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The soil fungus Rhizoctonia solani is an economically important pathogen of agricultural and forestry crops. Here, we present the complete sequence and analysis of the mitochondrial genome of R. solani , field isolate Rhs1AP. The genome (235 849 bp) is the largest mitochondrial genome of a filamentous fungus sequenced to date and exhibits a rich accumulation of introns, novel repeat sequences, homing endonuclease genes, and hypothetical genes. Stable secondary structures exhibited by repeat sequences suggest that they comprise functional, possibly catalytic RNA elements. RNA-Seq expression profiling confirmed that the majority of homing endonuclease genes and hypothetical genes are transcriptionally active. Comparative analysis suggests that the mitochondrial genome of R. solani is an example of a dynamic history of expansion in filamentous fungi.

A morphospecies is defined as a taxonomic species based wholly on morphology, but often morphospecies consist of clusters of cryptic species that can be identified genetically or molecularly. The nature of the evolutionary novelty that accompanies speciation in a morphospecies is an intriguing question. Morphospecies are particularly common among ciliates, a group of unicellular eukaryotes that separates 2 kinds of nuclei—the silenced germline nucleus (micronucleus [MIC]) and the actively expressed somatic nucleus (macronucleus [MAC])—within a common cytoplasm. Because of their very similar morphologies, members of the Tetrahymena genus are considered a morphospecies. We explored the hidden genomic evolution within this genus by performing a comprehensive comparative analysis of the somatic genomes of 10 species and the germline genomes of 2 species of Tetrahymena. These species show high genetic divergence; phylogenomic analysis suggests that the genus originated about 300 million years ago (Mya). Seven universal protein domains are preferentially included among the species-specific (i.e., the youngest) Tetrahymena genes. In particular, leucine-rich repeat (LRR) genes make the largest contribution to the high level of genome divergence of the 10 species. LRR genes can be sorted into 3 different age groups. Parallel evolutionary trajectories have independently occurred among LRR genes in the different Tetrahymena species. Thousands of young LRR genes contain tandem arrays of exactly 90-bp exons. The introns separating these exons show a unique, extreme phase 2 bias, suggesting a clonal origin and successive expansions of 90-bp–exon LRR genes. Identifying LRR gene age groups allowed us to document a Tetrahymena intron length cycle. The youngest 90-bp exon LRR genes in T. thermophila are concentrated in pericentromeric and subtelomeric regions of the 5 micronuclear chromosomes, suggesting that these regions act as genome innovation centers. Copies of a Tetrahymena Long interspersed element (LINE)-like retrotransposon are very frequently found physically adjacent to 90-bp exon/intron repeat units of the youngest LRR genes. We propose that Tetrahymena species have used a massive exon-shuffling mechanism, involving unequal crossing over possibly in concert with retrotransposition, to create the unique 90-bp exon array LRR genes., Genomic comparison of ten morphologically very similar species of ciliate from the genus Tetrahymena reveals how parallel microevolutionary processes have shaped their genomes and created unique genes through retrotransposition.

The germline genome of the binucleated ciliate Tetrahymena thermophila undergoes programmed chromosome breakage and massive DNA elimination to generate the somatic genome. Here, we present a complete sequence assembly of the germline genome and analyze multiple features of its structure and its relationship to the somatic genome, shedding light on the mechanisms of genome rearrangement as well as the evolutionary history of this remarkable germline/soma differentiation. Our results strengthen the notion that a complex, dynamic, and ongoing interplay between mobile DNA elements and the host genome have shaped Tetrahymena chromosome structure, locally and globally. Non-standard outcomes of rearrangement events, including the generation of short-lived somatic chromosomes and excision of DNA interrupting protein-coding regions, may represent novel forms of developmental gene regulation. We also compare Tetrahymena’s germline/soma differentiation to that of other characterized ciliates, illustrating the wide diversity of adaptations that have occurred within this phylum.

