| Grant number: | 98/16265-1 |
| Support Opportunities: | Genome Research Grants |
| Start date: | April 01, 1999 |
| End date: | March 31, 2002 |
| Field of knowledge: | Biological Sciences - Biochemistry |
| Principal Investigator: | Tsai Siu Mui |
| Grantee: | Tsai Siu Mui |
| Host Institution: | Centro de Energia Nuclear na Agricultura (CENA). Universidade de São Paulo (USP). Piracicaba , SP, Brazil |
| Associated scholarship(s): | 00/07007-0 - Identification of proteins and putative virulence related low molecular weight compounds, BP.TT |
Abstract
Virulence of pathogenic bacteria is known to be related to the availability of iron in the host organism. In order to establish infection, pathogenic bacteria must successfully compete for a limited iron pool. Five outer membrane receptors were reported in the Xylella fastidiosa genome (31b 9a5c-Xo-FAPESP-BRAZIL), including siderophores, ferrichrome-iron, and hemin receptors. These compounds are thought to be associated to iron transport and utilization. Most siderophore are either hydroxamates or catechols, based on such functional groups. The isolates were compared in four contaminated citrus orchards (Santa Rita do Passa Quatro, Neves Paulista, Gavião Peixoto, and Paraíso, state of São Paulo). Xylella fastidiosa strains (180) were isolated from these four orchards. All isolates were tested positive by specific PCR primers (CVC-1 and 272-2), confirming their classification as part of CVC group. Screening of 80 isolates for iron sequestration activity was performed by the chrome azurol S method. We have found that 10 of them did not form a halo (negative for siderophore production). Seven isolates of grape from U.S.A. (ATCC 6750, ATCC 6751, ATCC 6752, ATCC 6068, Fetzer, Temecula, Traver), also were used for iron sequestration activity, and all form strongly halo (positive for siderophore production). All of these isolates were further screened with PCR specific primers developed for the ferric enterobactin receptor (PfeA) from Escherichia coli and putative receptor protein sequences from Pseudomonas sp., Shewanella sp. and X. fastidiosa after comparative analysis by multiple alignment using ClustalW. We also developed PCR primers for the hydroxamate-type ferri-siderophore receptor gene (Ton-B) (pyoverdin) from Pseudomonas aeruginosa and X. fastidiosa. The specificity of amplified products was confirmed by subsequent sequencing of 50 PCR products followed by database search. The 10- siderophore negative CVC isolates did not form halo or yielded PCR amplification products (1,600 bp) for the putative enterobactin receptor. For primers developed against the putative pyoverdin receptor gene, in 10 isolates neither a halo was formed nor PCR products (1,000 bp) were obtained. In addition, we have developed two sets of oligonucleotides for synthetases identified in the X. fastidiosa genome, which could be involved in pathogenicity. One set produces a 0.3 kb fragment coding for a polyketide synthase, and the other amplifies a region (0.2 kb) coding for part of a putative non - ribosomal peptide synthetase. The strain 9a5c used for complete genome sequencing was used as our positive control. This is the first instance in which correlation between iron sequestration and genes involved on its transportation and reception is shown for X. fastidiosa. After several unsuccessful attempts to transform efficiently strain 9a5c, we tried to transform other Xylella strains, which were also not successful. After testing different plasmid vectors to obtain actable transformants, we have concentrated our efforts in transformation of strain J1a12, which was previously shown to be efficient for transformation (MONTEIRO et al., 2001). We are testing the plasmid vector called pSP3, and is a fusion between a colE1 plasmid (pBluescript KS) and the Xylella plasmid pXF1.3. (MARQUES et aI., 2002) (submitted). Plasmid pSP3 which contains the coding region of the nptll gene, was cloned after the promoter region of Xylella 16S rRNA gene, therefore conferring kanamycin resistance, being thus a shuttle vector. We have tested this plasmid for transformation of X. fastidiosa, either containing or not fragments of specific Xylella genes. In an iron-limited medium siderophores were detected in the supernatant of X. fastidiosa (X0) cultures. Furthermore, the isolate X0 was cultured in 50 ml of MM9 medium with different concentration of Fe3+ (0, 0.1, 10, 30, 50 and 100 µM). Maximum bacteria growth was observed when 100 µM of iron was supplemented to the medium. Other bacteria were also evaluated: X. fastidiosa from grape, Xanthomonas axonopodis pv. citri, X. campestris pv. campestris and Methylobacterium extorquens. The media supernatants were analyzed for siderophores production and in case of identified, they were characterized biochemically as either hydroxamate or catechol-type siderophores. All the isolates were negative for hydroxamate and catechol in the analyses of Csáky and Arnow, except for Methylobacterium extorquens that excreted hydroxamate-type siderophores. (AU)
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