Growth and siderophore production of under iron-limited conditions

ARTICLE IN PRESS Microbiological Research 160 (2005) 429—436 www.elsevier.de/micres Growth and siderophore production of Xylella fastidiosa under iron-limited conditions Maria Estela Silva-Stenicoa,, Flávia Tereza Hansen Pachecoa, Jorge Luiz Mazza Rodriguesa, Emanuel Carrilhob, Siu Mui Tsaia a Laboratório de Biologia Celular e Molecular, Centro de Energia Nuclear na Agricultura – Universidade de São Paulo, Piracicaba, SP, Brazil b Laboratório de Cromatografia, Instituto de Quı́mica de São Carlos – Universidade de São Paulo, São Carlos, SP, Brazil Accepted 21 March 2005 KEYWORDS Chromeazurol S; Iron chelation; Methylobacterium extorquens; Xylella fastidiosa; Siderophores Summary In this study, the production of siderophores by Xylella fastidiosa from the citrus bacteria isolate 31b9a5c (FAPESP – ONSA, Brazil) was investigated. The preliminary evidence supporting the existence of siderophore in X. fastidiosa was found during the evaluation of sequencing data generated in our lab using the BLAST-X tool, which indicated putative open reading frames (ORFs) associated with iron-binding proteins. In an iron-limited medium siderophores were detected in the supernatant of X. fastidiosa cultures. The endophytic bacterium Methylobacterium extorquens was also evaluated. Capillary electrophoresis was used to separate putative siderophores produced by X. fastidiosa. The bacterial culture supernatants of X. fastidiosa were identified negative for hydroxamate and catechol and positive for M. extorquens that secreted hydroxamate-type siderophores. & 2005 Elsevier GmbH. All rights reserved. Introduction Microorganisms need iron, which is an essential element for life (Loper and Buyer, 1991) and plays an important role in some host–bacteria interactions (Mila et al., 1996). Iron is a cofactor of several enzymes and acts in transport processes and redox reactions (Van Vliet et al., 1998). In addition, iron is required for a variety of functions in microorgan- isms that grow under aerobic conditions such as reduction of oxygen during ATP synthesis, reduction of ribonucleotide precursors of DNA, formation of heme (Neilands, 1995), oxidation–reduction in cellular reactions, which involve the activities of cytochromes, hydroperoxidases, non-iron nitrogenases, and ribonucleotide reductases (Robson et al., 1986). A concentration of, at least, 1 mM of iron is needed for optimum growth (Neilands, Corresponding author. E-mail address: estela@cena.usp.br (M. Estela Silva-Stenico). 0944-5013/$ - see front matter & 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2005.03.007 ARTICLE IN PRESS 430 1995); however, this concentration varies for different organisms. In an iron-limited condition, microorganisms produce siderophores (low molar mass biomolecules 0.5–1.5 kDa) to scavenge ferric ions, which bind to specific outer membrane receptors with high affinity. Some donor groups for the chelation of Fe3+ are hydroxamates, thiohydroxamates and catecholates (Richardson et al., 1999). Many bacteria can synthesize their own chelator or utilize other microbial- and plant-siderophores for iron acquisition (Grusak et al., 1999). The mechanism of iron acquisition is known to be a virulence factor for human and animal pathogenic bacteria (Neilands, 1995). In the case of plants (e.g. Saintpaulia ionantha leaves), studies conducted by Neema et al. (1993) confirm that the siderophore chrysobactin produced by Erwinia chrysanthemi was involved in the disease process. Xylella fastidiosa is a pathogenic bacterium in citrus and in a large variety of other hosts. In Brazil, this bacterium causes citrus variegated chlorosis (CVC), resulting in losses of US $ 100 million per year to the citrus industry (Coletta-Filho et al., 2001). Although the entire genome of X. fastidiosa was sequenced (Simpson et al., 2000), little is known about its genetics, pathogenesis, and ecological significance. The presence of putative genes in X. fastidiosa (Simpson et al., 2000), coding for iron uptake membrane receptors suggests a potential role for chelating agents during the development of disease symptoms such as chlorosis (Neema et al., 1993). Pacheco et al. (2001) have conducted a detailed molecular study relating the production of siderophores with the presence of putative iron uptake receptors. They have screened over 80 