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PubMed2002

Whole-genome comparative analysis of three phytopathogenic Xylella fastidiosa strains.

Xylella fastidiosa (Xf) causes wilt disease in plants and is responsible for major economic and crop losses globally. Owing to the public importance of this phytopathogen we embarked on a comparative analysis of the complete genome of Xf pv citrus and the partial genomes of two recently sequenced strains of this species: Xf pv almond and Xf pv oleander, which cause leaf scorch in almond and oleander plants, respectively. We report a reanalysis of the previously sequenced Xf 9a5c (CVC, citrus) strain and the two "gapped" Xf genomes revealing ORFs encoding critical functions in pathogenicity and conjugative transfer. Second, a detailed whole-genome functional comparison was based on the three sequenced Xf strains, identifying the unique genes present in each strain, in addition to those shared between strains. Third, an "in silico" cellular reconstruction of these organisms was made, based on a comparison of their core functional subsystems that led to a characterization of their conjugative transfer machinery, identification of potential differences in their adhesion mechanisms, and highlighting of the absence of a classical quorum-sensing mechanism. This study demonstrates the effectiveness of comparative analysis strategies in the interpretation of genomes that are closely related.

Bacterial Proteins↗
PubMed2000

Mapping analysis of the Xylella fastidiosa genome.

A cosmid library was made of the 2.7 Mb genome of the Gram-negative plant pathogenic bacterium Xylella fastidiosa and analysed by hybridisation mapping. Clones taken from the library as well as genomic restriction fragments of rarely cutting enzymes were used as probes. The latter served as a backbone for ordering the initial map contigs and thus facilitated gap closure. Also, the co-linearity of the cosmid map, and thus the eventual sequence, could be confirmed by this process. A subset of the eventual clone coverage was distributed to the Brazilian X.FASTIDIOSA: sequencing network. Data from this effort confirmed more quantitatively initial results from the hybridisation mapping that the redundancy of clone coverage ranged between 0 and 45-fold across the genome, while the average was 15-fold by experimental design. Reasons for this not unexpected fluctuation and the actual gaps are being discussed, as is the use of this effect for functional studies.

Brazil↗
PubMed2001

Transposon mutagenesis of Xylella fastidiosa by electroporation of Tn5 synaptic complexes.

Pierce's disease, a lethal disease of grapevine, is caused by Xylella fastidiosa, a gram-negative, xylem-limited bacterium that is transmitted from plant to plant by xylem-feeding insects. Strains of X. fastidiosa also have been associated with diseases that cause tremendous losses in many other economically important plants, including citrus. Although the complete genome sequence of X. fastidiosa has recently been determined, the inability to transform or produce transposon mutants of X. fastidiosa has been a major impediment to understanding pathogen-, plant-, and insect-vector interactions. We evaluated the ability of four different suicide vectors carrying either Tn5 or Tn10 transposons as well as a preformed Tn5 transposase-transposon synaptic complex (transposome) to transpose X. fastidiosa. The four suicide vectors failed to produce any detectable transposition events. Electroporation of transposomes, however, yielded 6 x 10(3) and 4 x 10(3) Tn5 mutants per microg of DNA in two different grapevine strains of X. fastidiosa. Molecular analysis showed that the transposition insertions were single, independent, stable events. Sequence analysis of the Tn5 insertion sites indicated that the transpositions occur randomly in the X. fastidiosa genome. Transposome-mediated mutagenesis should facilitate the identification of X. fastidiosa genes that mediate plant pathogenicity and insect transmission.

Animals↗
PubMed2002

A new regulatory DNA motif of the gamma subclass Proteobacteria: identification of the LexA protein binding site of the plant pathogen Xylella fastidiosa.

