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PubMed2006

Plasmid pBP136 from Bordetella pertussis represents an ancestral form of IncP-1beta plasmids without accessory mobile elements.

The complete 41,268 bp nucleotide sequence of the IncP-1beta plasmid pBP136 from the human pathogen Bordetella pertussis, the primary aetiological agent of whooping cough, was determined and analysed. This plasmid carried a total of 46 ORFs: 44 ORFs corresponding to the genes in the conserved IncP-1beta backbone, and 2 ORFs similar to the XF1596 and XF1597 genes with unknown function of the plant pathogen Xylella fastidiosa. Interestingly, pBP136 had no accessory genes carrying genetic traits such as antibiotic or mercury resistance and/or xenobiotic degradation. Moreover, pBP136 had only two of the kle genes (kleAE) that have been reported to be important for the stability of IncP-1 plasmid in Pseudomonas aeruginosa. Phylogenetic analysis of the Kle proteins revealed that the KleA and KleE of pBP136 were phylogenetically distant from those of the present IncP-1 plasmids. In contrast, IncC1 and KorC, encoded upstream and downstream of the kle genes respectively, and the replication-initiation protein, TrfA, were closely related to those of the IncP-1beta 'R751 group'. These results suggest that (i) pBP136 without any apparent accessory genes diverged early from an ancestor of the present IncP-1beta plasmids, especially those of the R751 group, and (ii) the kle genes might be incorporated independently into the backbone region of the IncP-1 plasmids for their stable maintenance in various host cells.

Bacterial Proteins↗
PubMed2004

Comparative genomics analyses of citrus-associated bacteria.

Xylella fastidiosa 9a5c (XF-9a5c) and Xanthomonas axonopodis pv. citri (XAC) are bacteria that infect citrus plants. Sequencing of the genomes of these strains is complete and comparative analyses are now under way with the genomes of other bacteria of the same genera. In this review, we present an overview of this comparative genomic work. We also present a detailed genomic comparison between XF-9a5a and XAC. Based on this analysis, genes and operons were identified that might be relevant for adaptation to citrus. XAC has two copies of a type II secretion system, a large number of cell wall-degrading enzymes and sugar transporters, a complete energy metabolism, a whole set of avirulence genes associated with a type III secretion system, and a complete flagellar and chemotatic system. By contrast, XF-9a5c possesses more genes involved with type IV pili biosynthesis than does XAC, contains genes encoding for production of colicins, and has 4 copies of Type I restriction/modification system while XAC has only one.

Animals↗
PubMed2004

Global features of sequences of bacterial chromosomes, plasmids and phages revealed by analysis of oligonucleotide usage patterns.

BACKGROUND: Oligonucleotide frequencies were shown to be conserved signatures for bacterial genomes, however, the underlying constraints have yet not been resolved in detail. In this paper we analyzed oligonucleotide usage (OU) biases in a comprehensive collection of 155 completely sequenced bacterial chromosomes, 316 plasmids and 104 phages. RESULTS: Two global features were analyzed: pattern skew (PS) and variance of OU deviations normalized by mononucleotide content of the sequence (OUV). OUV reflects the strength of OU biases and taxonomic signals. PS denotes asymmetry of OU in direct and reverse DNA strands. A trend towards minimal PS was observed for almost all complete sequences of bacterial chromosomes and plasmids, however, PS was substantially higher in separate genomic loci and several types of plasmids and phages characterized by long stretches of non-coding DNA and/or asymmetric gene distribution on the two DNA strands. Five of the 155 bacterial chromosomes have anomalously high PS, of which the chromosomes of Xylella fastidiosa 9a5c and Prochlorococcus marinus MIT9313 exhibit extreme PS values suggesting an intermediate unstable state of these two genomes. CONCLUSIONS: Strand symmetry as indicated by minimal PS is a universally conserved feature of complete bacterial genomes that results from the matching mutual compensation of local OU biases on both replichors while OUV is more a taxon specific feature. Local events such as inversions or the incorporation of genome islands are balanced by global changes in genome organization to minimize PS that may represent one of the leading evolutionary forces driving bacterial genome diversification.

Bacteriophages↗
PubMed2006

DBMODELING: a database applied to the study of protein targets from genome projects.