isolates by PCR-specific primers designed for the ferric enterobactin receptor (PfeA), primers for the hydroxamate-type ferrisiderophore receptor gene (fiuA) (pyoverdin), and also a new set of primers for a 300 bp fragment coding for a polyketide synthase (PKS). Of the 80 isolates, 70 were siderophore producers (chemical test) and also yielded positive PCR amplifications for all three putative genes tested; therefore the production of secondary metabolites may, in part, be involved in virulence of X. fastidiosa against host plants. In order to compare siderophores production by other bacteria found in contaminated citrus plants we included the endophytic bacterium Methylobacterium extorquens isolated from healthy and symptomatic plants of Citrus sinensis (Araujo et al., 2002). The purpose of this work was to investigate the effect of iron in the production of siderophores by X. fastidiosa, which could be M. Estela Silva-Stenico et al. involved in the iron-stress related virulence of this pathogen. Materials and methods Bacterial strains X. fastidiosa (9a5c – citrus), from the Genome Sequencing Project, was obtained from INRA – Institut National de la Recherche Agronomique et Université Victor Ségale, Bordeaux, France. M. extorquens was given by the Department of Genetics from Escola Superior de Agricultura Luiz de Queiroz – Universidade de São Paulo (ESALQ/USP), Brazil. Media and bacterial growth Strains were cultured three times in an irondeficient liquid medium (MM9) according to Payne (1994). The medium was composed of 0.3 g l 1 KH2PO4; 0.5 g l 1 NaCl; 1.0 g l 1 NH4Cl; 6.0 g l 1 NaOH, and 30.24 g l 1 PIPES. This solution was autoclaved and supplemented with 30 ml of 10% (m/v) deferrated casamino acids (contaminating iron was removed with 3% 8-hydroxyquinoline in chloroform), 2.0 g l 1 fructose, 1 ml of 1 M MgCl2 and 1 ml of 100 mM CaCl2. These solutions were prepared and sterilized separately. Glassware was cleaned with 6 M HCl prior to use. Growth was measured by monitoring the optical density at 600 nm (OD600) with a Perkin-Elmer spectrophotometer. Bacteria were grown from a standard inoculum (100 ml of a suspension with an absorbance at 600 nm of 0.5) in 200 ml flasks containing 50 ml MM9 medium and incubated at 28 1C on a rotary shaker at 150 rpm. Iron requirement To verify iron requirement, X. fastidiosa growth was examined as a function of iron concentration added to the MM9 medium. Iron (FeCl3) was added to 200 ml flasks containing 50 ml MM9 medium at the following concentrations: 0, 0.1, 10, 50 and 100 mmol l 1. The flasks were incubated at 28 1C for 12 days. Growth rate was measured as above. Siderophore-type assays The Chromeazurol S (CAS) assay (Schwyn and Neilands, 1987) was used to detect siderophores independent of their structure in the MM9 culture supernatants. The CAS liquid assay was performed as follows: a 6-ml volume of 10 mM hexadecyltri- ARTICLE IN PRESS Growth and siderophore production of Xylella fastidiosa under iron-limited conditions methylammonium bromide (HDTMA) solution was placed in a 100 ml volumetric flask and diluted with water. A mixture of 1.5 ml of 1 mM FeCl3
6H2O in 10 mM HCl and 7.5 ml 2 mM aqueous CAS solution was added slowly under stirring. A total of 4.3 g of anhydrous piperazine was dissolved in water and 6.25 ml 12 M hydrochloric acid was added. The flask was completed with distilled water to obtain 100 ml of CAS solution. The pH of the solution was 5.6. Cells were removed by centrifugation (12,000g for 20 min). A 0.5 ml aliquot of supernatant was mixed with 0.5 ml CAS assay solution. A reference was prepared using sterile MM9 medium. After reaching equilibrium the absorbance was measured at 630 nm. Positive reactions were recorded by a change in the color of the CAS reagent from blue to yellow or orange. CAS agar plates were also used, and positive results were recorded as a halo formation around the colonies. Catechol-type siderophores were measured in culture supernatants by the Arnow assay (Arnow, 1937) and hydroxamates were measured according to Csáky (1948), with 2,3-dihydroxybenzoic acid and hydroxylamine hydrochloride as standards, respectively. Each assay was performed in triplicate. Desferrioxamine B equivalent assay This assay was developed according to Shin et al. (2001). The CAS agar diffusion assay was performed as follows: 60.5 mg CAS were dissolved in 50 ml deionized water and mixed with 10 ml iron (III) solution (1 mM FeCl3
