, forward; R, reverse.

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Immunofluorescence of the target proteins in X. fastidiosa biofilms. For theimmunofluorescence analysis, biofilms were obtained using cover glasses placedin the bottom of 24-well Nunclon Delta SI Multidishes (Nunc) containing 1 mlof liquid PW medium in each well. One plate was used for each time point, and100 �l of a preinoculum containing X. fastidiosa 9a5c (optical density, 0.1) wasadded to four different wells. The culture plates were maintained statically at28°C and were analyzed after 3, 5, 10, 15, 20, and 30 days of growth. The PWmedium was gently removed from the wells with a pipette, and the biofilmadhering to cover glasses was washed as described by Roper et al. (49) butwithout fixation of the cells since the aim of this work was to evaluate just theadhering cells. The antibodies and the preimmune sera were used individually ina dilution of 1:1,000. For localization of proteins in the biofilms that formed, weused 1 ml of goat anti-rabbit rhodamine-conjugated IgG (Santa Cruz Biotechnl-ogy) at a 1:10,000 dilution in PBS. The biofilm cells were stained with Syto 9diluted 1:600 in autoclaved Milli-Q H2O. Images were obtained using an oilimmersion lens (numerical aperture, 1.0) with an Olympus UIS2 fluorescence mi-croscope; for Syto 9, a filter that permits excitation at wavelengths between 460 and490 nm and emission at wavelengths between 500 and 520 nm was used, and forrhodamine, a filter that allows excitation at wavelengths between 510 and 550 nmand emission at wavelengths between 570 and 590 nm was used. To verify that apossible nonspecific fluorescent signal was present, two negative controls were pre-pared from biofilms after 30 days of growth, one without secondary rhodamine-conjugated antibodies and the other without incubation with primary antibody.

Immunogold transmission electron microscopy of symptomatic plants. Smallfragments of petioles from citrus (sweet orange; Citrus sinensis cv. Pera), peri-winkle (Catharantus roseus), and hibiscus (Hibiscus schizopetalus) plants infectedby X. fastidiosa were fixed in a modified Karnovksy solution (2% paraformalde-hyde and 2.5% glutaraldehyde in 0.05 M cacodylate buffer [pH 7.2] with 0.001 MCaCl2) for 1 to 2 h. The plants were naturally infected with X. fastidiosa, whosepresence was confirmed by previous PCR assays. The citrus plants exhibitedcharacteristic symptoms of CVC, while the periwinkle plants exhibited generalchlorosis and stunting and the hibiscus plants had leaf scald symptoms (28).Fixed tissues were dehydrated using an ethanol series (30%, 50%, 70%, and 90%ethanol for about 10 min each) and then transferred to chilled 100% ethanol.Dehydrated tissues were infiltrated for 6 h with a mixture of 100% ethanol andLR White resin (1:1) at 4°C and overnight with pure LR White at 4°C. Infiltratedtissues were then transferred to size 3 gelatin capsules filled with pure LR Whiteand allowed to polymerize at 60°C for 2 days. Embedded tissues were cut intothin sections with a Leica UC 6 ultramicrotome equipped with a Diatomediamond knife, and the sections were mounted in 100-mesh nickel grids coveredwith Formvar films. Immunolocalization assays with antibodies against the targetproteins were performed using the protocol of Vandenbosch (57). As controls forthe experiment, images of noninfected xylem vessels of infected plants, the wallsof infected xylem vessels, and parenchyma were evaluated and compared. Thebackground was reduced by adjusting the antibody dilution to 1:250. About 40immunogold tests for each plant with at least 80 sections per test were done,which generated a minimum of 20 images for each protein in each plant speciesin the experiment. Transmission electron microscopic examination was doneusing a Zeiss EM 900 electron microscope at 80 kV, and the images wereregistered digitally with an F-view charge-coupled-device (CCD) monochromecamera (Soft Imaging System).

