Ana C. R. da Silva

The genome sequence of the gram-positive sugarcane pathogen Leifsonia xyli subsp. xyli.

The genome sequence of Leifsonia xyli subsp. xyli, which causes ratoon stunting disease and affects sugarcane worldwide, was determined. The single circular chromosome of Leifsonia xyli subsp. xyli CTCB07 was 2.6 Mb in length with a GC content of 68% and 2,044 predicted open reading frames. The analysis also revealed 307 predicted pseudogenes, which is more than any bacterial plant pathogen sequenced to date. Many of these pseudogenes, if functional, would likely be involved in the degradation of plant heteropolysaccharides, uptake of free sugars, and synthesis of amino acids. Although L. xyli subsp. xyli has only been identified colonizing the xylem vessels of sugarcane, the numbers of predicted regulatory genes and sugar transporters are similar to those in free-living organisms. Some of the predicted pathogenicity genes appear to have been acquired by lateral transfer and include genes for cellulase, pectinase, wilt-inducing protein, lysozyme, and desaturase. The presence of the latter may contribute to stunting, since it is likely involved in the synthesis of abscisic acid, a hormone that arrests growth. Our findings are consistent with the nutritionally fastidious behavior exhibited by L. xyli subsp. xyli and suggest an ongoing adaptation to the restricted ecological niche it inhabits.

Novel insights into the genomic basis of citrus canker based on the genome sequences of two strains of Xanthomonas fuscans subsp. aurantifolii

BackgroundCitrus canker is a disease that has severe economic impact on the citrus industry worldwide. There are three types of canker, called A, B, and C. The three types have different phenotypes and affect different citrus species. The causative agent for type A is Xanthomonas citri subsp. citri, whose genome sequence was made available in 2002. Xanthomonas fuscans subsp. aurantifolii strain B causes canker B and Xanthomonas fuscans subsp. aurantifolii strain C causes canker C.ResultsWe have sequenced the genomes of strains B and C to draft status. We have compared their genomic content to X. citri subsp. citri and to other Xanthomonas genomes, with special emphasis on type III secreted effector repertoires. In addition to pthA, already known to be present in all three citrus canker strains, two additional effector genes, xopE3 and xopAI, are also present in all three strains and are both located on the same putative genomic island. These two effector genes, along with one other effector-like gene in the same region, are thus good candidates for being pathogenicity factors on citrus. Numerous gene content differences also exist between the three cankers strains, which can be correlated with their different virulence and host range. Particular attention was placed on the analysis of genes involved in biofilm formation and quorum sensing, type IV secretion, flagellum synthesis and motility, lipopolysacharide synthesis, and on the gene xacPNP, which codes for a natriuretic protein.ConclusionWe have uncovered numerous commonalities and differences in gene content between the genomes of the pathogenic agents causing citrus canker A, B, and C and other Xanthomonas genomes. Molecular genetics can now be employed to determine the role of these genes in plant-microbe interactions. The gained knowledge will be instrumental for improving citrus canker control.

DNA Microarray-Based Genome Comparison of a Pathogenic and a Nonpathogenic Strain of Xylella fastidiosa Delineates Genes Important for Bacterial Virulence

Xylella fastidiosa is a phytopathogenic bacterium that causes serious diseases in a wide range of economically important crops. Despite extensive comparative analyses of genome sequences of Xylella pathogenic strains from different plant hosts, nonpathogenic strains have not been studied. In this report, we show that X. fastidiosa strain J1a12, associated with citrus variegated chlorosis (CVC), is nonpathogenic when injected into citrus and tobacco plants. Furthermore, a DNA microarray-based comparison of J1a12 with 9a5c, a CVC strain that is highly pathogenic and had its genome completely sequenced, revealed that 14 coding sequences of strain 9a5c are absent or highly divergent in strain J1a12. Among them, we found an arginase and a fimbrial adhesin precursor of type III pilus, which were confirmed to be absent in the nonpathogenic strain by PCR and DNA sequencing. The absence of arginase can be correlated to the inability of J1a12 to multiply in host plants. This enzyme has been recently shown to act as a bacterial survival mechanism by down-regulating host nitric oxide production. The lack of the adhesin precursor gene is in accordance with the less aggregated phenotype observed for J1a12 cells growing in vitro. Thus, the absence of both genes can be associated with the failure of the J1a12 strain to establish and spread in citrus and tobacco plants. These results provide the first detailed comparison between a nonpathogenic strain and a pathogenic strain of X. fastidiosa, constituting an important step towards understanding the molecular basis of the disease.

