FEMS Microbiology Letters 236 (2004) 313–318

www.fems-microbiology.org

A kinetic model for Xylella fastidiosa adhesion, biofilmformation, and virulence

Denise Osiro a,b, Luiz Alberto Colnago a,*, Alda Maria Machado Bueno Otoboni c,Eliana Gertrudes Macedo Lemos c, Alessandra Alves de Souza d,e,

Helv�ecio Della Coletta Filho d, Marcos Antonio Machado d

a Embrapa Instrumentac�~ao Agropecu�aria, Rua XV de Novembro 1452, S~ao Carlos SP 13560-970, Brazilb Instituto de Qu�ımica de S~ao Carlos, Universidade de S~ao Paulo, Avenida Trabalhador S~ao-carlense 400, S~ao Carlos, SP 13560-970, Brazil

c Departamento de Tecnologia, Faculdade de Ciencias Agr�arias e Veterin�arias de Jaboticabal, Universidade Estadual Paulista,

Via de Acesso Prof. Dr. Paulo Donato Castelane s/n, Km 5, Jaboticabal SP 14884-900, Brazild Centro APTA Citros ‘Sylvio Moreira’/Instituto Agronomico - CP 04, Cordeir�opolis SP 13490-970, Brazil

e Embrapa Recursos Gen�eticos e Biotecnologia, Parque Estac�~ao Biol�ogica W5 Norte Final, Bras�ılia, DF 70770-900, Brazil

Received 30 January 2004; received in revised form 23 April 2004; accepted 1 June 2004

First published online 11 June 2004

Abstract

Xylella fastidiosa is the causal agent of citrus variegated chlorosis and Pierce’s disease which are the major threat to the citrus and

wine industries. The most accepted hypothesis for Xf diseases affirms that it is a vascular occlusion caused by bacterial biofilm,

embedded in an extracellular translucent matrix that was deduced to be the exopolysaccharide fastidian. Fourier transform infrared

spectroscopy analysis demonstrated that virulent cells which form biofilm on glass have low fastidian content similar to the weak

virulent ones. This indicates that high amounts of fastidian are not necessary for adhesion. In this paper we propose a kinetic model

for X. fastidiosa adhesion, biofilm formation, and virulence based on electrostatic attraction between bacterial surface proteins and

xylem walls. Fastidian is involved in final biofilm formation and cation sequestration in dilute sap.

� 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Xylella fastidiosa; FTIR; Biofilm; Adhesion; Virulence

1. Introduction

The natural habitat for many bacterial species are

biofilm communities assembled at biotic and abiotic

surfaces [1,2]. Attachment of planktonic cells to a sub-stratum is the first and crucial step in all biofilm forma-

tion [1]. Adhesion and film formation on a substratum is

Abbreviations: Xf, Xylella fastidiosa; Xac, Xanthomonas axonopodis

pv. citri; FTIR, Fourier transform infrared spectroscopy; EPS,

exopolysaccharide; PD, Pierce’s disease; CVC, citrus variegated

chlorosis; PW, periwinkle wilt medium; XDM2, defined media for

Xf; SEM, scanning electron microscopy.* Corresponding author. Tel.: +55-16-2742477; fax: +55-16-2725958.

E-mail address: colnago@cnpdia.embrapa.br (L.A. Colnago).

0378-1097/$22.00 � 2004 Federation of European Microbiological Societies

doi:10.1016/j.femsle.2004.06.003

a spontaneous process observed not only with cells but

also in abiotic solutions [3]. Bacterial adhesion on sub-

strata is thought to be governed by physical–chemical

interactions [1], but it is a complicated process because

the cells can quickly adapt to different growth conditionsand substrata, changing their surface properties during

the adsorption process [1].