RESULTS

Protein purification, polyclonal antibodies, and Westernblot analysis. Clones containing recombinant plasmids pro-duced sufficient amounts of the proteins of interest for furtherpurification. The PilA2, XadA2, and XadA1 recombinant pro-teins were in the soluble fraction of the protein extracts. Incontrast, PilC was in only the insoluble fraction, even when lowinduction temperatures were used. The fractions containingPilA2, XadA2, and XadA1 were purified by IMAC, and it waspossible to recover large amounts of purified proteins afterelution with imidazole. For PilC, we excised the band corre-sponding to the target protein directly from a polyacrylamidegel. Induction and purification were monitored by SDS-PAGE(data not shown). The identities of the purified proteins wereconfirmed by LC-MS/MS (data not shown). The proteins wereinoculated into rabbits for antibody production.

The amounts of the fimbrial and afimbrial proteins in bio-films were verified by Western blotting using cells obtainedafter 3, 5, 10, 15, 20, and 30 days of growth (Fig. 1), whichcorresponded to the initial adhesion of the cells to the sub-strate (3 and 5 days), microcolony formation (10 days), earlydevelopment of the biofilm architecture (15 days), mature bio-film (20 days), and the biofilm after some apparent dispersionhad occurred (30 days) (unpublished data). Analysis of PilA2,PilC, and XadA2 revealed that these proteins had apparentmolecular masses of approximately 15 kDa, 55 kDa, and 200kDa, respectively. These masses are in accordance with thevalues expected for these proteins (http://www.lbi.ic.unicamp.br/xf/). For XadA1, Western blotting revealed a band at ap-proximately 70 kDa (Fig. 1D); the predicted molecular mass ofXadA1 from X. fastidiosa 9a5c is 98 kDa. Since the possibilityof contamination was excluded by the mass spectrometry anal-ysis (Table 2), there was apparently cleavage of the protein orits genome annotation is wrong.

The temporal patterns of abundance of the two fimbrialproteins (PilA2 and PilC) were similar, and statistically signif-icant differences between the initial and later phases of biofilmformation were observed (Fig. 1A and B). The patterns ofabundance for the afimbrial proteins were different from eachother in Western blot analyses. Very little XadA2 protein wasobserved at the beginning of biofilm formation. By 10 days, theamount of this protein had increased, but a statistically signif-icant increase compared with the amount in young biofilm cellswas observed only after 30 days of growth (Fig. 1C). XadA1was produced rather constitutively, and there was no statisti-cally significant change in the amount of the XadA1 protein atany biofilm phase (Fig. 1D).

To verify that these adhesins are associated with biofilmgrowth, we compared their abundance with that in planktoniccells. Under our experimental conditions, none of the adhesinsevaluated was detected in planktonic X. fastidiosa cells (datanot shown).

Immunofluorescence of adhesion proteins in biofilms grownin vitro. In order to localize the adhesion proteins during X.fastidiosa biofilm formation in vitro, we immunolabeled theproteins and visualized them by using fluorescence microscopy.The PilA2 and PilC fimbrial proteins were apparent through-out a biofilm by day 3. However, by days 20 and 30, theseproteins seemed to be more concentrated in some parts of thebiofilm than in other parts (Fig. 2 and 3). The XadA2 afimbrialprotein was not observed in small cell aggregates on day 3 ofgrowth but was seen by day 5, when cellular aggregates were alittle larger. By day 10, when the biofilm began to exhibit amultilayer organization, this protein was quite evident, suggest-ing that it may have a role in cell-cell adhesion and three-dimensional structuring (Fig. 4). The biofilm distribution ofXadA1 was quite different from that of the other proteins (Fig.5). At day 10, when biofilm maturation had started, this proteinwas present primarily in gaps in the biofilm structure. At days15 and 20, when the biofilm was thickest, the localization ofthis protein was somewhat different; it was located primarily indiscrete spots, forming an “island-like” pattern in the biofilm.No other protein exhibited this pattern. The XadA1 proteinremained localized in “island-like” inclusions on day 30, eventhough the spots were less numerous than they were in earlier

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phases of growth and the labeling seemed not to be as tightlylinked to the biofilm (Fig. 5).