6H2O, 10 mM HCl). While stirring, this solution was slowly mixed with 72.9 mg HDTMA dissolved in 40 ml water. The resultant dark blue solution was autoclaved and mixed with an autoclaved mixture of 900 ml water, 15 g agarose, 30.24 g PIPES, and 12 g of a solution with 50% (m/v) NaOH to raise the pH of the solution near the pKa of PIPES (6.8). Wells of 5 mm diameter were punched into agarose plates. Each well was filled with 100 mM of the supernatants and the haloes were measured. A range of desferrioxamine concentrations (0, 1, 5, 10, 50, 100, 500, 1000, 1500, 2000, and 2500 mM) were used to quantify siderophore production. A blank control of MM9 medium was used to identify any interference with the color development for this CAS agar assay. The assay was performed in triplicate. Capillary electrophoresis and sample preparation Bacteria were inoculated on agar plates containing MM9 medium plus Chromeazurol S. An agar 431 block (1 cm2) excised from the halo region was used for analysis and a corresponding agar block, in which no bacterial growth was observed, was used as control. Samples of the CAS agar inoculated with X. fastidiosa were extracted as shown in Fig. 3 following disruption with 2 ml 0.1 M NaOH (1 min shaking). After centrifugation, the supernatant was filtered (0.22 mm membrane) and injected in the capillary electrophoresis system. Electrophoresis separation was carried out on a P/ACE Beckman 5510 (Palo Alto, CA, USA). A fused-silica capillary with 75 mm inner diameter and 37 cm total length was used. The samples were injected for 5 s under 0.5 psi, the voltage was 30 kV. The temperature was held constant at 25 1C. The separation was monitored at 214 nm using a UV-detector. The capillary was conditioned after each run by the following sequence: 1 M HCl (1 min), 1 M NaOH (1 min), deionized water (1 min), 0.1 M NaOH (1 min), and 0.1 M potassium phosphate buffer pH 7 (2 min). The peaks obtained in the electropherogram were acquired from the P/ACE System (Beckman) software. MALDI-TOF-MS (matrix-assisted laser desorption–ionization time of flight mass spectrometry) analysis Strains were cultured three times in an irondeficient liquid medium (MM9) as described earlier. Siderophore purification was carried out as follows: the immobilization of Fe3+ ions in the Chelating Sepharose Fast Flow (CSFF) adsorbent was prepared using 3 ml of resin, which was washed twice with a solution of 0.1 mol l 1 iron (FeCl3
6H2O), followed by five washes with milli-Q water. The supernatant of each culture was added to the resin and centrifuged at 1200g for 3 min. The supernatant was discarded and a solution of 1 ml of 0.05 mM EDTA was added. The complex Fe–resin was disrupted and the supernatant containing Fe–siderophore complex was lyophilized. Freeze-dried samples were, then, prepared for MALDI-TOF on a Voyager-DE STR Bioworkstation (PerSeptive Biosystems, Framingham, MA, USA). Samples were dissolved in 1% aqueous trifluoroacetic acid and the matrix sinapinic acid added (a saturated solution dissolved in acetonitrile/0.1% TFA 1:1, v/v). The solution was then vortex mixed and aliquots of 1 ml were applied to the Voyager Bioworkstation sample plate. Samples were air-dried at room temperature. The spectrometer, equipped with a delayedextraction system, was operated in linear mode. Sample ions were evaporated by irradiation with a N2 laser at a wavelength of 337 nm, and ARTICLE IN PRESS 432 M. Estela Silva-Stenico et al. accelerated at 23 kV potential in the ion source with a delay of 150 ns. Samples were ionized with 100–200 shots of a 3 ns pulse width laser light. The signal was digitized at a rate of 500 MHz and averaged data was presented to a standard Voyager data system for manipulation. iron added to the medium. When 100 mM Fe3+ was added to the culture, optical density increased, but the presence of siderophores was not detected at this concentration (Fig. 2). Results The Csáky and Arnow reactions were used to identify the most common functional groups bound to iron in the siderophore extracts of X. fastidiosa and M. extorquens. The supernatant of X. fastidiosa was negative for hydroxamate or catechol-type siderophores, while M. extorquens showed a positive result for Csáky reaction, thus indicating that this siderophore has a hydroxamate donor group. The CAS agar assay also revealed