This fast adaptation process, which complicates ad-

hesion studies, is not observed in a fastidious bacterium

like Xylella fastidiosa (Xf), with a duplication time of

from 5 to 48 h [4]. The time necessary to adapt or change

its surface character is certainly much longer than the

adhesion time scale required for a good experimentaland theoretical model [1]. Xf is also an interesting

bacterium for adhesion studies because the genome of

several strains are known [6,7] and it is one of the

. Published by Elsevier B.V. All rights reserved.

314 D. Osiro et al. / FEMS Microbiology Letters 236 (2004) 313–318

smallest phytopathogens, with less than 3000 open-read

frames [4–6]. This simplicity reflects its limited habitat

within plant xylem and insect foregut [4–10] and, con-

sequently, range of adaptation processes, when com-

pared to bacteria that can live in open environments [1].The most accepted hypothesis for Xf diseases is that it

is caused by a vascular occlusion resulting from biofilm

formation and leading to water stress [4–10]. In symp-

tomatic plants, Xf is embedded in an extracellular

translucent matrix, was deduced to be its exopolysac-

charide (EPS). The EPS, named fastidian, has been

linked to the adhesion and biofilm formation that plays

a central role in xylem occlusion and pathogenicity [5,7].The present study analyzed the influence of growth

conditions on the concentration and acetylation degree

of fastidian in Xf cells, 9a5c (CVC), and Temecula (PD)

strains, as measured by Fourier transform infrared

(FTIR) spectroscopy. Based on these findings, we also

propose a kinetic model for Xf adhesion, biofilm for-

mation, and virulence.

2. Materials and methods

2.1. Growth conditions

The Xf 9a5c and Temecula strains were cultivated in

two different media: PW, the traditional medium for Xf

cultures [11], and XDM2 based on genome analysis [12].The cultures in PW were shaken at 28 �C for 20, 30, 40,

and 70 days and the cultures in XDM2 medium were

agitated at 28 �C for 10 days. After growth time, the

cultures were centrifuged at 4000g for 20 min. The pel-

lets were suspended in 0.5% NaCl aqueous solution and

centrifuged twice. The final pellets were lyophilized and

used in FTIR analysis.

To prepare fresh isolated bacteria, we infected youngCitrus sinensis plants (6 months) with 9a5c strain. Six

months later the cells were isolated from petioles and

stems of symptomatic plants. The first colonies were

observed between 10 and 15 days after inoculation. To

obtain cells from biofilms, individual colonies were

transferred to polypropylene tubes containing 3 mL of

PW broth. The tubes were vortexed and the inoculum

was transferred to flasks with a capacity of 1000 mL,containing 300 mL of PW broth. After 3 days of growth

at 28 �C in a rotary shaker at 120 rpm, a thin biofilm

formation attached to the glass was observed. However,

the biofilm was collected only after 20 days, when a

thicker layer of biofilm was observed. The biofilm layer

was scraped from the flasks, suspended in 0.5% NaCl

solution, centrifuged, and lyophilized.

The 9a5c strain that did not attach to the glass wasprepared by ten passages in PW broths. After this pro-

cedure the cells lost the capacity to form biofilm on the

glass surface, but small aggregates not attached to the

glass were observed at the bottom of the culture. The

cells were collected from the last culture after 5 days of

growth, suspended in 0.5% NaCl aqueous solution,

centrifuged, and lyophilized.

The Xanthomonas axonopodis pv. citri (Xac) strain306 was obtained by cultivating the cells in defined M9

medium [13] at 28 �C in a rotary shaker. The cells were

centrifuged after 24 h of culture, suspended in 0.5%

NaCl solution, and centrifuged. The pellets were ly-

ophilized and analyzed by FTIR.

