Author Correction: A transcriptomic analysis of the phylum Nematoda.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The genome sequence of ectromelia virus Naval and Cornell isolates from outbreaks in North America.

Ectromelia virus (ECTV) is the causative agent of mousepox, a disease of laboratory mouse colonies and an excellent model for human smallpox. We report the genome sequence of two isolates from outbreaks in laboratory mouse colonies in the USA in 1995 and 1999: ECTV-Naval and ECTV-Cornell, respectively. The genome of ECTV-Naval and ECTV-Cornell was sequenced by the 454-Roche technology. The ECTV-Naval genome was also sequenced by the Sanger and Illumina technologies in order to evaluate these technologies for poxvirus genome sequencing. Genomic comparisons revealed that ECTV-Naval and ECTV-Cornell correspond to the same virus isolated from independent outbreaks. Both ECTV-Naval and ECTV-Cornell are extremely virulent in susceptible BALB/c mice, similar to ECTV-Moscow. This is consistent with the ECTV-Naval genome sharing 98.2% DNA sequence identity with that of ECTV-Moscow, and indicates that the genetic differences with ECTV-Moscow do not affect the virulence of ECTV-Naval in the mousepox model of footpad infection.

Genomic analysis of the causative agents of coccidiosis in domestic chickens.

Global production of chickens has trebled in the past two decades and they are now the most important source of dietary animal protein worldwide. Chickens are subject to many infectious diseases that reduce their performance and productivity. Coccidiosis, caused by apicomplexan protozoa of the genus Eimeria, is one of the most important poultry diseases. Understanding the biology of Eimeria parasites underpins development of new drugs and vaccines needed to improve global food security. We have produced annotated genome sequences of all seven species of Eimeria that infect domestic chickens, which reveal the full extent of previously described repeat-rich and repeat-poor regions and show that these parasites possess the most repeat-rich proteomes ever described. Furthermore, while no other apicomplexan has been found to possess retrotransposons, Eimeria is home to a family of chromoviruses. Analysis of Eimeria genes involved in basic biology and host-parasite interaction highlights adaptations to a relatively simple developmental life cycle and a complex array of co-expressed surface proteins involved in host cell binding.

A conserved acetyl esterase domain targets diverse bacteriophages to the Vi capsular receptor of Salmonella enterica serovar Typhi.

A number of bacteriophages have been identified that target the Vi capsular antigen of Salmonella enterica serovar Typhi. Here we show that these Vi phages represent a remarkably diverse set of phages belonging to three phage families, including Podoviridae and Myoviridae. Genome analysis facilitated the further classification of these phages and highlighted aspects of their independent evolution. Significantly, a conserved protein domain carrying an acetyl esterase was found to be associated with at least one tail fiber gene for all Vi phages, and the presence of this domain was confirmed in representative phage particles by mass spectrometric analysis. Thus, we provide a simple explanation and paradigm of how a diverse group of phages target a single key virulence antigen associated with this important human-restricted pathogen.

New insights into the blood-stage transcriptome of Plasmodium falciparum using RNA-Seq.

Recent advances in high-throughput sequencing present a new opportunity to deeply probe an organism's transcriptome. In this study, we used Illumina-based massively parallel sequencing to gain new insight into the transcriptome (RNA-Seq) of the human malaria parasite, Plasmodium falciparum. Using data collected at seven time points during the intraerythrocytic developmental cycle, we (i) detect novel gene transcripts; (ii) correct hundreds of gene models; (iii) propose alternative splicing events; and (iv) predict 5' and 3' untranslated regions. Approximately 70% of the unique sequencing reads map to previously annotated protein-coding genes. The RNA-Seq results greatly improve existing annotation of the P. falciparum genome with over 10% of gene models modified. Our data confirm 75% of predicted splice sites and identify 202 new splice sites, including 84 previously uncharacterized alternative splicing events. We also discovered 107 novel transcripts and expression of 38 pseudogenes, with many demonstrating differential expression across the developmental time series. Our RNA-Seq results correlate well with DNA microarray analysis performed in parallel on the same samples, and provide improved resolution over the microarray-based method. These data reveal new features of the P. falciparum transcriptional landscape and significantly advance our understanding of the parasite's red blood cell-stage transcriptome.

Characterization of a novel wood mouse virus related to murid herpesvirus 4.

Two novel gammaherpesviruses were isolated, one from a field vole (Microtus agrestis) and the other from wood mice (Apodemus sylvaticus). The genome of the latter, designated wood mouse herpesvirus (WMHV), was completely sequenced. WMHV had the same genome structure and predicted gene content as murid herpesvirus 4 (MuHV4; murine gammaherpesvirus 68). Overall nucleotide sequence identity between WMHV and MuHV4 was 85 % and most of the 10 kb region at the left end of the unique region was particularly highly conserved, especially the viral tRNA-like sequences and the coding regions of genes M1 and M4. The partial sequence (71 913 bp) of another gammaherpesvirus, Brest herpesvirus (BRHV), which was isolated ostensibly from a white-toothed shrew (Crocidura russula), was also determined. The BRHV sequence was 99.2 % identical to the corresponding portion of the WMHV genome. Thus, WMHV and BRHV appeared to be strains of a new virus species. Biological characterization of WMHV indicated that it grew with similar kinetics to MuHV4 in cell culture. The pathogenesis of WMHV in wood mice was also extremely similar to that of MuHV4, except for the absence of inducible bronchus-associated lymphoid tissue at day 14 post-infection and a higher load of latently infected cells at 21 days post-infection.

