Whole-genome expression profiling of Xylella fastidiosa in response to growth on glucose

Xylella fastidiosa is the etiologic agent of diseases in a wide range of economically important crops including citrus variegated chlorosis, a major threat to the Brazilian citrus industry. The genomes of several strains of this phytopathogen have been completely sequenced enabling large-scale functional studies. In this work we used whole-genome DNA microarrays to investigate the transcription profile of X. fastidiosa grown in defined media with different glucose concentrations. Our analysis revealed that while transcripts related to fastidian gum production were unaffected, colicin-V-like and fimbria precursors were induced in high glucose medium. Based on these results, we suggest a model for colicin-defense mechanism in X. fastidiosa.

Genomics-based design of defined growth media for the plant pathogen Xylella fastidiosa

Based on the genetic analysis of the phytopathogen Xylella fastidiosa genome, five media with defined composition were developed and the growth abilities of this fastidious prokaryote were evaluated in liquid media and on solid plates. All media had a common salt composition and included the same amounts of glucose and vitamins but differed in their amino acid content. XDM1 medium contained amino acids threonine, serine, glycine, alanine, aspartic acid and glutamic acid, for which complete degradation pathways occur in X fastidiosa; XDM2 included serine and methionine, amino acids for which biosynthetic enzymes are absent, plus asparagine and glutamine, which are abundant in the xylem sap; XDM3 had the same composition as XDM2 but with asparagine replaced by aspartic acid due to the presence of complete degradation pathway for aspartic acid; XDM4 was a minimal medium with glutamine as a sole nitrogen source; XDM5 had the same composition as XDM4, plus methionine. The liquid and solidified XDM2 and XDM3 media were the most effective for the growth of X. fastidiosa. This work opens the opportunity for the in silico design of bacterial defined media once their genome is sequenced. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier B.V. B.V. All rights reserved.

Growth optimization procedures for the phytopathogen Xylella fastidiosa

For the first time, growth curves are shown for the phytopathogen Xylella fastidiosa on traditional growth media such as PW (periwinkle wilt), BCYE (buffered charcoal yeast extract), and on new ones such as GYE (glutamate yeast extract) and PYE (phosphate yeast extract) that were developed in this work. The optimal growth conditions on solid and liquid media as well as their measurements are presented, by using total protein content and turbidity determinations. The results demonstrated that yeast extract provided sufficient nutrients for X. fastidiosa, since the cells grew well on PYE medium.

Xylella fastidiosa gene expression analysis by DNA microarrays

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Crystallization and preliminary X-ray diffraction analysis of a glutathione S-transferase from Xylella fastidiosa

Glutathione S-transferases (GSTs) form a group of multifunctional isoenzymes that catalyze the glutathione-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GST from Xylella fastidiosa (xfGST) was overexpressed in Escherichia coli and purified by conventional affinity chromatography. In this study, the crystallization and preliminary X-ray analysis of xfGST is described. The purified protein was crystallized by the vapour-diffusion method, producing crystals that belonged to the triclinic space group P1. The unit-cell parameters were a = 47.73, b = 87.73, c = 90.74 angstrom, alpha = 63.45, beta = 80.66, gamma = 94.55 degrees. xfGST crystals diffracted to 2.23 angstrom resolution on a rotating-anode X-ray source.