The JCVI metagenomics analysis pipeline provides for the efficient and consistent annotation of shotgun metagenomics sequencing data for sampling communities of prokaryotic organisms. The process can be equally applied to individual sequence reads from traditional Sanger capillary electrophoresis sequences, newer technologies such as 454 pyrosequencing, or sequence assemblies derived from one or more of these data types. It includes the analysis of both coding and non-coding genes, whether full-length or, as is often the case for shotgun metagenomics, fragmentary. The system is designed to provide the best-supported conservative functional annotation based on a combination of trusted homology-based scientific evidence and computational assertions and an annotation value hierarchy established through extensive manual curation. The functional annotation attributes assigned by this system include gene name, gene symbol, GO terms [1], EC numbers [2], and JCVI functional role categories [3].

The Comprehensive Microbial Resource or CMR (http://cmr.jcvi.org) provides a web-based central resource for the display, search and analysis of the sequence and annotation for complete and publicly available bacterial and archaeal genomes. In addition to displaying the original annotation from GenBank, the CMR makes available secondary automated structural and functional annotation across all genomes to provide consistent data types necessary for effective mining of genomic data. Precomputed homology searches are stored to allow meaningful genome comparisons. The CMR supplies users with over 50 different tools to utilize the sequence and annotation data across one or more of the 571 currently available genomes. At the gene level users can view the gene annotation and underlying evidence. Genome level information includes whole genome graphical displays, biochemical pathway maps and genome summary data. Comparative tools display analysis between genomes with homology and genome alignment tools, and searches across the accessions, annotation, and evidence assigned to all genes/genomes are available. The data and tools on the CMR aid genomic research and analysis, and the CMR is included in over 200 scientific publications. The code underlying the CMR website and the CMR database are freely available for download with no license restrictions.

Pathema (http://pathema.jcvi.org) is one of the eight Bioinformatics Resource Centers (BRCs) funded by the National Institute of Allergy and Infectious Disease (NIAID) designed to serve as a core resource for the bio-defense and infectious disease research community. Pathema strives to support basic research and accelerate scientific progress for understanding, detecting, diagnosing and treating an established set of six target NIAID Category A-C pathogens: Category A priority pathogens; Bacillus anthracis and Clostridium botulinum, and Category B priority pathogens; Burkholderia mallei, Burkholderia pseudomallei, Clostridium perfringens and Entamoeba histolytica. Each target pathogen is represented in one of four distinct clade-specific Pathema web resources and underlying databases developed to target the specific data and analysis needs of each scientific community. All publicly available complete genome projects of phylogenetically related organisms are also represented, providing a comprehensive collection of organisms for comparative analyses. Pathema facilitates the scientific exploration of genomic and related data through its integration with web-based analysis tools, customized to obtain, display, and compute results relevant to ongoing pathogen research. Pathema serves the bio-defense and infectious disease research community by disseminating data resulting from pathogen genome sequencing projects and providing access to the results of inter-genomic comparisons for these organisms.

The complete genomes of three strains from the phylum Acidobacteria were compared. Phylogenetic analysis placed them as a unique phylum. They share genomic traits with members of the Proteobacteria , the Cyanobacteria , and the Fungi. The three strains appear to be versatile heterotrophs. Genomic and culture traits indicate the use of carbon sources that span simple sugars to more complex substrates such as hemicellulose, cellulose, and chitin. The genomes encode low-specificity major facilitator superfamily transporters and high-affinity ABC transporters for sugars, suggesting that they are best suited to low-nutrient conditions. They appear capable of nitrate and nitrite reduction but not N 2 fixation or denitrification. The genomes contained numerous genes that encode siderophore receptors, but no evidence of siderophore production was found, suggesting that they may obtain iron via interaction with other microorganisms. The presence of cellulose synthesis genes and a large class of novel high-molecular-weight excreted proteins suggests potential traits for desiccation resistance, biofilm formation, and/or contribution to soil structure. Polyketide synthase and macrolide glycosylation genes suggest the production of novel antimicrobial compounds. Genes that encode a variety of novel proteins were also identified. The abundance of acidobacteria in soils worldwide and the breadth of potential carbon use by the sequenced strains suggest significant and previously unrecognized contributions to the terrestrial carbon cycle. Combining our genomic evidence with available culture traits, we postulate that cells of these isolates are long-lived, divide slowly, exhibit slow metabolic rates under low-nutrient conditions, and are well equipped to tolerate fluctuations in soil hydration.

