Diversity of Secondary Metabolism in Aspergillus nidulans Clinical Isolates.

The filamentous fungus Aspergillus nidulans has been a primary workhorse used to understand fungal genetics. Much of this work has focused on elucidating the genetics of biosynthetic gene clusters (BGCs) and the secondary metabolites (SMs) they produce. SMs are both niche defining in fungi and of great economic importance to humans. Despite the focus on A. nidulans, very little is known about the natural diversity in secondary metabolism within this species. We determined the BGC content and looked for evolutionary patterns in BGCs from whole-genome sequences of two clinical isolates and the A4 reference genome of A. nidulans Differences in BGC content were used to explain SM profiles determined using liquid chromatography-high-resolution mass spectrometry. We found that in addition to genetic variation of BGCs contained by all isolates, nine BGCs varied by presence/absence. We discovered the viridicatumtoxin BGC in A. nidulans and suggest that this BGC has undergone a horizontal gene transfer from the Aspergillus section Nigri lineage into Penicillium sometime after the sections Nigri and Nidulantes diverged. We identified the production of viridicatumtoxin and several other compounds previously not known to be produced by A. nidulans One isolate showed a lack of sterigmatocystin production even though it contained an apparently intact sterigmatocystin BGC, raising questions about other genes and processes known to regulate this BGC. Altogether, our work uncovers a large degree of intraspecies diversity in BGC and SM production in this genetic model species and offers new avenues to understand the evolution and regulation of secondary metabolism.IMPORTANCE Much of what we know about the genetics underlying secondary metabolite (SM) production and the function of SMs in the model fungus Aspergillus nidulans comes from a single reference genome. A growing body of research indicates the importance of biosynthetic gene cluster (BGC) and SM diversity within a species. However, there is no information about the natural diversity of secondary metabolism in A. nidulans We discovered six novel clusters that contribute to the considerable variation in both BGC content and SM production within A. nidulans We characterize a diverse set of mutations and emphasize how findings of single nucleotide polymorphisms (SNPs), deletions, and differences in evolutionary history encompass much of the variation observed in nonmodel systems. Our results emphasize that A. nidulans may also be a strong model to use within-species diversity to elucidate regulatory cross talk, fungal ecology, and drug discovery systems.

Sequence-independent cloning methods for long DNA fragments applied to synthetic biology.

Simplified methods to assemble DNA fragments by independent cloning sequence have helped in the progress of synthetic biology, allowing some biotechnological processes to become economically viable by genetic improvement of microorganisms. We compared three methods of assembling six DNA fragments: PCR fusion-based, isothermal NEBuilder and circular polymerase extension cloning (CPEC). Double and triple fusion occurs directly with the PCR products using PCR fusion-based and NEBuilder methods. For multiple fragments the results showed higher efficiency by the CPEC method which allowed assembly of six fragments previously purified by agarose gel extraction, after a sequence of 20 annealing/extension cycles without any primer.

Functional characterization of the Aspergillus nidulans glucosylceramide pathway reveals that LCB Δ8-desaturation and C9-methylation are relevant to filamentous growth, lipid raft localization and Psd1 defensin activity.

C8-desaturated and C9-methylated glucosylceramide (GlcCer) is a fungal-specific sphingolipid that plays an important role in the growth and virulence of many species. In this work, we investigated the contribution of Aspergillus nidulans sphingolipid Δ8-desaturase (SdeA), sphingolipid C9-methyltransferases (SmtA/SmtB) and glucosylceramide synthase (GcsA) to fungal phenotypes, sensitivity to Psd1 defensin and Galleria mellonella virulence. We showed that ΔsdeA accumulated C8-saturated and unmethylated GlcCer, while gcsA deletion impaired GlcCer synthesis. Although increased levels of unmethylated GlcCer were observed in smtA and smtB mutants, ΔsmtA and wild-type cells showed a similar 9,Me-GlcCer content, reduced by 50% in the smtB disruptant. The compromised 9,Me-GlcCer production in the ΔsmtB strain was not accompanied by reduced filamentation or defects in cell polarity. When combined with the smtA deletion, smtB repression significantly increased unmethylated GlcCer levels and compromised filamentous growth. Furthermore, sdeA and gcsA mutants displayed growth defects and raft mislocalization, which were accompanied by reduced neutral lipids levels and attenuated G. mellonella virulence in the ΔgcsA strain. Finally, ΔsdeA and ΔgcsA showed increased resistance to Psd1, suggesting that GlcCer synthesis and fungal sphingoid base structure specificities are relevant not only to differentiation but also to proper recognition by this antifungal defensin.