that bacteria present a halo formation around the colonies (Table 1, Fig. 3). Cellular growth in different iron concentration Figure 1a shows growth rates of X. fastidiosa in MM9 medium with different concentrations of Fe3+. Cultures were grown to the stationary phase in which the maximum siderophore production occurred (Fig. 1b). Siderophore production by X. fastidiosa was regulated by the concentration of Capillary electrophoresis profile 0.16 0.14 Electrophoretic runs were accomplished to verify the excreted metabolite present in agar plates. The results of Fig. 4 present differences in the metabolites produced by X. fastidiosa in the MM9 medium. Comparing samples from CAS agar inoculated with X. fastidiosa, one can see that the agar sample where there was no bacterial growth (CAS agar in complex with iron) presented no peak and was used as control, while the yellowish sample (halo) presented three peaks (around 10, 15, and 20 min of migration time). 0.12 OD600 nm 0.10 0.08 0.06 0.0 0.1 10 50 100 0.04 0.02 0.00 0 2 4 6 10 18 16 16 14 14 12 12 A/Aref630 nm µmol l-1 DFX equivalent 8 period (d) (a) 10 8 6 10 8 6 4 4 2 2 0 0 -2 -1 (b) Siderophore-type classification and halo formation 0 1 2 3 4 5 6 7 8 9 10 11 12 period (d) Figure 1. (a) Growth curve of X. fastidiosa in MM9 medium containing different concentrations of iron (III): 0.0 (’), 0.1 (K), 10 (m), 50 (.) and 100 (~) mmol L 1. (b) CAS reactivity in X. fastidiosa supernatant. Where error bars are not shown, the standard error of triplicates is smaller than the size of the symbol. 0 20 40 60 [Fe(III)] (µmol 80 100 l-1) Figure 2. Relative absorbance at 630 nm from X. fastidiosa supernatant as a function of iron concentration. Values refer to the concentration before mixing with the assay solution. Where error bars are not shown, the standard error of triplicates is smaller than the size of the symbol. ARTICLE IN PRESS Growth and siderophore production of Xylella fastidiosa under iron-limited conditions Table 1. 433 Siderophore classification for the evaluated bacteria. Microorganisms Xylella fastidiosa Methylobacterium extorquens Siderophore type Hydroxamate Catechol Desferrioxamine equivalents (mmol l 1) ( ) (+) ( ) ( ) 15 26 CAS assay (halo formation) (+) (+) 0.05 Absorbance (mAU) 0.04 0.03 0.02 halo 0.01 Figure 3. Chromeazurol S agar test for X. fastidiosa. The frames show the sample areas used to capillary electrophoresis analysis. (a) Sample used as control (no bacterial growth) and (b) Sample showing siderophore production, formation of a halo around the colony. control 0.00 2 4 6 8 10 12 14 16 18 20 22 time (min) Figure 4. Electropherograms from CAS agar plate inoculated with X. fastidiosa and its control. MALDI-TOF profile After purification of the siderophores in the culture supernatant of X. fastidiosa, three fractions were obtained and analyzed by mass spectrometry, which confirmed the presence of the expected molar mass for such compounds in the range of 600 Da; Fig. 5). M. extorquens presented peaks around 1200 and 1400 Da (Fig. 6). Discussion Little is known about the consequences of siderophore production of phytopathogens in plants. This is due to the difficulty in establishing nutritional conditions for microorganisms that promote the production or limit the synthesis of different siderophore types (Teintze and Leong, 1981). Because of extensive knowledge that siderophores play an important role as determinants of virulence in animal pathogens, it has been suggested that siderophores may be important for phytopathogenic bacteria, as confirmed by the production of chrysobactin in Erwinia chrysanthemi (Neema et al., 1993). The presence of putative open reading frames (ORFs) encoding iron uptake (Table 2) in the genome of X. fastidiosa suggests a potential role for chelating agents in the development of the disease symptoms. Iron sequestration might play an important role in leaf chlorosis since this is the initial symptom of the disease caused by X. fastidiosa, as it occurs with other pathogens that can also be influenced by iron, such as Colletotrichum musae in banana (Brown and Swinburne, 1981). Using MM9 as an iron-free medium, X. fastidiosa grew 20% less than that observed in PW medium (Davis et al., 1981), a commonly used medium for this microorganism, due to its fastidious requirement for some amino acids and mineral elements for growth. Because the PW