Genome sequencing efforts are providing us with complete genetic blueprints for hundreds of organisms. We are now faced with assigning, understanding, and modifying the functions of proteins encoded by these genomes. DBMODELING is a relational database of annotated comparative protein structure models and their metabolic pathway characterization, when identified. This procedure was applied to complete genomes such as Mycobacterium tuberculosis and Xylella fastidiosa. The main interest in the study of metabolic pathways is that some of these pathways are not present in humans, which makes them selective targets for drug design, decreasing the impact of drugs in humans. In the database, there are currently 1,116 proteins from two genomes. It can be accessed by any researcher at http://www.biocristalografia.df.ibilce.unesp.br/tools/. This project confirms that homology modeling is a useful tool in structural bioinformatics and that it can be very valuable in annotating genome sequence information, contributing to structural and functional genomics, and analyzing protein-ligand docking.

Animals↗
PubMed2000

Effects of temperature on the flight activity of Graphocephala atropunctata (Hemiptera: Cicadellidae).

Graphocephala atropunctata (Signoret) is the principal vector of Xylella fastidiosa (Wells, Raju, Hung, Weisberg, Mandelco-Paul and Brenner), the bacterium that causes Pierce's disease of grapevine in coastal California. Monitoring the activity of C. atropunctata in the early spring is important for timing insecticide sprays and assessing the potential for disease spread to adjacent vineyards. Trapping studies with yellow sticky traps over 3 yr in Napa Valley, CA, established a significant correlation between early spring trap catch and temperature. Sticky trap catches of G. atropunctata occurred in the springs of 1996-1998 only when temperature was greater than or equal to 14.5 degrees C. In 1997 and 1998, the degree-hours (> 14.5 degrees C) per day from sunrise to sunset during March and April, but not in May, correlated significantly with trap catches. The temperature threshold of 14.5 degrees C in the early spring can be used to improve the timing of insecticidal applications aimed at reducing C. atropunctata populations in vineyards affected by Pierce's disease.

Animals↗
PubMed2006

Spatial and temporal dynamics of overwintering Homalodisca coagulata (Hemiptera: Cicadellidae).

A 4-yr landscape-scale study was conducted to investigate spatial and temporal dynamics of overwintering Homalodisca coagulata (Say) (Hemiptera: Cicadellidae) in the lower San Joaquin Valley, California. Spatial structures of H. coagulata distributions were characterized with Moran's I index, and spatial associations between H. coagulata and the surrounding environment were investigated with a geographic information system. H. coagulata was caught consistently with sticky traps throughout the winter, and trap catches formed a distinctive peak in December or January, indicating active flight of H. coagulata during the winter. In 2000-2001, the mean +/-SE trap count was 4.8 +/- 1.21 per trap per wk, and H. coagulata trap catches were spatially autocorrelated within approximately 1.3 km. Approximately 49% of H. coagulata were caught in citrus, 23% in stone fruit, and 11% in grape. After a control program began in spring 2001, the mean trap count was considerably lower (0.041 +/- 0.0004 per trap per wk), and no spatial autocorrelations were detected in 2001-2004. H. coagulata trap catch-crop associations also changed after initiation of the control program. Between 25 and 38% of H. coagulata trap catches were from citrus, between 8 and 20% were from stone fruit, and between 11 and 25% were from grape. Potential for winter-season spread and management of Xylella fastidiosa Wells et al., a pathogen causing Pierce's disease, are discussed.

Animals↗
PubMed2006

SpotWhatR: a user-friendly microarray data analysis system.

SpotWhatR is a user-friendly microarray data analysis tool that runs under a widely and freely available R statistical language (http://www.r-project.org) for Windows and Linux operational systems. The aim of SpotWhatR is to help the researcher to analyze microarray data by providing basic tools for data visualization, normalization, determination of differentially expressed genes, summarization by Gene Ontology terms, and clustering analysis. SpotWhatR allows researchers who are not familiar with computational programming to choose the most suitable analysis for their microarray dataset. Along with well-known procedures used in microarray data analysis, we have introduced a stand-alone implementation of the HTself method, especially designed to find differentially expressed genes in low-replication contexts. This approach is more compatible with our local reality than the usual statistical methods. We provide several examples derived from the Blastocladiella emersonii and Xylella fastidiosa Microarray Projects. SpotWhatR is freely available at http://blasto.iq.usp.br/~tkoide/SpotWhatR, in English and Portuguese versions. In addition, the user can choose between "single experiment" and "batch processing" versions.

Blastocladiella↗
PubMed2000

Application of bootstrap techniques to physical mapping.

