RESUMO
The genetic diversity among twenty three strains of Xylella fastidiosa, isolated from sweet orange citrus, was assessed by RFLP analysis of the 16S rDNA and 16S-23S intergenic spacer and by rep-PCR fingerprinting together with strains isolated from coffee, grapevine, plum and pear. The PCR products obtained by amplification of the 16S rDNA and 16S-23S spacer region were digested with restriction enzymes and a low level of polymorphism was detected. In rep-PCR fingerprinting, a relationship between the strains and their hosts was observed by using the BOX, ERIC and REP primers. Two major groups were obtained within the citrus cluster and relationships to the geographic origin of the strains revealed. Citrus strains isolated from the States of São Paulo and Sergipe formed one group and strains from the Southern States formed another group. Distinct origins of X. fastidiosa in the Southern and Southeastern States is postulated. The pear isolate was distantly related to all of the other X. fastidiosa strains.
Assuntos
Citrus/microbiologia , DNA Bacteriano/genética , DNA Espaçador Ribossômico/genética , Gammaproteobacteria/genética , Variação Genética/genética , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Brasil , Café , Impressões Digitais de DNA , Gammaproteobacteria/classificação , Filogenia , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNA , VitisRESUMO
The phylogenetic relationships of Xylella fastidiosa strains isolated from different hosts, including citrus trees, coffee, grapevine, plum and pear, were inferred by sequence analysis of the 16S rDNA and 16S-23S intergenic spacer region. A high level of similarity (97.1-100%) was found in the 16S rDNA of the Xylella fastidiosa strains. The 16S-23S region showed a higher level of variation, with similarity values ranging from 79.8 to 100%. Two tRNAs (tRNA(Ala) and tRNA(Ile)) were encountered within the spacer sequence. The phylogenetic trees, constructed using the neighbour-joining method, showed that the citrus, coffee, peach and plum strains were closely related and separate from grapevine strains. The pear strain remained isolated from all the other Xylella strains in both analyses and produced values of less than 20% in DNA-DNA hybridization experiments with a citrus strain. These results show that this strain does not belong to the Xylella fastidiosa genomic species.
Assuntos
DNA Espaçador Ribossômico/genética , Gammaproteobacteria/classificação , Pseudomonadaceae/classificação , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , DNA Bacteriano , Gammaproteobacteria/genética , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Filogenia , Doenças das Plantas/microbiologia , Pseudomonadaceae/genética , Análise de Sequência de DNARESUMO
The present study reports the expression of proteins of Xanthomonas axonopodis pv. citri in response to different growth conditions. The bacterium was cultured in the basal medium MM1 and in the presence of leaf extracts from a susceptible host plant (sweet orange) as well as a resistant (ponkan) and a nonhost plant (passion fruit). The protein profiles were analyzed by two-dimensional gel electrophoresis (2-DE). Twelve differential spots (induced, up- and down-regulated and repressed) were observed in the protein profiles of the bacterium cultivated in citrus extract (susceptible host) when compared to that of MM1. The 2-DE profile of the bacterium cultured in the complex medium nutrient yeast glycerol was also obtained and the comparison with that of MM1 revealed 36 differential spots. Five proteins from the different treatments were successfully N-terminally sequenced and the putative functions were assigned by homology searches in databases. Two constitutively expressed proteins, B4 and B5, were identified as pseudouridine synthase and elongation factor P, respectively. The large subunit of ribulose 1,5-biphosphate carboxylase/oxygenase and a sulfate binding protein were found as specifically up-regulated in the presence of citrus extracts. Finally, the heat shock protein G was found exclusively in the complex medium and repressed in all other media.
Assuntos
Proteínas de Bactérias/metabolismo , Extratos Vegetais/química , Folhas de Planta/química , Proteínas de Plantas/metabolismo , Xanthomonas/química , Proteínas de Bactérias/química , Citrus/química , Citrus/microbiologia , Eletroforese em Gel Bidimensional , Passiflora/química , Passiflora/microbiologia , Folhas de Planta/microbiologia , Proteínas de Plantas/química , Xanthomonas/genética , Xanthomonas/patogenicidadeRESUMO
The genetic diversity of 55 xanthomonad strains isolated from passion fruit plants (Passiflora spp.) and identified as Xanthomonas campestris pv. passiflorae was initially assessed by randomly amplified polymorphic DNA (RAPD) analysis. The strains showed a high level of polymorphism with almost unique fingerprints. Fifteen clusters with a similarity of approximately 70% were identified, three of which were prevalent. There was a correlation between the clusters and the geographic origin of the strains. A representative strain of each cluster, together with the pathovar reference strain, were used to verify the relationships of these strains to 18 Xanthomonas species and Pseudomonas syringae pv. passiflorae. All Xanthomonas species yielded a unique RAPD profile and no consistent relatedness to the X. campestris pv. passiflorae strains was observed. Amplification products were also analysed by repetitive (rep) primers (BOX, ERIC and REP), RFLP of the 16S-23S rDNA intergenic spacer and SDS-PAGE of whole-cell proteins. All of these approaches generated profiles characteristic for each Xanthomonas species but the taxonomic position of the X. campestris pv. passiflorae strains could not be unequivocally assigned. Finally, DNA-DNA hybridization allowed a sound taxonomic allocation of the strains to Xanthomonas axonopodis pv. passiflorae.