Escherichia coli LexA protein is the repressor of a gene network whose members are directly involved in the repair of damaged DNA and in the survival of bacterial cells until DNA lesions have been eliminated. The lexA gene is widely present in bacteria, although the sequences of only three LexA-binding sites are known: Gram-positive, alpha Proteobacteria and some members of gamma Proteobacteria represented by E. coli. Taking advantage of the fact that the genome sequence of the plant-pathogenic bacterium Xylella fastidiosa has been determined, its lexA gene has been cloned and overexpressed in E. coli to purify its product. After demonstration that X. fastidiosa lexA and recA genes are co-transcribed, gel mobility shift assays and directed mutagenesis experiments using the promoter of the lexA-recA transcriptional unit demonstrated that the X. fastidiosa LexA protein specifically binds the imperfect palindrome TTAGN(6)TACTA. This is the first LexA binding sequence identified in the gamma Proteobacteria differing from the E. coli-like LexA box. Although a computational search has revealed the presence of TTAGN(6)TACTA-like motifs upstream of X. fastidiosa genes other than lexA, X. fastidiosa LexA only binds the promoter of one of them, XF2313, encoding a putative DNA-modification methylase. Moreover, X. fastidiosa LexA protein does not bind any of the other genes whose homologues are regulated by the LexA repressor in E. coli (uvrA, uvrB, ssb, ruvAB, ftsK, dinG, recN and ybfE). RT-PCR quantitative analysis has also demonstrated that lexA-recA and XF2313 genes, as well as the X. fastidiosa genes which are homologues to those of E. coli belonging to the LexA regulon, with the exception of ssb, are DNA damage-inducible in X. fastidiosa.

Bacterial Proteins↗
PubMed1999

A simplified subtractive hybridization protocol used to isolate DNA sequences specific to Xylella fastidiosa.

A simplified protocol of subtractive hybridization based on the technique of L. M. Kunkel, A. P. Monaco, W. Middlesworth, H. D. Ochs & S. A. Latt (1985, Proc Natl Acad Sci USA, 82, 4778-4782) was used to obtain DNA sequences specific to Xylella fastidiosa isolated from diseased citrus plants. As a driver, DNA extracted from bacteria showing different degrees of relatedness was used: Xy. fastidiosa 788 isolated from another host (plum), Xanthomonas campestris pv. campestris and Burkholderia gladioli strains. A DNA fragment, f14, showing no hybridization to the driver DNA, was used as a probe specific to Xy. fastidiosa from citrus and oleander. This fragment was sequenced and the predicted protein showed 40% similarity to the central region of flagellin of Escherichia coli serotypes H1 and H12. A pair of internal primers (f14-1 and f14-2) was designed for amplification of Xy. fastidiosa DNA. These primers detected Xy. fastidiosa strains isolated from citrus and oleander and yielded an amplification product of about 600 bp. They were also able to detect the bacteria in extracts from citrus plants with or without symptoms of disease. No amplification reaction was obtained using DNA extracted from other species and pathovars of Xanthomonas, Pseudomonas cichorii, Erwinia carotovora, Agrobacterium tumefaciens and phytopathogens of citrus (Xanthomonas axonopodis pv. citri) and coffee (Burkholderia andropogonis, P. cichorii, Pseudomonas syringae pv. garcae). The isolation of a DNA fragment specific to Xy. fastidiosa from citrus showed that the simplified protocol of subtractive hybridization used in this work is potentially applicable to other micro-organisms.

Amino Acid Sequence↗
PubMed2003

Microarray analyses of Xylella fastidiosa provide evidence of coordinated transcription control of laterally transferred elements.

Genetically distinct strains of the plant bacterium Xylella fastidiosa (Xf) are responsible for a variety of plant diseases, accounting for severe economic damage throughout the world. Using as a reference the genome of Xf 9a5c strain, associated with citrus variegated chlorosis (CVC), we developed a microarray-based comparison involving 12 Xf isolates, providing a thorough assessment of the variation in genomic composition across the group. Our results demonstrate that Xf displays one of the largest flexible gene pools characterized to date, with several horizontally acquired elements, such as prophages, plasmids, and genomic islands (GIs), which contribute up to 18% of the final genome. Transcriptome analysis of bacteria grown under different conditions shows that most of these elements are transcriptionally active, and their expression can be influenced in a coordinated manner by environmental stimuli. Finally, evaluation of the genetic composition of these laterally transferred elements identified differences that may help to explain the adaptability of Xf strains to infect such a wide range of plant species.

Chromosome Mapping↗
PubMed2004

Crystallization and preliminary X-ray diffraction analysis of an oxidized state of Ohr from Xylella fastidiosa.

Xylella fastidiosa organic hydroperoxide-resistance protein (Ohr) is a dithiol-dependent peroxidase that is widely conserved in several pathogenic bacteria with high affinity for organic hydroperoxides. The protein was crystallized using the hanging-drop vapour-diffusion method in the presence of PEG 4000 as precipitant after treatment with organic peroxide (t-butyl hydroperoxide). X-ray diffraction data were collected to a maximum resolution of 1.8 A using a synchrotron-radiation source. The crystal belongs to the hexagonal space group P6(5)22, with unit-cell parameters a = b = 87.66, c = 160.28 A. The crystal structure was solved by molecular-replacement methods. The enzyme has a homodimeric quaternary structure similar to that observed for its homologue from Pseudomonas aeruginosa, but differs from the previous structure as the active-site residue Cys61 is oxidized. Structure refinement is in progress.