Ordering genetic markers or clones from a genomic library into a physical map is a central problem in genetics. In the presence of errors, there is no efficient algorithm known that solves this problem. Based on a standard heuristic algorithm for it, we present a method to construct a confidence neighborhood for a computed solution. We compute a confidence value for putative local solutions derived from bootstrap replicates of the original solution. In the reliable parts, the confidence neighborhood and the computed solution tend to coincide. In regions that are ill-defined by the data, the neighborhood contains additional reasonable alternatives. This offers the possibility of designing further experiments for the badly defined regions to improve the quality of the physical map. We analyze our approach by a simulation study and by application to a dataset of the genome of the bacterium Xylella fastidiosa.

Algorithms↗
PubMed2004

A genetic linkage map of grape, utilizing Vitis rupestris and Vitis arizonica.

A genetic linkage map of grape was constructed, utilizing 116 progeny derived from a cross of two Vitis rupestris x V. arizonica interspecific hybrids, using the pseudo-testcross strategy. A total of 475 DNA markers-410 amplified fragment length polymorphism, 24 inter-simple sequence repeat, 32 random amplified polymorphic DNA, and nine simple sequence repeat markers-were used to construct the parental maps. Markers segregating 1:1 were used to construct parental framework maps with confidence levels >90% with the Plant Genome Research Initiative mapping program. In the maternal (D8909-15) map, 105 framework markers and 55 accessory markers were ordered in 17 linkage groups (756 cM). The paternal (F8909-17) map had 111 framework markers and 33 accessory markers ordered in 19 linkage groups (1,082 cM). One hundred eighty-one markers segregating 3:1 were used to connect the two parental maps' parents. This moderately dense map will be useful for the initial mapping of genes and/or QTL for resistance to the dagger nematode, Xiphinema index, and Xylella fastidiosa, the bacterial causal agent of Pierce's disease.

Base Sequence↗
PubMed2006

Identification and molecular mapping of PdR1, a primary resistance gene to Pierce's disease in Vitis.

A major quantitative trait locus (QTL) controlling resistance to Pierce's disease (PD) of grape, caused by the bacterium Xylella fastidiosa (Xf), was identified on a Vitis linkage map and denoted as 'Pierce's disease resistance 1' (PdR1). Placement of the locus was accomplished by evaluating a family of full-sib progeny from a cross of two PD-resistant interspecific hybrids with resistance inherited from Vitis arizonica. Resistance was measured under greenhouse conditions by direct quantification of Xf numbers in stem tissues as well as by evaluation of disease symptoms based on leaf scorch and a cane maturation index (CMI). A large QTL (LOD 17.2) accounting for 72% of the phenotypic variance in bacterial numbers was localized to linkage group 14 of the male parent F8909-17. The approximate 95% confidence interval around the QTL peak extended 5.7 cM when using composite interval mapping. The other disease evaluation methods (leaf scorch and CMI, respectively) placed the resistance QTL to the same region on linkage group 14, although at wider 95% confidence intervals (6.0 and 7.5 cM), lower peak LOD scores (11.9 and 7.7) and accounting for less phenotypic variance (59 and 42%). This is the first report of an Xf resistance QTL mapped in any crop species. The relevance of the markers located in the region spanning the QTL will be discussed, addressing their usefulness for the development of PD-resistant grape cultivars.

Chromosome Mapping↗
PubMed2001

Characterization of stress-responsive genes, hrcA-grpE-dnaK-dnaJ, from phytopathogenic Xanthomonas campestris.

Sequencing of a 6.4-kb DNA fragment, cloned from the plant pathogenic bacterium Xanthomonas campestris pv. campestris 17 revealed five ORFs whose deduced amino acid sequences show strong similarities to the bacterial HrcA, GrpE, DnaK, DnaJ, and PdxK. The four heat shock genes are organized in the order hrcA-grpE-dnaK-dnaJ, a genome organization found in many gram-positive bacteria, but only in one gram-negative species (Xylella fastidiosa). These observations suggest that the HrcA-CIRCE system, comprising at least four genes arranged in this order, already existed for the regulation of stress responses before bacteria diverged into gram-negative and gram-positive groups. Primer-extension results suggested the presence of promoters at the regions upstream of grpE and dnaK. In the presence of stress, heat or ethanol (4%), the X. campestris pv. campestris 17 grpE and dnaK promoters were induced two- to three-fold over controls. Since the grpE and dnaK promoters possess E. coli sigma(32) promoter-like sequences, they are functional in E. coli, although at levels much lower than in X. campestris pv. campestris 17. Furthermore, expression of the X. campestris pv. campestris 17 dnaK promoter in E. coli was elevated by the cloned X. campestris sigma(32) gene, indicating that the cognate sigma(32) works more efficiently for the X. campestris promoters.