The complete genome sequence of Burkholderia mallei ATCC 23344 provides insight into this highly infectious bacterium's pathogenicity and evolutionary history. B. mallei , the etiologic agent of glanders, has come under renewed scientific investigation as a result of recent concerns about its past and potential future use as a biological weapon. Genome analysis identified a number of putative virulence factors whose function was supported by comparative genome hybridization and expression profiling of the bacterium in hamster liver in vivo . The genome contains numerous insertion sequence elements that have mediated extensive deletions and rearrangements of the genome relative to Burkholderia pseudomallei . The genome also contains a vast number (>12,000) of simple sequence repeats. Variation in simple sequence repeats in key genes can provide a mechanism for generating antigenic variation that may account for the mammalian host's inability to mount a durable adaptive immune response to a B. mallei infection.

Motivation: The presence or absence of metabolic pathways and structures provide a context that makes protein annotation far more reliable. Compiling such information across microbial genomes improves the functional classification of proteins and provides a valuable resource for comparative genomics. Results: We have created a Genome Properties system to present key aspects of prokaryotic biology using standardized computational methods and controlled vocabularies. Properties reflect gene content, phenotype, phylogeny and computational analyses. The results of searches using hidden Markov models allow many properties to be deduced automatically, especially for families of proteins (equivalogs) conserved in function since their last common ancestor. Additional properties are derived from curation, published reports and other forms of evidence. Genome Properties system was applied to 156 complete prokaryotic genomes, and is easily mined to find differences between species, correlations between metabolic features and families of uncharacterized proteins, or relationships among properties. Availability: Genome Properties can be found at http://www.tigr.org/Genome_Properties Contact: selengut@tigr.org Supplementary information: http://www.tigr.org/tigr-scripts/CMR2/genome_properties_references.spl

We report the complete genome sequence of the model bacterial pathogen Pseudomonas syringae pathovar tomato DC3000 (DC3000), which is pathogenic on tomato and Arabidopsis thaliana . The DC3000 genome (6.5 megabases) contains a circular chromosome and two plasmids, which collectively encode 5,763 ORFs. We identified 298 established and putative virulence genes, including several clusters of genes encoding 31 confirmed and 19 predicted type III secretion system effector proteins. Many of the virulence genes were members of paralogous families and also were proximal to mobile elements, which collectively comprise 7% of the DC3000 genome. The bacterium possesses a large repertoire of transporters for the acquisition of nutrients, particularly sugars, as well as genes implicated in attachment to plant surfaces. Over 12% of the genes are dedicated to regulation, which may reflect the need for rapid adaptation to the diverse environments encountered during epiphytic growth and pathogenesis. Comparative analyses confirmed a high degree of similarity with two sequenced pseudomonads, Pseudomonas putida and Pseudomonas aeruginosa , yet revealed 1,159 genes unique to DC3000, of which 811 lack a known function.

The soil fungus Rhizoctonia solani is a pathogen of agricultural crops. Here, we report on the 51,705,945 bp draft consensus genome sequence of R. solani strain Rhs1AP. A comprehensive understanding of the heterokaryotic genome complexity and organization of R. solani may provide insight into the plant disease ecology and adaptive behavior of the fungus.

Among the species that cause blue mold, isolates of Penicillium expansum are the most prevalent and virulent species, causing more than 50 percent of postharvest decay. We report the draft genome sequence of P. expansum R19 in order to identify fungal virulence factors and to understand the mechanism of infection.