Bacterial Proteins↗
PubMed2001

Fastidian gum: the Xylella fastidiosa exopolysaccharide possibly involved in bacterial pathogenicity.

The Gram-negative bacterium Xylella fastidiosa was the first plant pathogen to be completely sequenced. This species causes several economically important plant diseases, including citrus variegated chlorosis (CVC). Analysis of the genomic sequence of X. fastidiosa revealed a 12 kb DNA fragment containing an operon closely related to the gum operon of Xanthomonas campestris. The presence of all genes involved in the synthesis of sugar precursors, existence of exopolysaccharide (EPS) production regulators in the genome, and the absence of three of the X. campestris gum genes suggested that X. fastidiosa is able to synthesize an EPS different from that of xanthan gum. This novel EPS probably consists of polymerized tetrasaccharide repeating units assembled by the sequential addition of glucose-1-phosphate, glucose, mannose and glucuronic acid on a polyprenol phosphate carrier.

Bacterial Proteins↗
PubMed2002

Transposon Tn1721 distribution among strains of Xylella fastidiosa.

Transposons are mobile genetic elements found within the genomes of various organisms including bacteria, fungi, plants and animals. Fragments of the transposon Tn1721 were found included in the genome of Xylella fastidiosa strain 9a5c. Regions from such fragments were PCR-amplified using specially designed primers (TNP(1) and TNP(2)). In order to detect insertions of the Tn1721 element, both primers were used and one of them included a region of the transposon (TNP(1)) and the other one had the right repeat and part of the bacterial chromosome (TNP(2)). The PCR products obtained from strain 9a5c were used as a pattern for fragment size comparisons when DNA samples from other X. fastidiosa strains were used as template for the PCR assays. Differences were observed concerning the PCR products of such amplifications when some X. fastidiosa strains isolated from grapevine and plum were used. For the citrus-derived strains only the strains U187d and GP920b produced fragments with different sizes or weak band intensity. Such variations in the X. fastidiosa genome related to disrupted Tn1721 copies are probably due to the possibility of such a transposon element being still able to duplicate even after deletion events might have taken place and also because the bacterial strains in which the main differences were detected are derived from different host plants cultivated under different climate conditions from the one used as reference.

Base Sequence↗
PubMed2002

Competitive hybridization on spotted microarrays as a tool to conduct comparative genomic analyses of Xylella fastidiosa strains.

Xylella fastidiosa strains are responsible for several plant diseases and since such isolates display a broad host range and complex biological behavior, genomic comparisons employing microarray hybridizations may provide an effective method to compare them. Thus, we performed a thorough validation of this type of approach using two recently sequenced strains of this phytopathogen. By matching microarray hybridization results to direct sequence comparisons, we were able to establish precise cutoff ratios for common and exclusive sequences, allowing the identification of exclusive genes involved in important biological traits. This validation will enable the use of microarray-based comparisons across a wide variety of microorganisms

Genome, Bacterial↗
PubMed1995

Randomly amplified polymorphic DNA analysis of Xylella fastidiosa Pierce's disease and oak leaf scorch pathotypes.

Randomly amplified polymorphic DNA analysis was conducted with 14 primers to 17 strains of Xylella fastidiosa. There was a high degree of similarity among the seven Pierce's disease (PD) strains (Sxy > 0.93) and the seven oak leaf scorch (OLS) strains (Sxy > 0.96). However, the two groups were different, with a similarity index of 0.67, confirming the presence of a PD DNA cluster and suggesting the presence of a new OLS cluster. The control plum leaf scald strains (two strains) together with the periwinkle wilt strain had a much smaller similarity index (0.44) compared with the PD and OLS clusters.

Base Sequence↗
PubMed2001

Genetic diversity of Pierce's disease strains and other pathotypes of Xylella fastidiosa.