Bacterial Proteins↗
PubMed2005

Comparison of the yeast proteome to other fungal genomes to find core fungal genes.

The purpose of this research was to search for evolutionarily conserved fungal sequences to test the hypothesis that fungi have a set of core genes that are not found in other organisms, as these genes may indicate what makes fungi different from other organisms. By comparing 6355 predicted or known yeast (Saccharomyces cerevisiae) genes to the genomes of 13 other fungi using Standalone TBLASTN at an e-value <1E-5, a list of 3340 yeast genes was obtained with homologs present in at least 12 of 14 fungal genomes. By comparing these common fungal genes to complete genomes of animals (Fugu rubripes, Caenorhabditis elegans), plants (Arabidopsis thaliana, Oryza sativa), and bacteria (Agrobacterium tumefaciens, Xylella fastidiosa), a list of common fungal genes with homologs in these plants, animals, and bacteria was produced (938 genes), as well as a list of exclusively fungal genes without homologs in these other genomes (60 genes). To ensure that the 60 genes were exclusively fungal, these were compared using TBLASTN to the major sequence databases at GenBank: NR (nonredundant), EST (expressed sequence tags), GSS (genome survey sequences), and HTGS (unfinished high-throughput genome sequences). This resulted in 17 yeast genes with homologs in other fungal genomes, but without known homologs in other organisms. These 17 core, fungal genes were not found to differ from other yeast genes in GC content or codon usage patterns. More intensive study is required of these 17 genes and other common fungal genes to discover unique features of fungi compared to other organisms.

Computational Biology↗
PubMed2005

Gene expression analysis of six GC-rich Gram-negative phytopathogens.

Predicted highly expressed (PHX) genes are comparatively analyzed for six GC-rich Gram-negative phytopathogens, i.e., Ralstonia solanacearum, Agrobacterium tumefaciens, Xanthomonas campestris pv. campestris (Xcc), Xanthomonas axonopodis pv. citri (Xac), Pseudomonas syringae pv. tomato, and Xylella fastidiosa. Enzymes involved in energy metabolism, such as ATP synthase, and genes involved in TCA cycle, are PHX in most bacteria except X. fastidiosa, which prefers an anaerobic environment. Most pathogenicity-related factors, including flagellar proteins and some outer membrane proteins, are PHX, except that flagellar proteins are missing in X. fastidiosa which is spread by insects and does not need to move during invasion. Although type III secretion system apparatus are homologous to flagellar proteins, none of them is PHX, which support the viewpoint that the two types of genes have evolved independently. Furthermore, it is revealed that some biosynthesis-related enzymes are highly expressed in certain bacteria. The PHX genes may provide potential drug targets for the design of new bactericide.

Bacterial Proteins↗
PubMed2004

Quantifying intrachromosomal GC heterogeneity in prokaryotic genomes.

The sequencing of prokaryotic genomes covering a wide taxonomic range has sparked renewed interest in intrachromosomal compositional (GC) heterogeneity, largely in view of lateral transfers. We present here a brief overview of some methods for visualizing and quantifying GC variation in prokaryotes. We used these methods to examine heterogeneity levels in sequenced prokaryotes, for a range of scales or stringencies. Some species are consistently homogeneous, whereas others are markedly heterogeneous in comparison, in particular Aeropyrum pernix, Xylella fastidiosa, Mycoplasma genitalium, Enterococcus faecalis, Bacillus subtilis, Pyrobaculum aerophilum, Vibrio vulnificus chromosome I, Deinococcus radiodurans chromosome II and Halobacterium. As we discuss here, the wide range of heterogeneities calls for reexamination of an accepted belief, namely that the endogenous DNA of bacteria and archaea should typically exhibit low intrachromosomal GC contrasts. Supplementary results for all species analyzed are available at our website: http://bioinfo2.ugr.es/prok.

Algorithms↗
PubMed2002

Crystal structure of a transcarbamylase-like protein from the anaerobic bacterium Bacteroides fragilis at 2.0 A resolution.