Background The genera Aspergillus and Penicillium include some of the most beneficial as well as the most harmful fungal species such as the penicillin-producer Penicillium chrysogenum and the human pathogen Aspergillus fumigatus, respectively. Their mitochondrial genomic sequences may hold vital clues into the mechanisms of their evolution, population genetics, and biology, yet only a handful of these genomes have been fully sequenced and annotated. Results Here we report the complete sequence and annotation of the mitochondrial genomes of six Aspergillus and three Penicillium species: A. fumigatus, A. clavatus, A. oryzae, A. flavus, Neosartorya fischeri (A. fischerianus), A. terreus, P. chrysogenum, P. marneffei, and Talaromyces stipitatus (P. stipitatum). The accompanying comparative analysis of these and related publicly available mitochondrial genomes reveals wide variation in size (25–36 Kb) among these closely related fungi. The sources of genome expansion include group I introns and accessory genes encoding putative homing endonucleases, DNA and RNA polymerases (presumed to be of plasmid origin) and hypothetical proteins. The two smallest sequenced genomes (A. terreus and P. chrysogenum) do not contain introns in protein-coding genes, whereas the largest genome (T. stipitatus), contains a total of eleven introns. All of the sequenced genomes have a group I intron in the large ribosomal subunit RNA gene, suggesting that this intron is fixed in these species. Subsequent analysis of several A. fumigatus strains showed low intraspecies variation. This study also includes a phylogenetic analysis based on 14 concatenated core mitochondrial proteins. The phylogenetic tree has a different topology from published multilocus trees, highlighting the challenges still facing the Aspergillus systematics. Conclusions The study expands the genomic resources available to fungal biologists by providing mitochondrial genomes with consistent annotations for future genetic, evolutionary and population studies. Despite the conservation of the core genes, the mitochondrial genomes of Aspergillus and Penicillium species examined here exhibit significant amount of interspecies variation. Most of this variation can be attributed to accessory genes and mobile introns, presumably acquired by horizontal gene transfer of mitochondrial plasmids and intron homing.

Background The Amoebozoa constitute one of the primary divisio ns of eukaryotes encompassing taxa of both biomedic al and evolutionary importance, yet its genomic divers ity remains largely unsampled. Here we present an analysis of a whole genome assembly of Acanthamoeba castellanii ( Ac ) the first representative from a solitary free-living amoebozoan. Results Ac encodes 15,455 compact intron rich genes a signifi cant number of which are predicted to have arisen through interkingdom lateral gene transfer (LGT). A majority of the LGT candidates have undergone a substantial degree of intronization and Ac appears to have incorporated them into established transcriptional programs. Ac manifests a complex signaling and cell communicati on repertoire including a complete tyrosine kinase signaling toolkit and a comparable diversity of predicted extracellular receptors to that found in the facultatively multicellular dictyostelids. An impor tant environmental host of a diverse range of bacte ria and viruses, Ac utilizes a diverse repertoire of predicted pattern recognition receptors many with predicted orthologous functions in the innate immune systems of higher organisms. Conclusions Our analysis highlights the important role of LGT i n the biology of Ac and in the diversification of microbial eukaryotes. The early evolution of a key signaling facility implicated in the evolution of metazoan multicellularity strongly argues for its emergence early in the Unikont lineage. Overall the availabil ity of an Ac genome should aid in deciphering the biology of th e Amoebozoa and facilitate functional genomic studi es in this important model organism and environmental host.

Comparative genomics is enhanced by data mining the rapidly expanding DNA sequence databases. Because of the immense amount of data, computational tools and methods are needed to augment traditional manual visualizations and manipulations of these data. GeneOrder2.0, a Java-based interactive software programme, organizes genome sequence data into tabular and graphical visualizations of the extent of colinearity of genes between any two chromosome genomes of ≤250 kilobases. Both GenBank and proprietary data can be analyzed with this tool.

Anaplasma (formerly Ehrlichia) phagocytophilum, Ehrlichia chaffeensis, and Neorickettsia (formerly Ehrlichia) sennetsu are intracellular vector-borne pathogens that cause human ehrlichiosis, an emerging infectious disease. We present the complete genome sequences of these organisms along with comparisons to other organisms in the Rickettsiales order. Ehrlichia spp. and Anaplasma spp. display a unique large expansion of immunodominant outer membrane proteins facilitating antigenic variation. All Rickettsiales have a diminished ability to synthesize amino acids compared to their closest free-living relatives. Unlike members of the Rickettsiaceae family, these pathogenic Anaplasmataceae are capable of making all major vitamins, cofactors, and nucleotides, which could confer a beneficial role in the invertebrate vector or the vertebrate host. Further analysis identified proteins potentially involved in vacuole confinement of the Anaplasmataceae, a life cycle involving a hematophagous vector, vertebrate pathogenesis, human pathogenesis, and lack of transovarial transmission. These discoveries provide significant insights into the biology of these obligate intracellular pathogens., Synopsis Ehrlichiosis is an acute disease that triggers flu-like symptoms in both humans and animals. It is caused by a range of bacteria transmitted by ticks or flukes. Because these bacteria are difficult to culture, however, the organisms are poorly understood. The genomes of three emerging human pathogens causing ehrlichiosis were sequenced. A database was designed to allow the comparison of these three genomes to sixteen other bacteria with similar lifestyles. Analysis from this database reveals new species-specific and disease-specific genes indicating niche adaptations, pathogenic traits, and other features. In particular, one of the organisms contains more than 100 copies of a single gene involved in interactions with the host(s). These comparisons also enabled a reconstruction of the metabolic potential of five representative genomes from these bacteria and their close relatives. With this work, scientists can study these emerging pathogens in earnest.