medium interferes with CAS siderophore liquid assay, we have selected the MM9 as the culture growth medium. Our results corroborate with those documented by Payne (1994), who found that growth rates for other aerobic bacteria in iron-limited cultures were slower than cultures containing an excess of iron. While the iron requirement generally ranges from 0.4 to 1.8 mM for most Gram-negative bacteria, we found that when the iron concentration was raised ARTICLE IN PRESS 434 M. Estela Silva-Stenico et al. Figure 5. MALDI-TOF spectrum of X. fastidiosa siderophore. Figure 6. MALDI-TOF spectrum of M. extorquens siderophore. ARTICLE IN PRESS Growth and siderophore production of Xylella fastidiosa under iron-limited conditions Table 2. Putative ORFs encoding iron uptake in the genome of X. fastidiosa. Genbank accession numbers Gene function XF0599 TonB-dependent receptor for iron transport Peptide synthase PKS (polyketide synthase) Ferric enterobactin receptor Peptide synthase XF1038 XF2135 XF2137 XF2276 to 100 mM the optical density of X. fastidiosa cultures increased 66% (Fig. 1a). Siderophore synthesis is mainly regulated by iron and can be maximized under low iron concentration conditions, thus improving bacterial growth (Bagg and Neilands, 1987). The CAS assay, developed by Schwyn and Neilands (1987), is based on the chelation of iron by any class of siderophores. Both species X. fastidiosa and M. extorquens were tested positive for this assay (Table 1). The CAS-reactive peak from X. fastidiosa occurred during the late log to the early stationary phase of growth, around 4 days (Fig. 1a and b). The CAS assay was not intense for X. fastidiosa (15 mM desferrioxamine equivalents) when compared to animal and human pathogens such as Legionella pneumophila (Liles et al., 2000), Helicobacter hepaticus, and H. cianedi (Dhaenens et al., 1999). This could be due to the slow growth of X. fastidiosa, taking up to 5 days for a positive CAS reaction to occur. To verify the repression of siderophore production by iron availability, X. fastidiosa was grown in the presence of several iron concentrations. The results presented in Fig. 2 show that the optimal chelator production was reached when no iron was added to the medium. At 50 and 100 mM the production was poor or no production occurred. The supernatant of X. fastidiosa was tested negative for hydroxamate- or catechol-type siderophores, and that was positive in the Csáky test for M. extorquens. Negative results for X. fastidiosa supernatant suggests that the siderophore type has no hydroxamate- or catechol-type functional groups (Guan et al., 2000). Capillary electrophoresis provides a powerful tool for the separation of charged analytes and biomolecules at sub-picomole levels. It is applied as a fast and efficient method for analysis of compounds and the technique was chosen due to the low concentration of these metabolites produced by X. fastidiosa. Mucha et al. (1999) using CE demonstrated the presence of siderophores in the 435 subsurface of seawater, which makes CE a good way to detect such molecules. In the case of X. fastidiosa growing in agar plates, the metabolites were analyzed in complex with iron (III) (CAS complex) and electropherograms showed the presence of three compounds excreted by the bacteria. When the same samples were injected in HPLC, we also found three peaks with siderophore activity (data not shown). These compounds have not been previously detected in X. fastidiosa supernatants. MALDI-TOF spectra indicated a pseudo-molecular ion with m/z 656 for X. fastidiosa (Fig. 5) and m/z 1287 for M. extorquens (Fig. 6). The appearance of major peaks in both spectra ranging from 790 to 876 Da was also detected in the blank (culture medium with no bacteria), possibly due to the interference of matrix constituents. For the structural elucidation of such molecules further MS/MS fragmentation should be carried out and it will be pursued next. Such molecular characterization will be fundamental in the understanding of iron uptake metabolism and its relationship to virulence of X. fastidiosa strains. Acknowledgements We thank Dr. W.L. Araújo (Department of Genetics – ESALQ/USP) for providing us with the endophytic bacterium and Dr. C. Bloch Jr. (Mass spectrometry laboratory – EMBRAPA) for technical assistance with MALDI-TOF-MS analysis. 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