Localization of adhesion proteins in different host plantsinfected with X. fastidiosa. To verify the localization of theadhesion bacterial proteins in vivo, we analyzed X. fastidiosacells present in sections of xylem vessels of infected periwinkle,hibiscus, and citrus leaves exhibiting disease symptoms. PilA2is one of the rod-forming units of the type IV pili, and immu-nogold labeling demonstrated that it was present in cell mem-branes and outside the cells in all of the hosts tested (Fig. 6A,B, and C). The PilC protein, which is involved in pilus assem-bly, was also detected in the three hosts tested, but it wasmainly in the bacterial cell membrane (Fig. 6D, E, and F). Incontrast, the afimbrial proteins detected were outside the cells.

XadA2 was outside the cells but close to the outer membraneof cells in the three plant hosts (Fig. 7A, B, and C). The highlevel of background labeling for XadA1 in periwinkle did notallow analysis of this protein in this plant, but there was littlelabeling of host materials in hibiscus and citrus, which allowedXadA1 to be visualized in these species. Similar to the findingsfor XadA2, most of this protein was observed to be close to theouter membrane, but some labeling was also observed at moredistant sites (Fig. 7D, E, and F).

DISCUSSION

Hierarchically ordered gene expression circuits are charac-teristic of biofilms (40). In the present work we demonstrated

FIG. 1. Analysis of proteins in different biofilm growth phases. Total protein was isolated from biofilm cells, normalized by using Lowry quantification,and evaluated by Western blotting using (A) anti-PilA, (B) anti-PilC, (C) anti-XadA2, and (D) anti-XadA1 antibodies. The bands were quantified usingImageJ software. The bars indicate the averages for three independent experiments. The error bars indicate the standard errors of the means.

TABLE 2. XadA1 sequencing by quantitative time of flight electrospray ionization analysisa

Peptide matched to XadA1 Observed Mr Avg Mr (exptl) Avg Mr (calculated) Concn (ppm) Score

R.ATANAIGSSVLGVDSR.A 759.3655 1,516.7164 1,516.7845 �44.89 82R.TYEANVLSIGSGNGR.G 769.3839 1,536.7532 1,536.7532 0.02 39R.IVNVGDGIGNNDAVNK.S 799.9739 1,597.9332 1,597.8060 79.6 52K.SQLDGVTASVNDVAASVK.T 880.9922 1,759.9698 1,759.8952 42.4 84K.SQLDGVTASVNDVVASVK.N 894.9738 1,787.9330 1,787.9265 3.65 97K.VVSGVAVSDSSVAANAQVLSK.G 994.5337 1,987.0528 1,987.0586 �2.89 56

a For surface-exposed outer membrane protein XF1516 (imported) of X. fastidiosa the accession number is C826721, the score is 405, the Mr is 98,281, the isoelectricpoint is 7.17, the coverage is 10%, and the number of peptides matched is 6.

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that adhesion proteins are expressed temporally throughoutbiofilm development in an orderly pattern. X. fastidiosa biofilmdevelopment can be divided into five different phases, includ-ing initial attachment, microcolony formation, beginning ofmaturation, mature biofilm, and dispersion (13). Growth ofbiofilms is a well-known behavior in bacteria, and in X. fastid-iosa it is necessary for development of symptoms due to occlu-sion of the xylem vessels (1, 41). A study of the Pierce’s diseaseX. fastidiosa Temecula strain suggested that its colonization ofvessels is related to its adhesion capacity and twitching motility,which are mediated by adhesion proteins (9).

Type IV pili are important for adhesion and twitching mo-

tility in bacteria (2, 11, 18, 30, 36, 44, 46). As expected, thetemporal patterns of variations in the amounts of PilA2 andPilC were somewhat similar during X. fastidiosa biofilm forma-tion since these proteins are involved in type IV pilus assembly.Larger amounts were observed at the beginning of develop-ment and the dispersion phase, when cells might be expectedto be more motile. Thus, type IV pili must have different rolesduring biofilm development, and adhesion to the surface andtwitching motility are associated with biofilm spread early indevelopment. The observation that the PilA2 and PilC proteinswere present in spots in the biofilms is intriguing and suggeststhat labeled cells may be released from a biofilm not only asindividual cells but also as clusters. Twitching may take placevery early in biofilm development and may be necessary for theformation of macrocolonies. It is known that twitching motilityallows existing microcolonies to join and form macrocoloniesthat develop into a mature biofilm (56).