Assuntos
Variação Genética , Rosales/microbiologia , Xanthomonas/classificação , Xanthomonas/genética , Técnicas de Tipagem Bacteriana , Impressões Digitais de DNA , DNA Bacteriano/genética , DNA Ribossômico/genética , Eletroforese em Gel de Poliacrilamida , Genótipo , Hibridização de Ácido Nucleico , Doenças das Plantas/microbiologia , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Técnica de Amplificação ao Acaso de DNA PolimórficoRESUMO
The indigenous multicopy miniplasmid (pXG33) of Xanthomonas campestris pv. glycines was entirely sequenced and evaluated as a cloning vector for Xanthomonas. The pXG33 contains 1738 bp and the nucleotide sequence revealed a consensus nicking site (TGATA) described for the pC194 family of rolling-circle replicating (RCR) plasmids. This nicking site is embebbed in a region of high potential to form a number of stem-loop structures. The predicted protein (Rep) showed conserved amino acid residues and potential catalytic regions, containing conserved Tyr and Glu residues. These results indicate that pXG33 replicates by a rolling-circle mechanism. For use as a cloning vector for Xanthomonas, a fragment containing the kanamycin resistance gene (aphA) and the stabilization locus (parB) was inserted into pXG33. The new construct, of 3.4 kb, was designated pXG31. By deletion of the parB locus and using pBluescript KS(+) as an intermediate, pXG40 (2.8 kb), containing unique restriction sites for BamHI, EcoRI, SacI, and KpnI at the ends of the kanamycin resistance gene, was generated. Both constructs showed stability in Xanthomonas during 18 h of growth or 72 h of fermentation, high-copy number, and no interference with pathogenicity. pXG31 and pXG40, however, were incapable of duplication in Escherichia coli and a shuttle vector (pKX33) was constructed by inactivation of some restriction sites of pXG40 and ligation to the cloning vector pBluescript KS(+). pKX33 is nonconjugative, is multicopy, is of low molecular weight (5.7 kb), presents antibiotic resistance markers for ampicillin and kanamycin, has unique restriction sites for KpnI, SalI, EcoRV, EcoRI, BamHI, XbaI, and SacI, and can be used directly for sequencing with universal primers. It can be maintained in E. coli and several species and pathovars of Xanthomonas.
Assuntos
Vetores Genéticos , Plasmídeos , Xanthomonas campestris/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Bacteriano , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
Ribosomal RNA gene restriction patterns have been determined for 43 strains of Bacillus thuringiensis representing 10 serovars and eight reference strains of B. anthracis, B. cereus and B. mycoides. Strains within a B. thuringiensis serovar produced highly related or identical ribotype patterns: in particular, 12 strains of serovar israelensis, five strains of serovar kurstaki, two strains of serovar galleriae and three strains of serovar aizawa produced ribotype patterns consistent with serotype designations. Moreover, variety tenebrionis (serotype 8a8b), a coleopteran pathogen, could be distinguished from the more common lepidopteran pathogens of this serotype (serovar morrisoni) by ribotyping. The correlation of ribotype patterns with serotype suggests a clonal population structure for B. thuringiensis.
Assuntos
Bacillus thuringiensis/classificação , Bacillus thuringiensis/genética , Toxinas Bacterianas , Técnicas de Tipagem Bacteriana , DNA Ribossômico/genética , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 16S/genética , Bacillus/classificação , Bacillus/genética , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Sequência de Bases , Análise por Conglomerados , Endotoxinas/genética , Proteínas Hemolisinas , Dados de Sequência Molecular , SorotipagemRESUMO
The stabilization locus parB was subcloned into the broad host range plasmid pAP2, which contains the alpha-amylase gene from Bacillus subtilis, and introduced into Xanthomonas campestris pv campestris and X.c.pv manihotis. Analysis of the stability of plasmid pAP2 (parB-) and pAP23 (parB+) showed that the parB locus decreased significantly the plasmid loss rate mainly by X.c.pv campestris. The lower efficiency of stabilization in X.c.pv manihotis was probably due to the incompatibility system between the native plasmids and the newly introduced pAP23. Although parB had conferred higher stability, it determined a lower rate of alpha-amylase activity even by the strain Cm where its stabilization rate was higher.
Assuntos
Expressão Gênica/genética , Plasmídeos/genética , Xanthomonas campestris/genética , Alelos , Clonagem Molecular , Expressão Gênica/fisiologia , Xanthomonas campestris/crescimento & desenvolvimento , alfa-Amilases/biossínteseRESUMO
O plasmídio híbrido pAP1, contendo o gene da alfa-amilase de Bacillus subtilis inserido no plasmídio pMFY-40, foi introduzido em células amilolíticas e näo amilolíticas de Xanthomonas campestris pv. campestris e pv. manhihotis. Os transformantes obtidos foram analisados quanto à viscosidade produzida em caldos de fermentaçäo contendo scarose e/ou amido como fontes de carbono. os valores de viscosidade mais altos foram obtidos em meio de sacarose 2% e os mais baixos em emio de sacarose 1% para as linhagens Ca 1120 e meio de amido 2% para as linhagens Ma3281. O gene da alfa-amilase clonado apresentou boa expressäo em linhagem näo amilolíticas e o aumento da viscosidade foi altamente significativo em emio de sacarose + amido mas näo em meio contendo apenas amido