Jasmonates are phytohormones with multiple functions, including plant defense and reproduction.

The plant hormones jasmonic acid and methyl jasmonate, along with their intermediate compounds, produced in the octadecanoid pathway, are important signaling molecules that are collectively called jasmonates. These are widespread in the plant kingdom and play crucial roles in biotic/abiotic stress responses, as well as in processes related to plant growth and development. Recently, it has been shown that jasmonates are also involved in reproductive processes. We present the most recent findings related to the biosynthesis, regulation and signaling mechanisms of jasmonates. Additionally, we discuss the identification of [(+)-7-iso-JA-L-Ile] as the active biological hormonal form of jasmonate; this fills the greatest gap in our knowledge about the signaling mechanism that is responsible for the activation of downstream genes in the jasmonate-signaling cascade. The identification of several Arabidopsis thaliana mutants was crucial to the elucidation of the signaling mechanisms involved in jasmonate-mediated responses. Finally, the involvement of jasmonates in the reproductive process of Nicotiana tabacum L. is briefly discussed, since some of the main enzymes of the jasmonic acid biosynthesis pathway were identified in a stigma/style expressed sequence tag database (TOBEST) of this Solanaceae species.

Cdc42p controls yeast-cell shape and virulence of Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is characterized by a multiple budding phenotype and a polymorphic cell growth, leading to the formation of cells with extreme variations in shape and size. Since Cdc42 is a pivotal molecule in establishing and maintaining polarized growth for diverse cell types, as well as during pathogenesis of certain fungi, we evaluated its role during cell growth and virulence of the yeast-form of P. brasiliensis. We used antisense technology to knock-down PbCDC42's expression in P. brasiliensis yeast cells, promoting a decrease in cell size and more homogenous cell growth, altering the typical polymorphism of wild-type cells. Reduced expression levels also lead to increased phagocytosis and decreased virulence in a mouse model of infection. We provide genetic evidences underlying Pbcdc42p as an important protein during host-pathogen interaction and the relevance of the polymorphic nature and cell size in the pathogenesis of P. brasiliensis.

The ergosterol biosynthesis pathway, transporter genes, and azole resistance in Aspergillus fumigatus.

The continuous use of triazoles can result in the development of drug resistance. Azole-resistant clinical isolates, spontaneous and induced mutants of Aspergillus fumigatus have been documented. The azoles block the ergosterol biosynthesis pathway by inhibiting the enzyme 14-alpha-demethylase, product of the CYP51. Fungal azole resistance involves both amino acid changes in the target site that alter drug-target interactions and those that decrease net azole accumulation. The reduced intracellular accumulation has also been correlated with overexpression of multidrug resistance (MDR) efflux transporter genes of the ATP-binding cassette (ABC) and the major facilitator superfamily (MFS) classes. About 20 genes are involved in the A. fumigatus ergosterol biosynthesis pathway. There are several duplicated genes in this pathway. Interestingly, erg3 and erg11 showed two copies in A. fumigatus. In general, Aspergillus spp. have proportionally more MFS transporter encoding genes than Saccharomyces cerevisiae, S. pombe, and Neurospora crassa. The drug H+ (12 and 14 spanners) sub-families are also proportionally greater than in the other species. Although the numbers of ABC transporter encoding genes are comparable, again the Aspergillus spp. have more ABC transporters related to multidrug permease than the other fungal species.

Identification of genes preferentially expressed in the pathogenic yeast phase of Paracoccidioides brasiliensis, using suppression subtraction hybridization and differential macroarray analysis.

Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America. Pathogenicity appears to be intimately related to the dimorphic transition from the hyphal to the yeast form, which is induced by a shift from environmental temperature to the temperature of the mammalian host. Little information is available on the P. brasiliensis genes that are necessary during the pathogenic phase. We have therefore undertaken Suppression Subtraction Hybridization (SSH) and macroarray analyses with the aim of identifying genes that are preferentially expressed in the yeast phase. Genes identified by both procedures as being more highly expressed in the yeast phase are involved in basic metabolism, signal transduction, growth and morphogenesis, and sulfur metabolism. In order to test whether the observed changes in gene expression reflect the differences between the growth conditions used to obtain the two morphological forms rather than differences intrinsic to the cell types, we performed real-time RT-PCR experiments using RNAs derived from both yeast cells and mycelia that had been cultured at 37 degrees C and 26 degrees C in either complete medium (YPD or Sabouraud) or minimal medium. Twenty genes, including AGS1 (alpha-1,3-glucan synthase) and TSA1 (thiol-specific antioxidant), were shown to be more highly expressed in the yeast cells than in the hyphae. Although their levels of expression could be different in rich and minimal media, there was a general tendency for these genes to be more highly expressed in the yeast cells.

Comparative genomics of two Leptospira interrogans serovars reveals novel insights into physiology and pathogenesis.

Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in accidental hosts, including humans. Complete genome sequencing of Leptospira interrogans serovar Copenhageni and comparative analysis with the available Leptospira interrogans serovar Lai genome reveal that despite overall genetic similarity there are significant structural differences, including a large chromosomal inversion and extensive variation in the number and distribution of insertion sequence elements. Genome sequence analysis elucidates many of the novel aspects of leptospiral physiology relating to energy metabolism, oxygen tolerance, two-component signal transduction systems, and mechanisms of pathogenesis. A broad array of transcriptional regulation proteins and two new families of afimbrial adhesins which contribute to host tissue colonization in the early steps of infection were identified. Differences in genes involved in the biosynthesis of lipopolysaccharide O side chains between the Copenhageni and Lai serovars were identified, offering an important starting point for the elucidation of the organism's complex polysaccharide surface antigens. Differences in adhesins and in lipopolysaccharide might be associated with the adaptation of serovars Copenhageni and Lai to different animal hosts. Hundreds of genes encoding surface-exposed lipoproteins and transmembrane outer membrane proteins were identified as candidates for development of vaccines for the prevention of leptospirosis.

Identification of an unusual VanA element in glycopeptide-resistant Enterococcus faecium in Brazil following international transfer of a bone marrow transplant patient.

A vancomycin-resistant Enterococcus (VRE) was isolated from a blood culture of a patient in a Brazilian hospital who had a treatment history of a bone marrow transplant in the USA. The organism was identified as Enterococcus faecium, which exhibited an MIC (minimum inhibitory concentration) >or= 256 microg/mL for vancomycin. This was confirmed by E-test and the vanA gene was detected by PCR. Overlapping PCR revealed a left IR deletion and an additional 1.5 kb fragment between vanSH genes. DdeI digestion of vanRSHAX genes showed the determinant to be a T type variant, and the element was cloned and sequenced. These results revealed an IS1251 downstream of nucleotide 5820 of the VanA element. Insertions like this have not been reported previously in Brazil, but have been detected in the USA. The genotype and association with a patient previously treated in the USA suggest that this VRE was introduced from abroad, probably through inter-hospital strain spread.

Comparative analyses of the complete genome sequences of Pierce's disease and citrus variegated chlorosis strains of Xylella fastidiosa.

Xylella fastidiosa is a xylem-dwelling, insect-transmitted, gamma-proteobacterium that causes diseases in many plants, including grapevine, citrus, periwinkle, almond, oleander, and coffee. X. fastidiosa has an unusually broad host range, has an extensive geographical distribution throughout the American continent, and induces diverse disease phenotypes. Previous molecular analyses indicated three distinct groups of X. fastidiosa isolates that were expected to be genetically divergent. Here we report the genome sequence of X. fastidiosa (Temecula strain), isolated from a naturally infected grapevine with Pierce's disease (PD) in a wine-grape-growing region of California. Comparative analyses with a previously sequenced X. fastidiosa strain responsible for citrus variegated chlorosis (CVC) revealed that 98% of the PD X. fastidiosa Temecula genes are shared with the CVC X. fastidiosa strain 9a5c genes. Furthermore, the average amino acid identity of the open reading frames in the strains is 95.7%. Genomic differences are limited to phage-associated chromosomal rearrangements and deletions that also account for the strain-specific genes present in each genome. Genomic islands, one in each genome, were identified, and their presence in other X. fastidiosa strains was analyzed. We conclude that these two organisms have identical metabolic functions and are likely to use a common set of genes in plant colonization and pathogenesis, permitting convergence of functional genomic strategies.