Immunogold electron microscopy of the fimbrial proteins inperiwinkle, hibiscus, and citrus plants revealed that they were

FIG. 2. Fluorescence labeling of PilA in X. fastidiosa biofilmsgrown on glass slides. Cells in different growth phases of a biofilm werestained using the green nucleic acid dye Syto 9, and the red spotscorrespond to antibody recognition of PilA (secondary antibody la-beled with rhodamine). Cells were visualized with an oil immersionlens (numerical aperture, 1.0) using an Olympus UIS2 fluorescencemicroscopy; for Syto 9, excitation wavelengths between 460 and 490nm and emission wavelengths between 500 and 520 nm were used, andfor rhodamine, excitation wavelengths between 510 and 550 nm andemission wavelengths between 570 and 590 nm were used. The imagesin the column on the left show PilA labeling, and X. fastidiosa cells arered. The images in the column in the middle show Syto 9 staining, andX. fastidiosa cells are green. The images in the column on the right aresuperposed images for the two channels (green and red). The presenceof PilA is indicated by red or orange in small cell clusters and in thebiofilm structure in the column on the right. Bars � 10 �m.

FIG. 3. Fluorescence labeling of PilC in X. fastidiosa biofilmsgrown on glass slides. Cells were stained with Syto 9, labeled with ananti-PilC antibody, and visualized with the fluorescence microscope.The images in the column on the left show immunolabeling of PilC,and the images in the column in the middle show the green channel.The images in the column on the right are superposed images for thetwo channels (green and red). Bars � 10 �m.

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located in the same subcellular compartment of the X. fastid-iosa biofilms. PilA2 was located in the cell membrane, as wellas outside the cell, while PilC was preferentially close to themembrane. This observation is in agreement with the expectedlocalization for anchoring of immature pilus subunits and as-sembly of these subunits (24, 25). The small amount of PilA2observed was not unexpected since assembly of the rod couldblock the epitopes of this protein, so that only the epitopes ofproteins that are in the initial steps of assembly of the pili areavailable for interaction with the antibody (24).

The afimbrial protein XadA1 was detected in all phases ofbiofilm development, and XadA2 was detected mainly in laterphases of biofilm development. Western blot analysis ofXadA1 revealed a protein that was smaller than expected. Thisprotein is very similar to UspA1 of Moraxella catarrhalis, forwhich a reduction in size was reported previously; this reduc-tion in size was also dependent on the temperature to whichthe protein was subjected (21, 37). The discrepancy betweenthe expected and observed sizes of XadA1 could be due to thesame factors that apparently affect UspA1 processing. The

detection of XadA1 in all phases of biofilm formation suggeststhat it may have a role in the initial adhesion to surfaces, aswell as in cellular aggregation. UspA1 of M. catarrhalis is im-portant for adhesion to other cells and thus for biofilm forma-tion (45). It is noteworthy that XadA1 mutants of X. fastidiosaTemecula were reported to be defective in adhesion as singlecells to glass surfaces but that cell-cell aggregates could stillform. Thus, XadA1 is apparently involved in the initial adhe-sion of Temecula cells (15). In the mature biofilm phase,XadA1 is distributed in an “island-like” pattern, suggestingthat it may have a role in biofilm development by alteringcell-cell adhesion and aggregation. However, this hypothesisneeds to be investigated further.

