Origin and diversification of Xanthomonas citri subsp. citri pathotypes revealed by inclusive phylogenomic, dating, and biogeographic analyses.

BACKGROUND: Xanthomonas citri subsp. citri pathotypes cause bacterial citrus canker, being responsible for severe agricultural losses worldwide. The A pathotype has a broad host spectrum, while A* and Aw are more restricted both in hosts and in geography. Two previous phylogenomic studies led to contrasting well-supported clades for sequenced genomes of these pathotypes. No extensive biogeographical or divergence dating analytic approaches have been so far applied to available genomes. RESULTS: Based on a larger sampling of genomes than in previous studies (including six new genomes sequenced by our group, adding to a total of 95 genomes), phylogenomic analyses resulted in different resolutions, though overall indicating that A + AW is the most likely true clade. Our results suggest the high degree of recombination at some branches and the fast diversification of lineages are probable causes for this phylogenetic blurring effect. One of the genomes analyzed, X. campestris pv. durantae, was shown to be an A* strain; this strain has been reported to infect a plant of the family Verbenaceae, though there are no reports of any X. citri subsp. citri pathotypes infecting any plant outside the Citrus genus. Host reconstruction indicated the pathotype ancestor likely had plant hosts in the family Fabaceae, implying an ancient jump to the current Rutaceae hosts. Extensive dating analyses indicated that the origin of X. citri subsp. citri occurred more recently than the main phylogenetic splits of Citrus plants, suggesting dispersion rather than host-directed vicariance as the main driver of geographic expansion. An analysis of 120 pathogenic-related genes revealed pathotype-associated patterns of presence/absence. CONCLUSIONS: Our results provide novel insights into the evolutionary history of X. citri subsp. citri as well as a sound phylogenetic foundation for future evolutionary and genomic studies of its pathotypes.

Comparative Genomics of Xylella fastidiosa Explores Candidate Host-Specificity Determinants and Expands the Known Repertoire of Mobile Genetic Elements and Immunity Systems.

Xylella fastidiosa causes diseases in many plant species. Originally confined to the Americas, infecting mainly grapevine, citrus, and coffee, X. fastidiosa has spread to several plant species in Europe causing devastating diseases. Many pathogenicity and virulence factors have been identified, which enable the various X. fastidiosa strains to successfully colonize the xylem tissue and cause disease in specific plant hosts, but the mechanisms by which this happens have not been fully elucidated. Here we present thorough comparative analyses of 94 whole-genome sequences of X. fastidiosa strains from diverse plant hosts and geographic regions. Core-genome phylogeny revealed clades with members sharing mostly a geographic region rather than a host plant of origin. Phylogenetic trees for 1605 orthologous CDSs were explored for potential candidates related to host specificity using a score of mapping metrics. However, no candidate host-specificity determinants were strongly supported using this approach. We also show that X. fastidiosa accessory genome is represented by an abundant and heterogeneous mobilome, including a diversity of prophage regions. Our findings provide a better understanding of the diversity of phylogenetically close genomes and expand the knowledge of X. fastidiosa mobile genetic elements and immunity systems.

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

BACKGROUND: Citrus 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. RESULTS: We 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. CONCLUSION: We 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.

OVERFRAG: An overlapping DNA fragments generator for molecular cloning and synthetic biology.

DNA synthesis and homologous recombination can be used to simplify molecular cloning and to make synthetic biology easily accessible (M.J. Czar et al., 2009). However, the design of overlapping DNA fragments to construct large molecules is time-consuming and requires verification of several parameters to ensure that fragment synthesis is attainable, given the restrictions found in chemical synthesis of DNA. OVERFRAG is a web-based tool that generates overlapping DNA fragments to assemble either in yeast cells by Gap Repair (H. Ma et al., 1987) or in vitro by (D.G. Gibson et al., 2009) and In-Fusion (B. Zhu et al., 2007) methods. The fragments generated are suitable for chemical synthesis and molecular assembly. Some possible uses include cDNA cloning, design of chimeric antibodies and synthetic biology applications. Web tool is freely available at http://www.each.usp.br/digiampietri/overfrag.