A transcarbamylase-like protein essential for arginine biosynthesis in the anaerobic bacterium Bacteroides fragilis has been purified and crystallized in space group P4(3)2(1)2 (a=b=153.4 A, c=94.8 A). The structure was solved using a single isomorphous replacement with anomalous scattering (SIRAS) and was refined at 2.0 A resolution to an R-factor of 20.6% (R-free=25.2%). The molecular model is trimeric and comprises 960 amino acid residues, two phosphate groups and 422 water molecules. The monomer has the consensus transcarbamylase fold with two structural domains linked by two long interdomain helices: the putative carbamoyl phosphate-binding domain and a binding domain for the second substrate. Each domain has a central parallel beta-sheet surrounded by alpha-helices and loops with alpha/beta topology. The putative carbamoyl phosphate-binding site is similar to those in ornithine transcarbamylases (OTCases) and aspartate transcarbamylases (ATCases); however, the second substrate-binding site is strikingly different. This site has several insertions and deletions, and residues critical to substrate binding and catalysis in other known transcarbamylases are not conserved. The three-dimensional structure and the fact that this protein is essential for arginine biosynthesis suggest strongly that it is a new member of the transcarbamylase family. A similar protein has been found in Xylella fastidiosa, a bacterium that infects grapes, citrus and other plants.

Amino Acid Sequence↗
PubMed1998

A small-scale procedure for extracting nucleic acids from woody plants infected with various phytopathogens for PCR assay.

The complexity of most nucleic acid extraction procedures limits the number of samples that can be easily processed for analysis by polymerase chain reaction (PCR). A simple, small-scale procedure was developed which can be carried out entirely in 1.5-ml microfuge tubes whereby the container and contents are frozen with liquid nitrogen, tissue is pulverized, and targeted nucleic acids are extracted. DNA of bacterial and phytoplasmal plant pathogens was extracted in hot CTAB buffer followed by chloroform clarification. Following centrifugation, the DNA in the aqueous fraction was precipitated with isopropanol and resuspended in water. RNA originating from viruses and viroids was extracted from triturated tissue using STE buffer and phenol. The nucleic acid fraction was purified using CF-11 cellulose. All purified preparations were used as PCR or RT-PCR templates to detect DNA or RNA, respectively. These procedures were used to detect Xylella fastidiosa, peach yellow leaf roll phytoplasma, sour cherry green ring mottle virus, and peach latent mosaic viroid by agarose gel electrophoresis.

DNA, Bacterial↗
PubMed2002

HecA, a member of a class of adhesins produced by diverse pathogenic bacteria, contributes to the attachment, aggregation, epidermal cell killing, and virulence phenotypes of Erwinia chrysanthemi EC16 on Nicotiana clevelandii seedlings.

Erwinia chrysanthemi is representative of a broad class of bacterial pathogens that are capable of inducing necrosis in plants. The E. chrysanthemi EC16 hecA gene predicts a 3,850-aa member of the Bordetella pertussis filamentous hemagglutinin family of adhesins. A hecATn7 mutant was reduced in virulence on Nicotiana clevelandii seedlings after inoculation without wounding. Epifluorescence and confocal laser-scanning microscopy observations of hecA and wild-type cells expressing the green fluorescent protein revealed that the mutant is reduced in its ability to attach and then form aggregates on leaves and to cause an aggregate-associated killing of epidermal cells. Cell killing also depended on production of the major pectate lyase isozymes and the type II, but not the type III, secretion pathway in E. chrysanthemi. HecA homologs were found in bacterial pathogens of plants and animals and appear to be unique to pathogens and universal in necrogenic plant pathogens. Phylogenetic comparison of the conserved two-partner secretion domains in the proteins and the 16S rRNA sequences in respective bacteria revealed the two datasets to be fundamentally incongruent, suggesting horizontal acquisition of these genes. Furthermore, hecA and its two homologs in Yersinia pestis had a G+C content that was 10% higher than that of their genomes and similar to that of plant pathogenic Ralstonia, Xylella, and Pseudomonas spp. Our data suggest that filamentous hemagglutinin-like adhesins are broadly important virulence factors in both plant and animal pathogens.

Adhesins, Bacterial↗
PubMed2000

Porphyromonas gingivalis DPP-7 represents a novel type of dipeptidylpeptidase.

A novel dipeptidylpeptidase (DPP-7) was purified from the membrane fraction of Porphyromonas gingivalis. This enzyme, with an apparent molecular mass of 76 kDa, has the specificity for both aliphatic and aromatic residues in the P1 position. Although it belongs to the serine class of peptidases, it does not resemble other known dipeptidylpeptidases. Interestingly, the amino acid sequence around the putative active site serine residue shows significant similarity to the C-terminal region of the Staphylococcus aureus V-8 endopeptidase. The genes encoding homologues of DPP-7 were found in genomes of Xylella fastidiosa, Shewanella putrefaciens, and P. gingivalis. It is likely that at least in P. gingivalis, DPP-7 and its homologue, in concert with other di- and tripeptidases, serve nutritional functions by providing dipeptides to this asaccharolytic bacterium.

Amino Acid Sequence↗