2.2. FTIR analysis

The FTIR spectra were obtained with a Paragon

1000, Perkin–Elmer spectrometer (4000–400 cm�1, 4

cm�1 of spectral resolution, and 64 scans). The samples

were prepared as KBr pellets, using 2 mg of bacteria and

100 mg of KBr. The spectra were baseline and offset

corrected from 3000 to 2800 cm�1 and from 1800 to 900

cm�1 [14], and normalized by the intensity of the amide

II peak (intensity¼ 100) at 1550 cm�1, which is relatedto biomass [15]. To check dependence on growth con-

ditions [15,16], we analyzed three cultures for each ex-

perimental condition. The spectra for each culture were

obtained in triplicate (mean of nine spectra per experi-

mental condition) to minimize variation due to sample

thickness, non-uniformity, as well as incomplete baseline

correction [15,16]. Triplicate spectra for each sample

were used only when the error for intensities at 2930,1740, and 1180 cm�1 were within �2 of normalized

absorbance. The maximum standard error obtained,

including culture variability, was smaller than 7 units of

normalized absorbances.

3. Results and discussion

Fig. 1 shows some of the FTIR spectra of lyophi-

lized Xf, 9a5c, and Temecula strains and the related

bacterium X. axonopodis pv. citri (Xac), prepared un-

der several experimental conditions. The spectra from

3000 to 2800 cm�1 and from 1800 to 800 cm�1 were

normalized by a signal at 1550 cm�1, proportional to

biomass [15].

The four signals from 3000 to 2800 cm�1 (intensitymultiplied by 3) are assigned to the asymmetric and

symmetric stretch of methyl groups (2960 and 2875

cm�1) and asymmetric and symmetric stretch of meth-

ylene groups (2930 and 2850 cm�1). The signals from

methyl groups are due to their presence in aliphatic

amino acid side chains of proteins, in terminal carbon of

fatty acids from lipids, and in ester groups of polysac-

charides. The methylene signals are related to proteinsand polysaccharides (weak signals) but they are very

strong for fatty acid and can be used to monitor its

concentration in the sample [14–16].

Fig. 1. FTIR spectra of lyophilized Xf and Xac samples. The spectra

from 3000 to 2800 cm�1 (intensity multiplied by 3) and from 1800 to

800 cm�1 were normalized by the protein signal at 1550 cm�1. (a)

Spectrum of Xf sample, 9a5c, incubated in PW broth for 20 days. (b)

Spectrum of Xf, 9a5c, incubated for 70 days in PW broth. (c) Spectrum

of Xf, 9a5c, prepared as biofilm. (d) Spectrum of Xf, Temecula, in-

cubated in XDM2 medium for 10 days. (e) Spectrum of Xac incubated

in M9 broth for 24 h.

D. Osiro et al. / FEMS Microbiology Letters 236 (2004) 313–318 315

The weak signal at 1740 cm�1 is assigned to stretching

of the C@O bond found in fatty acids, phospholipids,lipopolysaccharides, and in the acetate group of xanthan

or fastidian [14–16]. The signal at 1650 cm�1 is assigned

to the amide I band of proteins [14–16], carboxylate

anions, and OH absorption of polysaccharides [14]. The

signals from 950 to 1150 cm�1 are assigned to stretching

of C–O of polysaccharide bonds [14].

Table 1 shows the intensity of the FTIR bands at

1080 (I1080), 1740 (I1740), and 2930 cm�1 (I2930), whichare proportional, respectively, to polysaccharides, and

Table 1

Mean values and standard error (SE) of the intensity of FTIR bands at

1080 (I1080), 1740 (I1740), and 2930 cm�1 (I2930), and the ratio

a ¼ 100� ðI1740=I2930Þ=I1080 calculated from the Xf and Xac samples

Samples I1080 I1740 I2930 a ratio

9a5c (PW) 20 days 33(2) 13(1) 24(2) 1.6

9a5c (PW) 30 days 34(3) 13(2) 23(3) 1.6

9a5c (PW) 40 days 41(3) 14(3) 20(2) 1.7

9a5c (PW) 70 days 148(7) 14(3) 17(3) 0.6

9a5c (XDM2) 10 days 38(2) 26(4) 33(2) 2.1

Temecula (XDM2) 10 days 41(3) 24(3) 30(3) 1.9

9a5c (PW) 5 days 33(2) 17(1) 29(4) 1.8

9a5c (PW-biofilm) 20 days 32(2) 17(2) 25(4) 2.1

Xac (M9) 1 day 71(3) 43(4) 20(3) 3.0

The Xf 9a5c samples were cultivated in PW medium for 5, 20, 30,

40, and 70 days, as biofilm on glassware (20 days) and in XDM2

medium for 10 days. The Xf Temecula was cultivated in XDM2 me-

dium for 10 days. All the spectra were normalized to signal in 1550

cm�1 (intensity¼ 100).