Characterization of Mycobacterium chelonae-Like Strains by Comparative Genomics.

Isolates of the Mycobacterium chelonae-M. abscessus complex are subdivided into four clusters (CHI to CHIV) in the INNO-LiPA® Mycobacterium spp DNA strip assay. A considerable phenotypic variability was observed among isolates of the CHII cluster. In this study, we examined the diversity of 26 CHII cluster isolates by phenotypic analysis, drug susceptibility testing, whole genome sequencing and single-gene analysis. Pairwise genome comparisons were performed using several approaches, including average nucleotide identity (ANI) and genome-to-genome distance (GGD) among others. Based on ANI and GGD the isolates were identified as M. chelonae (14 isolates), M. franklinii (2 isolates) and M. salmoniphium (1 isolate). The remaining 9 isolates were subdivided into three novel putative genomospecies. Phenotypic analyses including drug susceptibility testing, as well as whole genome comparison by TETRA and delta differences, were not helpful in separating the groups revealed by ANI and GGD. The analysis of standard four conserved genomic regions showed that rpoB alone and the concatenated sequences clearly distinguished the taxonomic groups delimited by whole genome analyses. In conclusion, the CHII INNO-LiPa is not a homogeneous cluster; on the contrary, it is composed of closely related different species belonging to the M. chelonae-M. abscessus complex and also several unidentified isolates. The detection of these isolates, putatively novel species, indicates a wider inner variability than the presently known in this complex.

Comparative analyses of Xanthomonas and Xylella complete genomes.

Computational analyses of four bacterial genomes of the Xanthomonadaceae family reveal new unique genes that may be involved in adaptation, pathogenicity, and host specificity. The Xanthomonas genus presents 3636 unique genes distributed in 1470 families, while Xylella genus presents 1026 unique genes distributed in 375 families. Among Xanthomonas-specific genes, we highlight a large number of cell wall degrading enzymes, proteases, and iron receptors, a set of energy metabolism genes, second copy of the type II secretion system, type III secretion system, flagella and chemotactic machinery, and the xanthomonadin synthesis gene cluster. Important genes unique to the Xylella genus are an additional copy of a type IV pili gene cluster and the complete machinery of colicin V synthesis and secretion. Intersections of gene sets from both genera reveal a cluster of genes homologous to Salmonella's SPI-7 island in Xanthomonas axonopodis pv citri and Xylella fastidiosa 9a5c, which might be involved in host specificity. Each genome also presents important unique genes, such as an HMS cluster, the kdgT gene, and O-antigen in Xanthomonas axonopodis pv citri; a number of avrBS genes and a distinct O-antigen in Xanthomonas campestris pv campestris, a type I restriction-modification system and a nickase gene in Xylella fastidiosa 9a5c, and a type II restriction-modification system and two genes related to peptidoglycan biosynthesis in Xylella fastidiosa temecula 1. All these differences imply a considerable number of gene gains and losses during the divergence of the four lineages, and are associated with structural genome modifications that may have a direct relation with the mode of transmission, adaptation to specific environments and pathogenicity of each organism.

A framework based on Web service orchestration for bioinformatics workflow management.

Bioinformatics activities are growing all over the world, with proliferation of data and tools. This brings new challenges: how to understand and organize these resources and how to provide interoperability among tools to achieve a given goal. We defined and implemented a framework to help meet some of these challenges. Four issues were considered: the use of Web services as a basic unit, the notion of a Semantic Web to improve interoperability at the syntactic and semantic levels, and the use of scientific workflows to coordinate services to be executed, including their interdependencies and service orchestration.

Analysis of an advisor-advisee relationship: an exploratory study of the area of exact and Earth sciences in Brazil.