Strains of Xylella fastidiosa isolated from grape, almond, maple, and oleander were characterized by enterobacterial repetitive intergenic consensus sequence-, repetitive extragenic palindromic element (REP)-, and random amplified polymorphic DNA (RAPD)-PCR; contour-clamped homogeneous electric field (CHEF) gel electrophoresis; plasmid content; and sequencing of the 16S-23S rRNA spacer region. Combining methods gave greater resolution of strain groupings than any single method. Strains isolated from grape with Pierce's disease (PD) from California, Florida, and Georgia showed greater than previously reported genetic variability, including plasmid contents, but formed a cluster based on analysis of RAPD-PCR products, NotI and SpeI genomic DNA fingerprints, and 16S-23S rRNA spacer region sequence. Two groupings of almond leaf scorch (ALS) strains were distinguished by RAPD-PCR and CHEF gel electrophoresis, but some ALS isolates were clustered within the PD group. RAPD-PCR, CHEF gel electrophoresis, and 16S-23S rRNA sequence analysis produced the same groupings of strains, with RAPD-PCR resolving the greatest genetic differences. Oleander strains, phony peach disease (PP), and oak leaf scorch (OLS) strains were distinct from other strains. DNA profiles constructed by REP-PCR analysis were the same or very similar among all grape strains and most almond strains but different among some almond strains and all other strains tested. Eight of 12 ALS strains and 4 of 14 PD strains of X. fastidiosa isolated in California contained plasmids. All oleander strains carried the same-sized plasmid; all OLS strains carried the same-sized plasmid. A plum leaf scald strain contained three plasmids, two of which were the same sizes as those found in PP strains. These findings support a division of X. fastidiosa at the subspecies or pathovar level.

Bacterial Typing Techniques↗
PubMed2001

Stable transformation of the Xylella fastidiosa citrus variegated chlorosis strain with oriC plasmids.

Xylella fastidiosa is a gram-negative, xylem-limited bacterium affecting economically important crops (e.g., grapevine, citrus, and coffee). The citrus variegated chlorosis (CVC) strain of X. fastidiosa is the causal agent of this severe disease of citrus in Brazil and represents the first plant-pathogenic bacterium for which the genome sequence was determined. Plasmids for the CVC strain of X. fastidiosa were constructed by combining the chromosomal replication origin (oriC) of X. fastidiosa with a gene which confers resistance to kanamycin (Kan(r)). In plasmid p16KdAori, the oriC fragment comprised the dnaA gene as well as the two flanking intergenic regions, whereas in plasmid p16Kori the oriC fragment was restricted to the dnaA-dnaN intergenic region, which contains dnaA-box like sequences and AT-rich clusters. In plasmid p16K, no oriC sequence was present. In the three constructs, the promoter region of one of the two X. fastidiosa rRNA operons was used to drive the transcription of the Kan(r) gene to optimize the expression of kanamycin resistance in X. fastidiosa. Five CVC X. fastidiosa strains, including strain 9a5c, the genome sequence of which was determined, and two strains isolated from coffee, were electroporated with plasmid p16KdAori or p16Kori. Two CVC isolates, strains J1a12 and B111, yielded kanamycin-resistant transformants when electroporated with plasmid p16KdAori or p16Kori but not when electroporated with p16K. Southern blot analyses of total DNA extracted from the transformants revealed that, in all clones tested, the plasmid had integrated into the host chromosome at the promoter region of the rRNA operon by homologous recombination. To our knowledge, this is the first report of stable transformation in X. fastidiosa. Integration of oriC plasmids into the X. fastidiosa chromosome by homologous recombination holds considerable promise for functional genomics by specific gene inactivation.

Base Sequence↗
PubMed2001

Differentiation of strains of Xylella fastidiosa by a variable number of tandem repeat analysis.

Short sequence repeats (SSRs) with a potential variable number of tandem repeat (VNTR) loci were identified in the genome of the citrus pathogen Xylella fastidiosa and used for typing studies. Although mono- and dinucleotide repeats were absent, we found several intermediate-length 7-, 8-, and 9-nucleotide repeats, which we examined for allelic polymorphisms using PCR. Five genuine VNTR loci were highly polymorphic within a set of 27 X. fastidiosa strains from different hosts. The highest average Nei's measure of genetic diversity (H) estimated for VNTR loci was 0.51, compared to 0.17 derived from randomly amplified polymorphic DNA (RAPD) analysis. For citrus X. fastidiosa strains, some specific VNTR loci had a H value of 0.83, while the maximum value given by specific RAPD loci was 0.12. Our approach using VNTR markers provides a high-resolution tool for epidemiological, genetic, and ecological analysis of citrus-specific X. fastidiosa strains.