ester and methylene groups. The a ratio (Table 1),

proportional to the acetyl group amount in the EPS side

chain, was calculated by the intensity of the signal at

1740 cm�1 (�100), divided by the intensity of the signals

at 2930 and at 1080 cm�1, (a ¼ 100� ðI1740=I2930Þ=I1080).The data in Fig. 1 and Table 1 show that under

several experimental conditions the two strains of Xf

9a5c of CVC and Temecula of PD produced a lower

amount of EPS than the related Xac bacterium. They

also demonstrate that both Xf strains have a similar

EPS/protein composition, which can be related to their

similar genomes [5,6] and the ability of CVC and

coffee strains to induce PD symptoms in grapevines[17]. The capsular EPS content in Xf, 9a5c, and

Temecula were similar, varying from I1080 ¼ 32–41 in

samples at 5 and 40 days, respectively, in PW medium

and 10 days in XDM2 medium. The similar fastidian

content in the 9a5c biofilm sample, and the 9a5c

sample that does not attach to negative glass surfaces

(Table 1), contradicts the hypothesis that a large fas-

tidian amount is required for bacterial attachment andsurvival in the turbulent environment of xylem and

insect foregut [5,7]. Low fastidian amounts in Xf bio-

film were also recently observed using scanning elec-

tron microscopy [18].

The FTIR spectra did not show any thiol peaks from

2600 to 2550 cm�1 and do not indicate the presence of a

large amount of this component required by the model

proposed by Leite et al. [18]. The sulfur signal observedin their energy dispersive X-rays spectrometry analysis

[18] could be related to sulfate anions, normally the

major sulfur source for plants [23] and Xf [5]. These

anions could be concentrated in the infected xylem to

balance the cations (counterions) sequestered by the

bacteria [1]. Part of the sulfur detected can be related to

bacterial proteins in general and the Xf response to or-

ganic oxidative stress by ohr gene products that involvethiols [21].

The amount of EPS is higher in 70-day Xf culture and

the Xac sample, as seen in Fig. 1(b) and (e) and Table 1.

The large EPS amount in older cultures, also observed

in other bacterial species [20], indicates participation in

the final process of biofilm assembly.

The a ratio (Table 1), proportional to the acetate

amount in the EPS, is almost constant from 5 to 40 daysand is reduced in 70 days. The influence of the experi-

mental parameter in EPS acetate content was also ob-

served in Xanthomonas campestris pv. campestris [20].

The lower acetate content in fastidian in all condi-

tions studied (Table 1) than in xanthan could be ex-

plained by low activity of Xf acetyltransferase I, coded

by gumF gene, which has only 42% of homology to Xac

gum F gene [5,7]. It is known that acetate groups inhibitthe interaction between EPS and cations [22]. Therefore,

a lower amount of acetylated fastidian could be involved

in both the adhesion process and biofilm formation

316 D. Osiro et al. / FEMS Microbiology Letters 236 (2004) 313–318

through calcium bridges, but it certainly is not the de-

terminant factor.

These results support the importance of surface pro-

teins in the adhesion mechanism. These proteins are

positive in pH values from 5.5 to 6.5 in xylem sap [19].The number of positive amino acids (lysines, arginines,

and hystidines) exceed those of negative ones (glutamic

and aspartic acids) in Xf surface protein sequences [5].