Scientific collaboration has been studied by researchers for decades. Several approaches have been adopted to address the question of how collaboration has evolved in terms of publication output, numbers of coauthors, and multidisciplinary trends. One particular type of collaboration that has received very little attention concerns advisor and advisee relationships. In this paper, we examine this relationship for the researchers who are involved in the area of Exact and Earth Sciences in Brazil and its eight subareas. These pairs are registered in the Lattes Platform that manages the individual curricula vitae of Brazilian researchers. The individual features of these academic researchers and their coauthoring relationships were investigated. We have found evidence that there exists positive correlation between time of advisor-advisee relationship with the advisee's productivity. Additionally, there has been a gradual decline in advisor-advisee coauthoring over a number of years as measured by the Kulczynski index, which could be interpreted as decline of the dependence.

BraX-Ray: an X-ray of the Brazilian computer science graduate programs.

Research productivity assessment is increasingly relevant for allocation of research funds. On one hand, this assessment is challenging because it involves both qualitative and quantitative analysis of several characteristics, most of them subjective in nature. On the other hand, current tools and academic social networks make bibliometric data web-available to everyone for free. Those tools, especially when combined with other data, are able to create a rich environment from which information on research productivity can be extracted. In this context, our work aims at characterizing the Brazilian Computer Science graduate programs and the relationship among themselves. We (i) present views of the programs from different perspectives, (ii) rank the programs according to each perspective and a combination of them, (iii) show correlation between assessment metrics, (iv) discuss how programs relate to another, and (v) infer aspects that boost programs' research productivity. The results indicate that programs with a higher insertion in the coauthorship network topology also possess a higher research productivity between 2004 and 2009.

Metagenomic analysis of a tropical composting operation at the são paulo zoo park reveals diversity of biomass degradation functions and organisms.

Composting operations are a rich source for prospection of biomass degradation enzymes. We have analyzed the microbiomes of two composting samples collected in a facility inside the São Paulo Zoo Park, in Brazil. All organic waste produced in the park is processed in this facility, at a rate of four tons/day. Total DNA was extracted and sequenced with Roche/454 technology, generating about 3 million reads per sample. To our knowledge this work is the first report of a composting whole-microbial community using high-throughput sequencing and analysis. The phylogenetic profiles of the two microbiomes analyzed are quite different, with a clear dominance of members of the Lactobacillus genus in one of them. We found a general agreement of the distribution of functional categories in the Zoo compost metagenomes compared with seven selected public metagenomes of biomass deconstruction environments, indicating the potential for different bacterial communities to provide alternative mechanisms for the same functional purposes. Our results indicate that biomass degradation in this composting process, including deconstruction of recalcitrant lignocellulose, is fully performed by bacterial enzymes, most likely by members of the Clostridiales and Actinomycetales orders.

An ontology-based framework for bioinformatics workflows.

The proliferation of bioinformatics activities brings new challenges - how to understand and organise these resources, how to exchange and reuse successful experimental procedures, and to provide interoperability among data and tools. This paper describes an effort toward these directions. It is based on combining research on ontology management, AI and scientific workflows to design, reuse and annotate bioinformatics experiments. The resulting framework supports automatic or interactive composition of tasks based on AI planning techniques and takes advantage of ontologies to support the specification and annotation of bioinformatics workflows. We validate our proposal with a prototype running on real data.

Gene projects: a genome web tool for ongoing mining and annotation applied to CitEST

Genome projects, both genomic DNA and ESTs (cDNA), generate a large amount of information, demanding time and a well-structured bioinformatics laboratory to manage these data. These genome projects use information available in heterogeneous formats from different sources. The amount and heterogeneity of this information, as well as the absence of a world consensus pattern, make the integration of these data a difficult task. At the same time, sub-tasks, such as microarray analyses of these projects, are very complex. This creates a demand for the development of creative solutions for ongoing annotation, thematic projects, microarray experiments, etc. This paper presents Gene Projects, a system developed to integrate all kinds of solutions.