Bacterial Typing Techniques↗
PubMed2002

Diversity of endophytic bacterial populations and their interaction with Xylella fastidiosa in citrus plants.

Citrus variegated chlorosis (CVC) is caused by Xylella fastidiosa, a phytopathogenic bacterium that can infect all Citrus sinensis cultivars. The endophytic bacterial communities of healthy, resistant, and CVC-affected citrus plants were studied by using cultivation as well as cultivation-independent techniques. The endophytic communities were assessed in surface-disinfected citrus branches by plating and denaturing gradient gel electrophoresis (DGGE). Dominant isolates were characterized by fatty-acid methyl ester analysis as Bacillus pumilus, Curtobacterium flaccumfaciens, Enterobacter cloacae, Methylobacterium spp. (including Methylobacterium extorquens, M. fujisawaense, M. mesophilicum, M. radiotolerans, and M. zatmanii), Nocardia sp., Pantoea agglomerans, and Xanthomonas campestris. We observed a relationship between CVC symptoms and the frequency of isolation of species of Methylobacterium, the genus that we most frequently isolated from symptomatic plants. In contrast, we isolated C. flaccumfaciens significantly more frequently from asymptomatic plants than from those with symptoms of CVC while P. agglomerans was frequently isolated from tangerine (Citrus reticulata) and sweet-orange (C. sinensis) plants, irrespective of whether the plants were symptomatic or asymptomatic or showed symptoms of CVC. DGGE analysis of 16S rRNA gene fragments amplified from total plant DNA resulted in several bands that matched those from the bacterial isolates, indicating that DGGE profiles can be used to detect some endophytic bacteria of citrus plants. However, some bands had no match with any isolate, suggesting the occurrence of other, nonculturable or as yet uncultured, endophytic bacteria. A specific band with a high G+C ratio was observed only in asymptomatic plants. The higher frequency of C. flaccumfaciens in asymptomatic plants suggests a role for this organism in the resistance of plants to CVC.

Bacillus cereus↗
PubMed2002

Evaluation of the genetic structure of Xylella fastidiosa populations from different Citrus sinensis varieties.

Xylella fastidiosa was isolated from sweet orange plants (Citrus sinensis) grown in two orchards in the northwest region of the Brazilian state of São Paulo. One orchard was part of a germ plasm field plot used for studies of citrus variegated chlorosis resistance, while the other was an orchard of C. sinensis cv. Pêra clones. These two collections of strains were genotypically characterized by using random amplified polymorphic DNA (RAPD) and variable number of tandem repeat (VNTR) markers. The genetic diversity (H(T)) values of X. fastidiosa were similar for both sets of strains; however, H(T)(RAPD) values were substantially lower than H(T)(VNTR) values. The analysis of six strains per plant allowed us to identify up to three RAPD and five VNTR multilocus haplotypes colonizing one plant. Molecular analysis of variance was used to determine the extent to which population structure explained the genetic variation observed. The genetic variation observed in the X. fastidiosa strains was not related to or dependent on the different sweet orange varieties from which they had been obtained. A significant amount of the observed genetic variation could be explained by the variation between strains from different plants within the orchards and by the variation between strains within each plant. It appears, therefore, that the existence of different sweet orange varieties does not play a role in the population structure of X. fastidiosa. The consequences of these results for the management of sweet orange breeding strategies for citrus variegate chlorosis resistance are also discussed.

Bacterial Typing Techniques↗
PubMed2002

Gene disruption by homologous recombination in the Xylella fastidiosa citrus variegated chlorosis strain.

Mutagenesis by homologous recombination was evaluated in Xylella fastidiosa by using the bga gene, coding for beta-galactosidase, as a model. Integration of replicative plasmids by homologous recombination between the cloned truncated copy of bga and the endogenous gene was produced by one or two crossover events leading to beta-galactosidase mutants. A promoterless chloramphenicol acetyltransferase gene was used to monitor the expression of the target gene and to select a cvaB mutant.

Alleles↗
PubMed2003

Genetic differences between two strains of Xylella fastidiosa revealed by suppression subtractive hybridization.

Suppression subtractive hybridization was used to rapidly identify 18 gene differences between a citrus variegated chlorosis (CVC) strain and a Pierce's disease of grape (PD) strain of Xylella fastidiosa. The results were validated as being highly representative of actual differences by comparison of the completely sequenced genome of a CVC strain with that of a PD strain.

Bacterial Proteins↗