The transcriptone and proteomic analyses of the virulent

bacteria show high expression of the fimbriae gene fimA

and adhesin genes uspA1 and hsp [23,24]. Scanning

electron microscopy analysis also shows the presence of

large numbers of fimbriae in bacteria in the xylem andinsect foregut, with less or no fimbriae after several

passages in liquid media [5]. The suppression of fimA

and fimF genes maintains pathogenicity, with reduced

fimbriae size and number, cell aggregation, and cell

size [25], indicating the importance of adhesins in the

process.

Based on analysis of the composition of Xf and host

surfaces, sap pH, ionic strength, flow velocity, time foradapting to host, and host defense mechanisms, we

propose a kinetic model (Fig. 2) for Xf adhesion, biofilm

formation, and virulence. In our model, adhesion is

dependent on the electrostatic attraction between posi-

tive surface proteins and negative host surfaces.

Strength and range of the electrostatic attraction force

are known to be very efficient in polymeric adsorption in

self-assembly processes [3] and also in bacterial adhesion[1].

In our model a bacterial population (B1) with two

phenotypes, one virulent (Bþ) with more positive surface

proteins, and one avirulent (B�) with less of these pro-

teins, is introduced into flowing xylem sap (Fig. 2). Both

cells can collide with the negative xylem wall at rate

constant K1 and return to the sap at rate constant K2.

The bacterium Bþ2 has a high probability of quickly

Fig. 2. Kinetic model for Xf adhesion, biofilm formation, and virulence. The

more positive surface proteins and one avirulent B�) with less of these protein

to the sap at rate constant K2. The Bþ2 cells growths and form biofilm (Bþ

4 ) at

quickly adhering to the host returning to sap (B�3 ). The B

�3 can slowly adapt

flow (B�4 ). This cell can also be killed (Bd) by plant defense mechanisms at a

binding to the host surfaces, and returning to the sap

(Bþ3 ) at rate constant K2�K2 of B�

2 . The Bþ2 cell can

start to digest the xylem wall [5] that provides a con-

tinuous sugar supply. It can grow, duplicate, and form

biofilm (Bþ4 ) at rate constant K4. Compared to cells in

the sap, this cell is highly protected by biofilm against

plant defense mechanisms like organic oxidative stress.

Bacterial biofilm can also produce larger amounts of

enzymes, necessary for degradation of pit membranes

[8], than do planktonic cells, allowing the vessel-to-ves-

sel movement necessary for systemic infection [8].

The cell in the sap (B�3 ) needs a very long time to

change its surface character (rate constant K3) and isdragged by the sap flow (B�

4 ). The long K3 rate makes Xf

a good model for adhesion studies because it behaves as

an inert particle in the adhesion time scale and, there-

fore, is a better model for these studies than the cell with

short K3 values normally cited in literature [1]. The B�4

cell in the dilute xylem sap can also be killed (Bd) at a

rate Kd by plant defense mechanisms.

Without fast adhesion, Xf is not also able to adhere tothe insect foregut. It is dragged by the sap flow to the

digestive tract of the insect and cannot infect other

plants. Therefore, in our model the fast electrostatic

adhesion process is the determinant step in plant viru-

lence and insect transmission. The model reflects the Xf

life cycle, infection strategy, and limited environment. It

also explains most of the observations about Xf–Xf

interactions, Xf-host interactions, pathogenicity, andvirulence.

After adhesion, low acetylated fastidian becomes

important in multiple biofilm layers. It has been shown

that negative polysaccharides increase by a factor of 2.5

ionic transport through membranes, compared to that

produced by apolar ones [26]. Therefore, similar faster

cation transport to Xf biofilm, than that predicted by the

diffusion process, can quickly sequester the cations

bacterial population (B1) with two phenotypes, one virulent (Bþ) withs can collide with the negative xylem wall at rate constant K1 and return

rate constant K4. The B�2 cell with K2�K2 of B

þ2 has low probability of

to more positive surface at rate constant K3 and is dragged by fast sap

rate constant Kd.