Development of a scar marker for Pierce's Disease strains of Xylella fastidiosa

O objetivo deste trabalho foi desenvolver um oligonucleotídeo iniciador para reação em cadeia da polimerase (PCR) específico para as estirpes de Xylella fastidiosa que causam o mal de Pierce (PD) em videira (Vitis vinifera). Amplificações de DNA de 23 diferentes hospedeiros, usando o conjunto de oligonucleotídeos REP1-R (5'-IIIICGICGIATCCIGGC-3') e REP 2 (5'-ICGICTTATCI GGCCTAC-3') utilizando o programa: 94 ºC/2 min; 35 X (94 ºC/1 min, 45 ºC/1 min; 72 ºC/1 min and 30 s) 72 ºC/5 min, produziu um fragmento de 630 pb que diferenciou as estirpes de videiras dos demais. Entretanto, padrões de bandeamento REP não são considerados confiáveis para detecção devido ao par de oligonucleotídeos REP 1 e REP 2 corresponderem a seqüências repetitivas encontradas por todo o genoma bacteriano. Desse modo, o produto amplificado de 630 pb foi eluído do gel de agarose, purificado e seqüenciado. A informação da seqüência nucleotídica foi usada para identificar e sintetizar um oligonucleotídeo específico para o isolado de X. fastidiosa causadora do mal de Pierce denominado Xf-1 (5'-CGGGGGTGTAGGAGGGGTTGT-3'), que foi utilizado juntamente com o oligonucleotídeo REP-2 nas condições 94 ºC/2 min; 35 X (94 ºC/1 min, 62 ºC/1 min; 72 ºC/1 min and 30 s) 72 ºC/10 min. Os DNAs das estirpes de X. fastidiosa de outros hospedeiros [amêndoa (Prumus amygdalus), citros (Citrus spp.), café (Coffea arabica), olmo (Ulmus americana), amora (Morus rubra), carvalho (Quercus rubra), vinca (Catharantus roseus), ameixa (Prunus salicina) e ragweed (Ambrosia artemisiifolia)] e de bactérias Gram negativas e positivas foram submetidos a amplificação com o conjunto de oligonucleotídeos Xf-1/REP 2. Um fragmento, de aproximadamente 350 pb, foi amplificado apenas com o DNA de X. fastidiosa isolada de videira.

Carbohydrate metabolism of Xylella fastidiosa: Detection of glycolytic and pentose phosphate pathway enzymes and cloning and expression of the enolase gene

The objective of this work was to assess the functionality of the glycolytic pathways in the bacterium Xylella fastidiosa. To this effect, the enzymes phosphoglucose isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase of the glycolytic pathway, and glucose 6-phosphate dehydrogenase of the Entner-Doudoroff pathway were studied, followed by cloning and expression studies of the enolase gene and determination of its activity. These studies showed that X. fastidiosa does not use the glycolytic pathway to metabolize carbohydrates, which explains the increased duplication time of this phytopatogen. Recombinant enolase was expressed as inclusion bodies and solubilized with urea (most efficient extractor), Triton X-100, and TCA. Enolase extracted from X. fastidiosa and from chicken muscle and liver is irreversibly inactivated by urea. The purification of enolase was partial and resulted in a low yield. No enzymatic activity was detected for either recombinant and native enolases, aldolase, and glyceraldehyde-3-phosphate dehydrogenase, suggesting that X. fastidiosa uses the Entner-Doudoroff pathway to produce pyruvate. Evidence is presented supporting the idea that the regulation of genes and the presence of isoforms with regulation patterns might make it difficult to understand the metabolism of carbohydrates in X. fastidiosa.

Strains of Xylella fastidiosa rapidly distinguished by arbitrarily primed-PCR

Genomic DNAs isolated from strains of Xylella fastidiosa that caused citrus variegated chlorosis, coffee leaf scorch, Pierce's Disease of grapevine, and plum leaf scorch were analyzed by arbitrarily primed polymerase chain reaction. Purified DNA was amplified under nonstringent conditions with single primers 21 nucleotides (nt) long. Thirty-nine amplification products were observed that were useful to distinguish among the strains and to derive a similarity matrix and construct a phenogram showing possible relationships among the strains. Strains isolated from diseased coffee and citrus in Brazil were closely related to each other (coefficient of similarity of 0.872), but only distantly related to a strain isolated from diseased grapevine in the USA (coefficient of similarity of 0.650). Strains of Xylella fastidiosa isolated from diseased plums in the USA and Brazil clustered with strains from different hosts isolated from their respective countries of origin. Thus, there may be two quite dissimilar clusters of strains of Xylella fastidiosa, one in North America and the other in South America. Each cluster contains strains that can cause disease in plum. The methods described provide a convenient and rapid method to distinguish between strains of Xylella fastidiosa that cause diseases of coffee and citrus in the same region of Brazil. This has not been possible previously. This will potentially enable the two strains to be distinguished in alternate hosts or in insect vectors.