The other afimbrial protein evaluated in this work, XadA2,was observed mainly only after 10 days, indicating that it islikely not involved in adherence to the substrate but instead isinvolved in cell-cell adhesion associated with higher cell den-

FIG. 4. Fluorescence labeling of XadA2 in X. fastidiosa biofilmsgrown on glass slides. Cells were stained with Syto 9, labeled with ananti-XadA2 antibody, and visualized with the fluorescence microscope.The images in the column on the left show immunolabeling of XadA2,and the images in the column in the middle show the green channel.The images in the column on the right are superposed images for thetwo channels (green and red). Bars � 10 �m.

FIG. 5. Fluorescence labeling of XadA1 in X. fastidiosa biofilmsgrown on glass slides. Cells were stained with Syto 9, labeled with ananti-XadA1 antibody, and visualized with the fluorescence microscope.The images in the column on the left show immunolabeling of XadA1,and the images in the column in the middle show the green channel.The images in the column on the right are superposed images for thetwo channels (green and red). The arrows indicate gaps in the biofilmat 10 days after inoculation, in which immunolabeling revealed thepresence of the XadA1 protein, as shown in panels H and I. Bars �10 �m.

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sity. A protein similar to XadA2 from Haemophilus influenzaeis known to have specific domains (Hia) that contribute toadherence to mammalian cells in vitro (6) and direct attach-ment to specific proteins (19, 55). These domains are presentin the X. fastidiosa protein (Fig. 8), and they could mediateprotein-protein interactions. XadA1 and XadA2 belong to thefamily of trimeric autotransporters, and thus they may haveadhesive activity that mediates bacterial interactions eitherwith host cells, which have their own extracellular matrix pro-teins, or with other unknown proteins (6, 20, 48). Even thoughthe X. fastidiosa XadA1 protein resembles UspA1 and YadA,two of the most-studied proteins of the family, it contains moreHep-Hag repetitive regions (Fig. 8). XadA2 exhibits similarityto Hsf of H. influenzae, but in X. fastidiosa there are many moredomains that are related to adhesive functions, such as theHep-Hag and hemagglutinin domains (Pfam domains PF05658and PF05662) (Fig. 8). Additionally, analysis of XadA2 re-vealed the presence of motifs that are unique to proteins from

X. fastidiosa (Pfam domain PF06669) (3) (Fig. 8). These struc-tural characteristics suggest that XadA1 and XadA2 may havedistinct functions in biofilm formation and host interactions.

These afimbrial adhesins may complement the role of otherafimbrial adhesins, such as the hemagglutinins. In X. fastidiosastrain Temecula, the hxfA gene encodes one of the hemagglu-tinins. A mutant with a mutation in this gene showed increasedvirulence and monolayer biofilm formation in vivo, suggestingthat the mutation allowed faster colonization of the xylemvessels, resulting in more severe symptoms (17). This observa-tion suggests that in X. fastidiosa Hxf may be related to aggre-gation and therefore biofilm growth may be a way to avoidrapid colonization of the host, which is undesirable for thepathogen since it could ultimately lead to the death of the planthost.

XadA1 and XadA2 immunogold labeling was detected closeto the cell membrane in a pattern similar to that reported forYadA of Yersinia enterocolitica, in which aggregation by theprotein head domain located outside the cell directed similarlocalization (22). However, some XadA1 was also detected farfrom the cells, and together with immunofluorescence obser-

FIG. 6. Immunogold electron microscopy of plant xylem vesselsinfected with X. fastidiosa. Sections were labeled with the anti-PilA2and anti-PilC antibodies and visualized by transmission electron mi-croscopy using a Zeiss EM 900 microscope. (A) X. fastidiosa cellslabeled with anti-PilA antibodies inside citrus vessels. (B) X. fastidiosacells labeled with anti-PilA antibodies inside periwinkle vessels. (C) X.fastidiosa cells labeled with anti-PilA antibodies inside hibiscus vessels.(D) X. fastidiosa cells labeled with anti-PilC antibodies inside citrusvessels. (E) X. fastidiosa cells labeled with anti-PilC antibodies insideperiwinkle vessels. (F) X. fastidiosa cells labeled with anti-PilC anti-bodies inside hibiscus vessels. The arrowheads indicate labeling ofPilA2 or PilC proteins.