Evidence for niche adaptation in the genome of the bovine pathogen Streptococcus uberis.

BACKGROUND: Streptococcus uberis, a Gram positive bacterial pathogen responsible for a significant proportion of bovine mastitis in commercial dairy herds, colonises multiple body sites of the cow including the gut, genital tract and mammary gland. Comparative analysis of the complete genome sequence of S. uberis strain 0140J was undertaken to help elucidate the biology of this effective bovine pathogen. RESULTS: The genome revealed 1,825 predicted coding sequences (CDSs) of which 62 were identified as pseudogenes or gene fragments. Comparisons with related pyogenic streptococci identified a conserved core (40%) of orthologous CDSs. Intriguingly, S. uberis 0140J displayed a lower number of mobile genetic elements when compared with other pyogenic streptococci, however bacteriophage-derived islands and a putative genomic island were identified. Comparative genomics analysis revealed most similarity to the genomes of Streptococcus agalactiae and Streptococcus equi subsp. zooepidemicus. In contrast, streptococcal orthologs were not identified for 11% of the CDSs, indicating either unique retention of ancestral sequence, or acquisition of sequence from alternative sources. Functions including transport, catabolism, regulation and CDSs encoding cell envelope proteins were over-represented in this unique gene set; a limited array of putative virulence CDSs were identified. CONCLUSION: S. uberis utilises nutritional flexibility derived from a diversity of metabolic options to successfully occupy a discrete ecological niche. The features observed in S. uberis are strongly suggestive of an opportunistic pathogen adapted to challenging and changing environmental parameters.

The genome of Burkholderia cenocepacia J2315, an epidemic pathogen of cystic fibrosis patients.

Bacterial infections of the lungs of cystic fibrosis (CF) patients cause major complications in the treatment of this common genetic disease. Burkholderia cenocepacia infection is particularly problematic since this organism has high levels of antibiotic resistance, making it difficult to eradicate; the resulting chronic infections are associated with severe declines in lung function and increased mortality rates. B. cenocepacia strain J2315 was isolated from a CF patient and is a member of the epidemic ET12 lineage that originated in Canada or the United Kingdom and spread to Europe. The 8.06-Mb genome of this highly transmissible pathogen comprises three circular chromosomes and a plasmid and encodes a broad array of functions typical of this metabolically versatile genus, as well as numerous virulence and drug resistance functions. Although B. cenocepacia strains can be isolated from soil and can be pathogenic to both plants and man, J2315 is representative of a lineage of B. cenocepacia rarely isolated from the environment and which spreads between CF patients. Comparative analysis revealed that ca. 21% of the genome is unique in comparison to other strains of B. cenocepacia, highlighting the genomic plasticity of this species. Pseudogenes in virulence determinants suggest that the pathogenic response of J2315 may have been recently selected to promote persistence in the CF lung. The J2315 genome contains evidence that its unique and highly adapted genetic content has played a significant role in its success as an epidemic CF pathogen.

Genome-wide discovery and verification of novel structured RNAs in Plasmodium falciparum.

We undertook a genome-wide search for novel noncoding RNAs (ncRNA) in the malaria parasite Plasmodium falciparum. We used the RNAz program to predict structures in the noncoding regions of the P. falciparum 3D7 genome that were conserved with at least one of seven other Plasmodium spp. genome sequences. By using Northern blot analysis for 76 high-scoring predictions and microarray analysis for the majority of candidates, we have verified the expression of 33 novel ncRNA transcripts including four members of a ncRNA family in the asexual blood stage. These transcripts represent novel structured ncRNAs in P. falciparum and are not represented in any RNA databases. We provide supporting evidence for purifying selection acting on the experimentally verified ncRNAs by comparing the nucleotide substitutions in the predicted ncRNA candidate structures in P. falciparum with the closely related chimp malaria parasite P. reichenowi. The high confirmation rate within a single parasite life cycle stage suggests that many more of the predictions may be expressed in other stages of the organism's life cycle.

Genome plasticity of BCG and impact on vaccine efficacy.