Molecular characterization of ABC transporter-encoding genes in Aspergillus nidulans

As a preliminary step towards characterizing genes encoding ATP-binding cassette (ABC) transporters that confer pleiotropic drug resistance in Aspergillus, we used a PCR-based approach to isolate four DNA fragments corresponding to different ABC type transporter genes. DNA sequencing and Southern blot analysis confirmed that they were distinct genes, which were designated abcA-D. One of these genes, abcD, was cloned and characterized. It was found to have a predicted 1,452-amino acid translation product with a calculated molecular mass of 147,467 kDa. The abcD gene specifies a single transcript of approximately 5.0 kb; there was a two- to six-fold enhancement of mRNA levels following exposure to miconazole, camptothecin, methotrexate, and ethidium bromide

A tobacco cDNA reveals two different transcription patterns in vegetative and reproductive organs.

In order to identify genes expressed in the pistil that may have a role in the reproduction process, we have established an expressed sequence tags project to randomly sequence clones from a Nicotiana tabacum stigma/style cDNA library. A cDNA clone (MTL-8) showing high sequence similarity to genes encoding glycine-rich RNA-binding proteins was chosen for further characterization. Based on the extensive identity of MTL-8 to the RGP-1a sequence of N. sylvestris, a primer was defined to extend the 5' sequence of MTL-8 by RT-PCR from stigma/style RNAs. The amplification product was sequenced and it was confirmed that MTL-8 corresponds to an mRNA encoding a glycine-rich RNA-binding protein. Two transcripts of different sizes and expression patterns were identified when the MTL-8 cDNA insert was used as a probe in RNA blots. The largest is 1,100 nucleotides (nt) long and markedly predominant in ovaries. The smaller transcript, with 600 nt, is ubiquitous to the vegetative and reproductive organs analyzed (roots, stems, leaves, sepals, petals, stamens, stigmas/styles and ovaries). Plants submitted to stress (wounding, virus infection and ethylene treatment) presented an increased level of the 600-nt transcript in leaves, especially after tobacco necrosis virus infection. In contrast, the level of the 1,100-nt transcript seems to be unaffected by the stress conditions tested. Results of Southern blot experiments have suggested that MTL-8 is present in one or two copies in the tobacco genome. Our results suggest that the shorter transcript is related to stress while the larger one is a flower predominant and nonstress-inducible messenger.

A tobacco cDNA reveals two different transcription patterns in vegetative and reproductive organs

In order to identify genes expressed in the pistil that may have a role in the reproduction process, we have established an expressed sequence tags project to randomly sequence clones from a Nicotiana tabacum stigma/style cDNA library. A cDNA clone (MTL-8) showing high sequence similarity to genes encoding glycine-rich RNA-binding proteins was chosen for further characterization. Based on the extensive identity of MTL-8 to the RGP-1a sequence of N. sylvestris, a primer was defined to extend the 5' sequence of MTL-8 by RT-PCR from stigma/style RNAs. The amplification product was sequenced and it was confirmed that MTL-8 corresponds to an mRNA encoding a glycine-rich RNA-binding protein. Two transcripts of different sizes and expression patterns were identified when the MTL-8 cDNA insert was used as a probe in RNA blots. The largest is 1,100 nucleotides (nt) long and markedly predominant in ovaries. The smaller transcript, with 600 nt, is ubiquitous to the vegetative and reproductive organs analyzed (roots, stems, leaves, sepals, petals, stamens, stigmas/styles and ovaries). Plants submitted to stress (wounding, virus infection and ethylene treatment) presented an increased level of the 600-nt transcript in leaves, especially after tobacco necrosis virus infection. In contrast, the level of the 1,100-nt transcript seems to be unaffected by the stress conditions tested. Results of Southern blot experiments have suggested that MTL-8 is present in one or two copies in the tobacco genome. Our results suggest that the shorter transcript is related to stress while the larger one is a flower predominant and nonstress-inducible messenger

The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome.

Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription-PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning.

New restriction fragment length polymorphism (RFLP) markers for Aspergillus fumigatus.

In this study, we isolated and tested restriction fragment length polymorphism (RFLP) markers for Aspergillus fumigatus based on PCR products amplified by the random amplified polymorphic DNA (RAPD) primer R108. Four DNA fragments, Afd, Af5, Af4, and Af4A, were amplified. Fragments Afd and Af5 were 85% and 88% identical at the DNA level to part of the Afut1 retrotransposon from A. fumigatus. Fragment Af4A is a duplication of fragment Af4 and both showed similarity at the amino acid level with endonucleases from other fungal retrotransposons. We used both RAPD with primer R108 and RFLP assays with Afut1, Afd, and Af4A, to determine the genetic relatedness of clinical isolates of A. fumigatus isolated sequentially from four patients colonized with A. fumigatus. The combination of these different methods suggested that the isolates infecting the four patients were not identical.

Sensitivity to camptothecin in Aspergillus nidulans identifies a novel gene, scaA+, related to the cellular DNA damage response.

The anti-cancer drug camptothecin targets eukaryotic DNA topoisomerase I by trapping the covalent complex formed between the catalytically active enzyme and DNA. We are interested in identifying factors, other than topoisomerase I, that are involved in mediating cellular sensitivity to camptothecin. To this end, we have isolated eighteen mutants that are sensitive to camptothecin (sca) in the filamentous fungus Aspergillus nidulans and characterised one of them, sca299. The mutant sca299 is hypersensitive to camptothecin, and sensitive to several different mutagenic agents and to actinomycin D. Using temperature-sensitive mutations in genes that are known to regulate the cell cycle, we showed that the camptothecin sensitivity of the mutant sca299 is not affected by a mitotic block. The abnormal nuclear morphology observed in the sca299 mutant strain suggests that the germlings might be undergoing mitosis in the presence of unrepaired DNA damage, which would result in mitotic catastrophe. The hypersensitivity of the sca299 mutant to camptothecin does not result from elevated levels of topoisomerase I mRNA or from alterations in enzyme activity. Using DNA-mediated complementation of the sca299 mutant phenotype, the scaA+ gene was cloned. This gene encodes a 594-amino acid product; moderate structural similarity suggests that the scaA gene product may be related to the human nibrin gene which encodes a product involved in DNA double-strand break repair. Strains disrupted in the scaA gene were sensitive to the anti-topoisomerase I agent berberine, the DNA crosslinking agents mitomycin C and cis-platinum, and also to t-butyl hydroperoxide, which is an inducer of oxidative stress.

Dissecting the sugarcane expressed sequence tag (SUCEST) database: unraveling flower-specific genes

Existem quase 260.000 clones independentes, seqüenciados a partir da extremidade 5', no banco de dados do SUCEST (Sugarcane Expressed Sequence Tag), os quais foram obtidos a partir de 37 bibliotecas de cDNA preparadas de diferentes tecidos. Este grande número de etiquetas de seqüências expressas (ESTs) fornece uma oportunidade, sem precedentes em plantas, de realizar um 'digital differential screening' em bibliotecas de cDNA selecionadas. Geralmente, a freqüência de um determinado EST está correlacionada ao acúmulo de transcritos nos tecidos dos quais as bibliotecas de cDNA foram construídas, e desta forma, é possível comparar o transcriptoma completo de diferentes tecidos, usando uma análise computacional de um banco de dados de ESTs. Em nossa pesquisa, analisamos os ESTs de cana-de-açúcar de acordo com sua expressäo tecidual e identificamos mais de 1.000 putativos genes específicos de flor. O fato de que usando esta técnica fomos capazes de identificar homólogos em cana-de-açúcar, de vários genes previamente descritos como específicos de pólen, sustenta este método de estimar especificidade tecidual. Além disto, ESTs com similaridade a genes específicos de órgäos reprodutivos foram revelados, como por exemplo, o gene que codifica uma proteína meiótica essencial para a montagem do complexo sinaptonêmico e sinapse normal. Esta abordagem também permitiu a identificaçäo de muitas seqüências anônimas, específicas de flor, que säo boas candidatas para novos genes envolvidos com a reproduçäo de plantas. Este trabalho descreve a análise dos níveis de expressäo gênica de 24 clusters de ESTs, durante o desenvolvimento floral, usando um 'northern blot digital' construído a partir da contagem direta dos ESTs das bibliotecas näo-normalizadas de cDNAs de cana-de-açúcar.

Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags.

Transcribed sequences in the human genome can be identified with confidence only by alignment with sequences derived from cDNAs synthesized from naturally occurring mRNAs. We constructed a set of 250,000 cDNAs that represent partial expressed gene sequences and that are biased toward the central coding regions of the resulting transcripts. They are termed ORF expressed sequence tags (ORESTES). The 250,000 ORESTES were assembled into 81,429 contigs. Of these, 1, 181 (1.45%) were found to match sequences in chromosome 22 with at least one ORESTES contig for 162 (65.6%) of the 247 known genes, for 67 (44.6%) of the 150 related genes, and for 45 of the 148 (30.4%) EST-predicted genes on this chromosome. Using a set of stringent criteria to validate our sequences, we identified a further 219 previously unannotated transcribed sequences on chromosome 22. Of these, 171 were in fact also defined by EST or full length cDNA sequences available in GenBank but not utilized in the initial annotation of the first human chromosome sequence. Thus despite representing less than 15% of all expressed human sequences in the public databases at the time of the present analysis, ORESTES sequences defined 48 transcribed sequences on chromosome 22 not defined by other sequences. All of the transcribed sequences defined by ORESTES coincided with DNA regions predicted as encoding exons by genscan. (http://genes.mit.edu/GENSCAN.html).

A genomic approach to the understanding of Xylella fastidiosa pathogenicity.

Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes several economically important plant diseases, including citrus variegated chlorosis (CVC). X. fastidiosa is the first plant pathogen to have its genome completely sequenced. In addition, it is probably the least previously studied of any organism for which the complete genome sequence is available. Several pathogenicity-related genes have been identified in the X. fastidiosa genome by similarity with other bacterial genes involved in pathogenesis in plants, as well as in animals. The X. fastidiosa genome encodes different classes of proteins directly or indirectly involved in cell-cell interactions, degradation of plant cell walls, iron homeostasis, anti-oxidant responses, synthesis of toxins, and regulation of pathogenicity. Neither genes encoding members of the type III protein secretion system nor avirulence-like genes have been identified in X. fastidiosa.

The genome sequence of the plant pathogen Xylella fastidiosa. The Xylella fastidiosa Consortium of the Organization for Nucleotide Sequencing and Analysis.

Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes a range of economically important plant diseases. Here we report the complete genome sequence of X. fastidiosa clone 9a5c, which causes citrus variegated chlorosis--a serious disease of orange trees. The genome comprises a 52.7% GC-rich 2,679,305-base-pair (bp) circular chromosome and two plasmids of 51,158 bp and 1,285 bp. We can assign putative functions to 47% of the 2,904 predicted coding regions. Efficient metabolic functions are predicted, with sugars as the principal energy and carbon source, supporting existence in the nutrient-poor xylem sap. The mechanisms associated with pathogenicity and virulence involve toxins, antibiotics and ion sequestration systems, as well as bacterium-bacterium and bacterium-host interactions mediated by a range of proteins. Orthologues of some of these proteins have only been identified in animal and human pathogens; their presence in X. fastidiosa indicates that the molecular basis for bacterial pathogenicity is both conserved and independent of host. At least 83 genes are bacteriophage-derived and include virulence-associated genes from other bacteria, providing direct evidence of phage-mediated horizontal gene transfer.