Hypoactivity of the Central Dopaminergic System and Autistic-Like Behavior Induced by a Single Early Prenatal Exposure to Lipopolysaccharide

The aim of the present study was to evaluate the behavioral patterns associated with autism and the prevalence of these behaviors in males and females, to verify whether our model of lipopolysaccharide (LPS) administration represents an experimental model of autism. For this, we prenatally exposed Wistar rats to LPS (100 μg/kg, intraperitoneally, on gestational day 9.5), which mimics infection by gram‐negative bacteria. Furthermore, because the exact mechanisms by which autism develops are still unknown, we investigated the neurological mechanisms that might underlie the behavioral alterations that were observed. Because we previously had demonstrated that prenatal LPS decreases striatal dopamine (DA) and metabolite levels, the striatal dopaminergic system (tyrosine hydroxylase [TH] and DA receptors D1a and D2) and glial cells (astrocytes and microglia) were analyzed by using immunohistochemistry, immunoblotting, and real‐time PCR. Our results show that prenatal LPS exposure impaired communication (ultrasonic vocalizations) in male pups and learning and memory (T‐maze spontaneous alternation) in male adults, as well as inducing repetitive/restricted behavior, but did not change social interactions in either infancy (play behavior) or adulthood in females. Moreover, although the expression of DA receptors was unchanged, the experimental animals exhibited reduced striatal TH levels, indicating that reduced DA synthesis impaired the striatal dopaminergic system. The expression of glial cell markers was not increased, which suggests that prenatal LPS did not induce permanent neuroinflammation in the striatum. Together with our previous finding of social impairments in males, the present findings demonstrate that prenatal LPS induced autism‐like effects and also a hypoactivation of the dopaminergic system.

Identification and Genomic Characterization of a New Virus (Tymoviridae Family) Associated with Citrus Sudden Death Disease

ABSTRACT Citrus sudden death (CSD) is a new disease that has killed approximately 1 million orange trees in Brazil. Here we report the identification of a new virus associated with the disease. RNAs isolated from CSD-affected and nonaffected trees were used to construct cDNA libraries. A set of viral sequences present exclusively in libraries of CSD-affected trees was used to obtain the complete genome sequence of the new virus. Phylogenetic analysis revealed that this virus is a new member of the genus Marafivirus. Antibodies raised against the putative viral coat proteins allowed detection of viral antigens of expected sizes in affected plants. Electron microscopy of purified virus confirmed the presence of typical isometric Marafivirus particles. The screening of 773 affected and nonaffected citrus trees for the presence of the virus showed a 99.7% correlation between disease symptoms and the presence of the virus. We also detected the virus in aphids feeding on affected trees. These results suggest that this virus is likely to be the causative agent of CSD. The virus was named Citrus sudden death-associated virus.

Identification of New Protein-Protein Interactions Involving the Products of the Chromosome- and Plasmid-Encoded Type IV Secretion Loci of the Phytopathogen Xanthomonas axonopodis pv. citri

The recently sequenced genome of the bacterial plant pathogen Xanthomonas axonopodis pv. citri contains two virB gene clusters, one on the chromosome and one on a 64-kb plasmid, each of which codes for a previously uncharacterized type IV secretion system (T4SS). Here we used a yeast two-hybrid assay to identify protein-protein interactions in these two systems. Our results revealed interactions between known T4SS components as well as previously uncharacterized interactions involving hypothetical proteins coded by open reading frames in the two X. axonopodis pv. citri virB loci. Our results indicate that both loci may code for previously unidentified VirB7 proteins, which we show interact with either VirB6 or VirB9 or with a hypothetical protein coded by the same locus. Furthermore, a set of previously uncharacterized Xanthomonas proteins have been found to interact with VirD4, whose gene is adjacent to the chromosomal virB locus. The gene for one member of this family is found within the chromosomal virB locus. All these uncharacterized proteins possess a conserved 120-amino-acid domain in their C termini and may represent a family of cofactors or substrates of the Xanthomonas T4SS.