Strain variability in the DNA immigration control region (ICR) of Xylella fastidiosa

The genome of the bacterium Xylella fastidiosa contains four ORFs (XF2721, XF2725, XF2739 and XF0295) related to the restriction modification type I system, ordinarily named R-M. This system belongs to the DNA immigration control region (ICR). Each CIRF is related to different operon structures, which are homologues among themselves and with subunit Hsd R from the endonuclease coding genes. In addition, these ORFs are highly homologous to genes in Pseudomonas aeruginosa, Methylococcus capsulatus str. Bath, Legionella pneumophila, Helicobacter pylori, Xanthomonas oryzae pv. Oryzae and Silicibacter pomeroyi, as well as to genes from X. fastidiosa strains that infect grapevine, almond and oleander plants. This study was carried out on R-M ORFs from forty-three X. fastidiosa strains isolated from citrus, coffee, grapevine, periwinkle, almond and plum trees, in order to assess the genetic diversity of these loci through PCR-RFLP. PCR-RFLP analysis of the four ORFs related to the R-M system from these strains enabled the detection of haplotypes for these loci. When the haplotypes were defined, wide genetic diversity and a large range of similar strains originating from different hosts were observed. This analysis also provided information indicating differences in population genetic structures, which led to detection of different levels of gene transfer among the groups of strains. (c) 2005 Elsevier SAS. All rights reserved.

Molecular models for shikimate pathway enzymes of Xylella fastidiosa

The Xylella fastidiosa is a bacterium that is the cause of citrus variegated chlorosis (CVC). The shikimate pathway is of pivotal importance for production of a plethora of aromatic compounds in plants, bacteria, and fungi. Putative structural differences in the enzymes from the shikimate pathway, between the proteins of bacterial origin and those of plants, could be used for the development of a drug for the control of CVC. However, inhibitors for shikimate pathway enzymes should have high specificity for X. fastidiosa enzymes, since they are also present in plants. In order to pave the way for structural and functional efforts towards antimicrobial agent development, here we describe the molecular modeling of seven enzymes of the shikimate pathway of X. fastidiosa. The structural models of shikimate pathway enzymes, complexed with inhibitors, strongly indicate that the previously identified inhibitors may also inhibit the X. fastidiosa enzymes. (C) 2004 Elsevier B.V. All rights reserved.

Production of DNA microarray and expression analysis of genes from Xylella fastidiosa in different culture media

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

CO2 assimilation, photosynthetic light response curves, and water relations of 'Pêra' sweet orange plants infected with Xylella fastidiosa

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparative analysis of differentially expressed sequence tags of sweet orange and mandarin infected with Xylella fastidiosa

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

A triply cloned strain of Xylella fastidiosa multiplies and induces symptoms of citrus variegated chlorosis in sweet orange

Xylella fastidiosa isolate 8.1,b obtained from a sweet orange tree affected by citrus variegated chlorosis in the state of Sb Paulo, Brazil, and shown in 1993 to be the causal agent of the disease, was cloned by repeated culture in liquid and on solid PW medium, yielding triply cloned strain 9a5c. The eighth and the 16th passages of strain 9a5c were mechanically inoculated into sweet orange plants. Presence of X. fastidiosa in sweet orange leaves of shoots having grown after inoculation (first-flush shoots) was detected by DAS-ELISA and PCR. Thirty-eight days after inoculation, 70% of the 20 inoculated plants rested positive, and all plants gave strong positive reactions 90 days after inoculation. Symptoms first appeared after 3 months and were conspicuous after 5 months. X. fastidiosa was reisolated from sweet orange leaves, 44 days after inoculation. These results indicate that X. fastidiosa strain 9a5c, derived from pathogenic isolate 8.1.b by triply cloning, is also pathogenic, Strain 9a5c is now used for the X. fastidiosa genome sequencing project undertaken on a large scale in Brazil.

Expression of pathogenicity-related genes of Xylella fastidiosa in vitro and in planta

Xylella fastidiosa is responsible for several economically important plant diseases. It is currently assumed that the symptoms are caused by vascular occlusion due to biofilm formation. Microarray technology was previously used to examine the global gene expression profile of X. filstidiosa freshly isolated from symptomatic plants or after several passages by axenic culture medium, and different pathogenicity profiles have been obtained. In the present study the expression of some pathogenicity-related genes was evaluated in vitro and in planta by RT-PCR. The results suggest that adhesion is important at the beginning of biofilm formation, while the genes related to adaptation are essential for the organism's maintenance in planta. Similar results were observed in vitro mainly for the adhesion genes. The pattern of expression observed suggests that adhesion modulates biofilm formation whereas the expression of some adaptation genes may be related to the environment in which the organism is living.