D. Osiro et al. / FEMS Microbiology Letters 236 (2004) 313–318 317

needed for cell crosslinks and their metabolism in dilute

xylem sap. When sequestering cations, fastidian also

transports counterions, like sulfate, nitrate, and chlorite

anions, necessary for bacterial growth. These negative

polysaccharides on bacterial surfaces, and also thosereleased as slime and crosslinked by calcium and other

cations, certainly compose the fibrous material observed

in some SEM images [5,8]. In conclusion, we have

demonstrated that Xf produces large amounts of fas-

tidian only in old cultures with low acetate content that

together form efficient sinks for cations, reducing their

availability to plants and explaining the zinc (or other

cation) deficiency symptoms [5,18] observed in plantswith CVC.

Acknowledgements

We thank FAPESP and CNPq (Brazilian agencies)

for financial support.

References

[1] Hermansson, M. (1999) The DLVO theory in microbial adhesion.

Colloids Surf. B 14, 105–119.

[2] Marques, L.L.R., Ceri, H., Manfio, G.P., Reid, D.M. and Olson,

M.E. (2002) Characterization of biofilm formation by Xylella

fastidiosa in vitro. Plant Dis. 86, 633.

[3] Borato, C.E., Herrmann, P.S.P., Colnago, L.A., Oliveira Jr., O.N.

and Mattosso, L.H.C. (1997) Using the self-assembly technique

for the fabrication of ultra-thin films of a protein. Braz. J. Chem.

Eng. 14, 367–373.

[4] Scarpari, L.M., Lambais, M.R., Silva, D.S., Carraro, D.M. and

Carrer, H. (2003) Expression of putative pathogenicity-related

genes in Xylella fastidiosa grown at low and high cell density

conditions in vitro. FEMS Microbiol. Lett. 222, 83–92.