New Protein-Protein Interactions Identified for the Regulatory and Structural Components and Substrates of the Type III Secretion System of the Phytopathogen Xanthomonas axonopodis Pathovar citri

We have initiated a project to identify protein-protein interactions involved in the pathogenicity of the bacterial plant pathogen Xanthomonas axonopodis pv. citri. Using a yeast two-hybrid system based on Gal4 DNA-binding and activation domains, we have focused on identifying interactions involving subunits, regulators, and substrates of the type III secretion system coded by the hrp (for hypersensitive response and pathogenicity), hrc (for hrp conserved), and hpa (for hrp associated) genes. We have identified several previously uncharacterized interactions involving (i) HrpG, a two-component system response regulator responsible for the expression of X. axonopodis pv. citri hrp operons, and XAC0095, a previously uncharacterized protein encountered only in Xanthomonas spp.; (ii) HpaA, a protein secreted by the type III secretion system, HpaB, and the C-terminal domain of HrcV; (iii) HrpB1, HrpD6, and HrpW; and (iv) HrpB2 and HrcU. Homotropic interactions were also identified for the ATPase HrcN. These newly identified protein-protein interactions increase our understanding of the functional integration of phytopathogen-specific type III secretion system components and suggest new hypotheses regarding the molecular mechanisms underlying Xanthomonas pathogenicity.

Parallel Measurement of Ca2+ Binding and Fluorescence Emission upon Ca2+ Titration of Recombinant Skeletal Muscle Troponin C MEASUREMENT OF SEQUENTIAL CALCIUM BINDING TO THE REGULATORY SITES

Calcium binding to chicken recombinant skeletal muscle TnC (TnC) and its mutants containing tryptophan (F29W), 5-hydroxytryptophan (F29HW), or 7-azatryptophan (F29ZW) at position 29 was measured by flow dialysis and by fluorescence. Comparative analysis of the results allowed us to determine the influence of each amino acid on the calcium binding properties of the N-terminal regulatory domain of the protein. Compared with TnC, the Ca2+ affinity of N-terminal sites was: 1) increased 6-fold in F29W, 2) increased 3-fold in F29ZW, and 3) decreased slightly in F29HW. The Ca2+titration of F29ZW monitored by fluorescence displayed a bimodal curve related to sequential Ca2+ binding to the two N-terminal Ca2+ binding sites. Single and double mutants of TnC, F29W, F29HW, and F29ZW were constructed by replacing aspartate by alanine at position 30 (site I) or 66 (site II) or both. Ca2+ binding data showed that the Asp → Ala mutation at position 30 impairs calcium binding to site I only, whereas the Asp → Ala mutation at position 66 impairs calcium binding to both sites I and II. Furthermore, the Asp → Ala mutation at position 30 eliminates the differences in Ca2+ affinity observed for replacement of Phe at position 29 by Trp, 5-hydroxytryptophan, or 7-azatryptophan. We conclude that position 29 influences the affinity of site I and that Ca2+ binding to site I is dependent on the previous binding of metal to site II.

Cloning and expression of soluble recombinant protein comprising the extracellular domain of the human type I interferon receptor 2c subunit (IFNAR-2c) in E. coli

An optimized procedure was developed for production of the extracellular domain encoding amino acids 1–243 of the human type I interferon receptor 2c subunit (IFNAR-2c) as a fusion protein with glutathione S-transferase (GST-IFNAR2cEC) in E. coli to obtain active, soluble protein. Induction of protein expression at 37 °C resulted in a formation of inclusion body. Co-expression with bacterial chaperones, GroEL and GroES, failed to support the folding of GST-IFNAR2cEc under IPTG induction at 37 °C. Expression induced at 25 °C decreased the inclusion body formation up to 62% and cell disruption by a French press provided higher recovery of the recombinant protein than cell disruption by sonication.

In silico identification of potential chaperone genes that belong to type III and type IV secretion systems in Xanthomonas axonopodis pv citri

The secretion of bacterial virulence factors and flagellar components requires the assistance of specific type III and flagellar chaperones. Standard computational annotation of the genome of Xanthomonas axonopodis pv citri, a plant pathogen that causes citrus canker, initially did not identify any genes belonging to these chaperone categories since the primary sequence homology between them was very low. However, in a search for hypothetical proteins with characteristics similar to these chaperones, we have now identified 30 chromosomal and 10 plasmidial potential genes encoding chaperones belonging to types III/IV, and flagellar secretion systems in this organism. The significance of these findings is discussed.