The development of efficient and inexpensive genome sequencing methods has revolutionized the study of human bacterial pathogens and improved vaccine design. Unfortunately, the sequence of a single genome does not reflect how genetic variability drives pathogenesis within a bacterial species and also limits genome-wide screens for vaccine candidates or for antimicrobial targets. We have generated the genomic sequence of six strains representing the five major disease-causing serotypes of Streptococcus agalactiae , the main cause of neonatal infection in humans. Analysis of these genomes and those available in databases showed that the S. agalactiae species can be described by a pan-genome consisting of a core genome shared by all isolates, accounting for ≈80% of any single genome, plus a dispensable genome consisting of partially shared and strain-specific genes. Mathematical extrapolation of the data suggests that the gene reservoir available for inclusion in the S. agalactiae pan-genome is vast and that unique genes will continue to be identified even after sequencing hundreds of genomes.

Improvements in DNA sequencing technology and methodology have led to the rapid expansion of databases comprising DNA sequence, gene and genome data. Lower operational costs and heightened interest resulting from initial intriguing novel discoveries from genomics are also contributing to the accumulation of these data sets. A major challenge is to analyze and to mine data from these databases, especially whole genomes. There is a need for computational tools that look globally at genomes for data mining. CoreGenes is a global JAVA-based interactive data mining tool that identifies and catalogs a "core" set of genes from two to five small whole genomes simultaneously. CoreGenes performs hierarchical and iterative BLASTP analyses using one genome as a reference and another as a query. Subsequent query genomes are compared against each newly generated "consensus." These iterations lead to a matrix comprising related genes from this set of genomes, e. g., viruses, mitochondria and chloroplasts. Currently the software is limited to small genomes on the order of 330 kilobases or less. A computational tool CoreGenes has been developed to analyze small whole genomes globally. BLAST score-related and putatively essential "core" gene data are displayed as a table with links to GenBank for further data on the genes of interest. This web resource is available at http://pumpkins.ib3.gmu.edu:8080/CoreGenes or http://www.bif.atcc.org/CoreGenes .

Pseudomonas syringae pv. phaseolicola, a gram-negative bacterial plant pathogen, is the causal agent of halo blight of bean. In this study, we report on the genome sequence of P. syringae pv. phaseolicola isolate 1448A, which encodes 5,353 open reading frames (ORFs) on one circular chromosome (5,928,787 bp) and two plasmids (131,950 bp and 51,711 bp). Comparative analyses with a phylogenetically divergent pathovar, P. syringae pv. tomato DC3000, revealed a strong degree of conservation at the gene and genome levels. In total, 4,133 ORFs were identified as putative orthologs in these two pathovars using a reciprocal best-hit method, with 3,941 ORFs present in conserved, syntenic blocks. Although these two pathovars are highly similar at the physiological level, they have distinct host ranges; 1448A causes disease in beans, and DC3000 is pathogenic on tomato and Arabidopsis . Examination of the complement of ORFs encoding virulence, fitness, and survival factors revealed a substantial, but not complete, overlap between these two pathovars. Another distinguishing feature between the two pathovars is their distinctive sets of transposable elements. With access to a fifth complete pseudomonad genome sequence, we were able to identify 3,567 ORFs that likely comprise the core Pseudomonas genome and 365 ORFs that are P. syringae specific.