[5] Simpson, A.J.G., Reinach, F.C., Arruda, P., Abreu, F.A.,

Acencio, M., Alvarenga, R., Alves, L.M.C., Araya, J.E., Baia,

G.S., Baptista, C.S., Barros, M.H., Bonaccorsi, E.D., Bordin, S.,

Bove, J.M., Briones, M.R.S., Bueno, M.R.P., Camargo, A.A.,

Camargo, L.E.A., Carraro, D.M., Carrer, H., Colauto, N.B.,

Colombo, C., Costa, F.F., Costa, M.C.R., Costa-Neto, C.M.,

Coutinho, L.L., Cristofani, M., Dias-Neto, E., Docena, C., El

Dorry, H., Facincani, A.P., Ferreira, A.J.S., Ferreira, V.C.A.,

Ferro, J.A., Fraga, J.S., Franca, S.C., Franco, M.C., Frohme, M.,

Furlan, L.R., Garnier, M., Goldman, G.H., Goldman, M.H.S.,

Gomes, S.L., Gruber, A., Ho, P.L., Hoheisel, J.D., Junqueira,

M.L., Kemper, E.L., Kitajima, J.P., Krieger, J.E., Kuramae, E.E.,

Laigret, F., Lambais, M.R., Leite, L.C.C., Lemos, E.G.M.,

Lemos, M.V.F., Lopes, S.A., Lopes, C.R., Machado, J.A.,

Machado, M.A., Madeira, A.M.B.N., Madeira, H.M.F., Marino,

C.L., Marques, M.V., Martins, E.A.L., Martins, E.M.F., Matsu-

kuma, A.Y., Menck, C.F.M., Miracca, E.C., Miyaki, C.Y.,

Monteiro-Vitorello, C.B., Moon, D.H., Nagai, M.A., Nasci-

mento, A.L.T.O., Netto, L.E.S., Nhani, J., Nobrega, F.G., Nunes,

L.R., Oliveira, M.A., de Oliveira, M.C., de Oliveira, R.C.,

Palmieri, D.A., Paris, A., Peixoto, B.R., Pereira, G.A.G., Pereira,

J., Pesquero, J.B., Quaggio, R.B., Roberto, P.G., Rodrigues, V.,

Rosa, A.J.D., de Rosa, V.E., de Sa, R.G., Santelli, R.V.,

Sawasaki, H.E., da Silva, A.C.R., da Silva, F.R., da Silva,

A.M., Silva, J., da Silveira, J.F., Silvestri, M.L.Z., Siqueira, W.J.,

de Souza, A.A., de Souza, A.P., Terenzi, M.F., Truffi, D., Tsai,

S.M., Tsuhako, M.H., Vallada, H., Van Sluys, M.A., Verjovski-

Almeida, S., Vettore, A.L., Zago, M.A., Zatz, M., Meidanis, J.

and Setubal, J.C. (2000) The genome sequence of the plant

pathogen Xylella fastidiosa. Nature 406, 151–157.

[6] Van Sluys, M.A., de Oliveira, M.C., Monteiro-Vitorello, C.B.,

Miyaki, C.Y., Furlan, L.R., Camargo, L.E., da Silva, A.C.,

Moon, D.H., Takita, M.A., Lemos, E.G., Machado, M.A., Ferro,

M.I., da Silva, F.R., Goldman, M.H., Goldman, G.H., Lemos,

M.V., El-Dorry, H., Tsai, S.M., Carrer, H., Carraro, D.M., de

Oliveira, R.C., Nunes, L.R., Siqueira, W.J., Coutinho, L.L.,

Kimura, E.T., Ferro, E.S., Harakava, R., Kuramae, E.E., Marino,

C.L., Giglioti, E., Abreu, I.L., Alves, L.M., do Amaral, A.M.,

Baia, G.S., Blanco, S.R., Brito, M.S., Cannavan, F.S., Celestino,

A.V., da Cunha, A.F., Fenille, R.C., Ferro, J.A., Formighieri,

E.F., Kishi, L.T., Leoni, S.G., Oliveira, A.R., Rosa Jr., V.E.,

Sassaki, F.T., Sena, J.A., de Souza, A.A., Truffi, D., Tsukumo, F.,

Yanai, G.M., Zaros, L.G., Civerolo, E.L., Simpson, A.J., Almeida

Jr., N.F., Setubal, J.C. and Kitajima, J.P. (2003) Comparative

analyses of the complete genome sequences of Pierce’s disease and

citrus variegated chlorosis strains of Xylella fastidiosa. J. Bacteriol.

185, 1018–1026.

[7] da Silva, F.R., Vettore, A.L., Kemper, E.L., Leite, A. and Arruda,

P. (2001) Fastidian gum: the Xylella fastidiosa exopolysaccharide

possibly involved in bacterial pathogenicity. FEMS Microbiol.

Lett. 203, 165–171.

[8] Hopkins, D.L. (1989) Xylella fastidiosa: a xylem-limited bacterial

pathogen of plants. Ann. Rev. Phytopathol. 27, 271–290.

[9] Machado, M.A., Souza, A.A., Coletta-Filho, H.D., Kuramae,

E.E. and Takita, M.A. (2001) Genome and pathogenicity of

Xylella fastidiosa. Mol. Biol. Today 2, 33–43.

[10] Nunes, L.R., Rosato, Y.B., Muto, N.H., Yanai, G.M., da Silva,

V.S., Leite, D.B., Goncalves, E.R., da Souza, A.A., Coleta-Filho,

H.D., Machado, M.A., Lopes, S.A. and de Oliveira, R.C. (2003)

Microarray analyses of Xylella fastidiosa provide evidence of

coordinated transcription control of laterally transferred elements.

Genome Res. 13, 570–578.

[11] Davis, M.J., French, W.J. and Schaad, N.W. (1981) Axenic

culture of the bacteria associated with phony disease of peach and

plum leaf scald. Curr. Microbiol. 6, 309–314.

[12] Lemos, E.G.de.M., Alves, L.M.C. and Campanharo, J.C. (2003)

Genomics based design of defined growth media for the plant

pathogen Xylella fastidiosa. FEMS Microbiol. Lett. 219, 39–45.