FIG. 7. Immunogold electron microscopy of plant xylem vesselsinfected with X. fastidiosa. Portions of different plant hosts were sec-tioned, and the X. fastidiosa cells were labeled with either anti-XadA2or XadA1 antibodies. (A) Labeling of citrus vessels with the anti-XadA2 antibody. (B) Labeling of periwinkle vessels with the anti-XadA2 antibody. (C) Labeling of hibiscus vessels with the anti-XadA2antibody. (D) Labeling of citrus vessels with the anti-XadA1 antibody.(E and F) Labeling of hibiscus vessels with the anti-XadA1 antibody.The arrowheads indicate labeling of XadA2 or XadA1 proteins.

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vation of apparent protein aggregates far from cells, this sug-gests that this protein is secreted into the environment. Thishypothesis and the possible roles of such secreted proteinsneed to be investigated further.

Interestingly, the expression of most of the genes encodingthe proteins studied in this work during biofilm formation invitro (12) does not correlate well with the accumulation ofproteins at a given time that was detected by Western blotanalysis. Expression of xadA2, for instance, was detectedmainly on days 5 and 10 during biofilm formation in vitro, yetwe observed that the proteins were present mainly after day 10.This lag between gene expression and protein appearance isintriguing. It seems likely that different patterns of transcrip-tion, translation, and protein degradation occur during biofilmdevelopment. Translation could be affected by small regulatoryRNAs mediated by gacA. Activity of small RNAs related togacA has been demonstrated for Pseudomonas aeruginosa andalso for X. fastidiosa (26, 47, 52). In the latter case, regulationvia gacA directly affects xadA1 and xadA2 expression and thuscell-cell adhesion and biofilm formation (52). These smallRNAs could posttranscriptionally regulate the expression of avariety of proteins, including afimbrial adhesins, thus explain-ing the delay in the production of these adhesins during biofilmformation. The regulation of these genes could also be medi-ated through a diffusible signal factor (DSF)-dependent sig-naling system. Mutations in rpfF, which is responsible for thesynthesis of DSF in X. fastidiosa, impairs biofilm formation inan insect vector (42) and leads to greater virulence in plantsdue to an increased ability to move in the xylem vessels (5, 42).

Both fimbrial and afimbrial proteins are involved in biofilmformation in X. fastidiosa in vitro. The expression of theseadhesins seems to be substantially different at different phasesof biofilm development, suggesting that there are different

contributions to various biofilm processes. The variation in thespatial distribution pattern of these proteins in the biofilm alsosuggests that they contribute in different ways to biofilm func-tions. The detection of these proteins in xylem vessels of in-fected plants indicates that the biofilms in plants may resemblethose produced in vitro and that these structures likely contrib-ute to the infection process.

ACKNOWLEDGMENTS

We thank Steven Lindow for critical revision of the manuscript andRenato Salaroli for transmission electron microscopy technical sup-port.

This work was supported by research grants from Fundacao deAmparo a Pesquisa do Estado de Sao Paulo (FAPESP) and ConselhoNacional de Desenvolvimento Científico e Tecnologico (CNPq)(grants 04/14576-2, 06/52681-8 and INCT-Citros 08/57909-2, 573848/08-4). M.A.T., A.P.D.S., E.W.K., C.A.L., M.A.M., and A.A.D.S. wererecipients of research fellowships from CNPq.

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FIG. 8. Comparison of the XadA2 and XadA1 proteins. The struc-tures of the XadA2 proteins from the X. fastidiosa strains 9a5c (A) andTemecula (B) were compared with the structure of the Hsf proteinfrom H. influenzae (C), and the structures of the XadA1 proteins fromX. fastidiosa strains 9a5c (D) and Temecula (E) were compared withthe structures of UspA1 from M. catarrhalis (F) and YadA from Y.enterocolitica (G). All of the C termini are YadA-like domains; redboxes indicate HIM domains; light green boxes indicate Hep-Hagrepetitive regions; dark blue boxes indicate X. fastidiosa surface pro-tein-related motifs; and yellow boxes indicate domains with unknownfunctions.

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