[13] Miller, J.H. (1972) In Experiments in Molecular Genetics. Cold

Spring Harbor Laboratories, New York.

[14] Forato, L.A., Bicudo, T.C. and Colnago, L.A. (2003) Conforma-

tion of a zeins in solid state by Fourier transform IR. Biopolymers

72, 421–426.

[15] Naumann, D. (2000) Infrared Spectroscopy in Microbiology. In:

Encyclopedia of Analytical Chemistry (Meyers, R.A., Ed.), pp.

102–131. Wiley, Chichester.

[16] Kansiz, M., Heraud, P., Wood, B., Burden, F., Beardall, J. and

McNaughton, D. (1999) Fourier transform infrared microspec-

troscopy and chemometrics as a tool for the discrimination of

cyanobacterial strain. Phytochemistry 52, 407–417.

[17] Li, W.-B., Zhou, C.-H., Pria, W.D., Teixeira, D.C., Miranda,

V.S., Pereira, E.O., Ayres, A.J., He, C.-X., Costa, P.I. and

Hartung, J.S. (2002) Citrus and coffee strains of Xylella fastidiosa

induce Pierce’s disease in grapevine. Plant Dis. 86, 1206–1210.

[18] Leite, B., Ishida, M.I., Alves, E., Carrer, H., Pascholati, S.F. and

Kitajima, E.W. (2002) Genomics and X-ray microanalysis indicate

that Ca2þ and thiols mediate the aggregation and adhesion of

Xylella fastidiosa. Braz. J. Med. Biol. Res. 35, 645–650.

[19] Wutscher, H.K. and McDonald, R.E. (1986) Mineral elements

and organic acids in branch and root xylem sap of healthy and

blight-affected sweet orange trees. J. Am. Soc. Hortic. Sci. 111,

426–429.

318 D. Osiro et al. / FEMS Microbiology Letters 236 (2004) 313–318

[20] Garc�ıa-Ochoa, F., Santos, V.E., Casas, J.A. and G�omez, E. (2000)

Xanthan gum: production, recovery, and properties. Biotechnol.

Adv. 18, 549–579.

[21] Cussiol, J.R.R., Alves, S.V., de Oliveira, M.A. and Netto, L.E.S.

(2003) Organic hydroperoxide resistance gene encodes a thiol-

dependent peroxidase. J. Biol. Chem. 278, 11570–11578.

[22] Sutherland, I.W. (2001) Biofilm exopolysaccharides: a strong and

sticky framework. Microbiology 147, 3–9.

[23] de Souza, A.A., Takita, M.A., Coleta-Filho, H.D., Caldana, C.,

Goldman, G.H., Yanai, G.M., Muto, N.H., de Oliveira, R.C.,

Nunes, L.R. and Machado, M.A. (2003) Analysis of gene

expression in two growth states of Xylella fastidiosa and its

relationship with pathogenicity. Mol. Plant Microbe Interact. 16,

867–875.

[24] Smolka, M.B., Martins, D., Winck, F.V., Santoro, C.E.,

Castellari, R.R., Ferrari, F., Brum, I.J., Galembeck, E.,

Coletta-Filho, H.D., Machado, M.A., Marangoni, S. and

Novello, J.C. (2003) Proteome analysis of the plant pathogen

Xylella fastidiosa reveals major cellular and extracellular pro-

teins and a peculiar codon bias distribution. Proteomics 3, 224–

237.

[25] Feil, H., Feil, W.S., Detter, J.C., Purcell, A.H. and Lindow,

S.E. (2003) Site-Directed Disruption of the fimA and fimF

Fimbrial Genes of Xylella fastidiosa. Phytopathology 93, 675–

682.

[26] Junior, W. and Engelsberg, M. (2003) Barros Enhanced migration

and ionic transport through membranes. Phys. Rev. E 67,

01–05.