To understand the evolution, attenuation, and variable protective efficacy of bacillus Calmette-Guérin (BCG) vaccines, Mycobacterium bovis BCG Pasteur 1173P2 has been subjected to comparative genome and transcriptome analysis. The 4,374,522-bp genome contains 3,954 protein-coding genes, 58 of which are present in two copies as a result of two independent tandem duplications, DU1 and DU2. DU1 is restricted to BCG Pasteur, although four forms of DU2 exist; DU2-I is confined to early BCG vaccines, like BCG Japan, whereas DU2-III and DU2-IV occur in the late vaccines. The glycerol-3-phosphate dehydrogenase gene, glpD2, is one of only three genes common to all four DU2 variants, implying that BCG requires higher levels of this enzyme to grow on glycerol. Further amplification of the DU2 region is ongoing, even within vaccine preparations used to immunize humans. An evolutionary scheme for BCG vaccines was established by analyzing DU2 and other markers. Lesions in genes encoding sigma-factors and pleiotropic transcriptional regulators, like PhoR and Crp, were also uncovered in various BCG strains; together with gene amplification, these affect gene expression levels, immunogenicity, and, possibly, protection against tuberculosis. Furthermore, the combined findings suggest that early BCG vaccines may even be superior to the later ones that are more widely used.

Meningococcal genetic variation mechanisms viewed through comparative analysis of serogroup C strain FAM18.

The bacterium Neisseria meningitidis is commonly found harmlessly colonising the mucosal surfaces of the human nasopharynx. Occasionally strains can invade host tissues causing septicaemia and meningitis, making the bacterium a major cause of morbidity and mortality in both the developed and developing world. The species is known to be diverse in many ways, as a product of its natural transformability and of a range of recombination and mutation-based systems. Previous work on pathogenic Neisseria has identified several mechanisms for the generation of diversity of surface structures, including phase variation based on slippage-like mechanisms and sequence conversion of expressed genes using information from silent loci. Comparison of the genome sequences of two N. meningitidis strains, serogroup B MC58 and serogroup A Z2491, suggested further mechanisms of variation, including C-terminal exchange in specific genes and enhanced localised recombination and variation related to repeat arrays. We have sequenced the genome of N. meningitidis strain FAM18, a representative of the ST-11/ET-37 complex, providing the first genome sequence for the disease-causing serogroup C meningococci; it has 1,976 predicted genes, of which 60 do not have orthologues in the previously sequenced serogroup A or B strains. Through genome comparison with Z2491 and MC58 we have further characterised specific mechanisms of genetic variation in N. meningitidis, describing specialised loci for generation of cell surface protein variants and measuring the association between noncoding repeat arrays and sequence variation in flanking genes. Here we provide a detailed view of novel genetic diversification mechanisms in N. meningitidis. Our analysis provides evidence for the hypothesis that the noncoding repeat arrays in neisserial genomes (neisserial intergenic mosaic elements) provide a crucial mechanism for the generation of surface antigen variants. Such variation will have an impact on the interaction with the host tissues, and understanding these mechanisms is important to aid our understanding of the intimate and complex relationship between the human nasopharynx and the meningococcus.

Complete genome of acute rheumatic fever-associated serotype M5 Streptococcus pyogenes strain manfredo.

Comparisons of the 1.84-Mb genome of serotype M5 Streptococcus pyogenes strain Manfredo with previously sequenced genomes emphasized the role of prophages in diversification of S. pyogenes and the close relationship between strain Manfredo and MGAS8232, another acute rheumatic fever-associated strain.

Common inheritance of chromosome Ia associated with clonal expansion of Toxoplasma gondii.

Toxoplasma gondii is a globally distributed protozoan parasite that can infect virtually all warm-blooded animals and humans. Despite the existence of a sexual phase in the life cycle, T. gondii has an unusual population structure dominated by three clonal lineages that predominate in North America and Europe, (Types I, II, and III). These lineages were founded by common ancestors approximately10,000 yr ago. The recent origin and widespread distribution of the clonal lineages is attributed to the circumvention of the sexual cycle by a new mode of transmission-asexual transmission between intermediate hosts. Asexual transmission appears to be multigenic and although the specific genes mediating this trait are unknown, it is predicted that all members of the clonal lineages should share the same alleles. Genetic mapping studies suggested that chromosome Ia was unusually monomorphic compared with the rest of the genome. To investigate this further, we sequenced chromosome Ia and chromosome Ib in the Type I strain, RH, and the Type II strain, ME49. Comparative genome analyses of the two chromosomal sequences revealed that the same copy of chromosome Ia was inherited in each lineage, whereas chromosome Ib maintained the same high frequency of between-strain polymorphism as the rest of the genome. Sampling of chromosome Ia sequence in seven additional representative strains from the three clonal lineages supports a monomorphic inheritance, which is unique within the genome. Taken together, our observations implicate a specific combination of alleles on chromosome Ia in the recent origin and widespread success of the clonal lineages of T. gondii.

The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mosaic genome.

We determined the complete genome sequence of Clostridium difficile strain 630, a virulent and multidrug-resistant strain. Our analysis indicates that a large proportion (11%) of the genome consists of mobile genetic elements, mainly in the form of conjugative transposons. These mobile elements are putatively responsible for the acquisition by C. difficile of an extensive array of genes involved in antimicrobial resistance, virulence, host interaction and the production of surface structures. The metabolic capabilities encoded in the genome show multiple adaptations for survival and growth within the gut environment. The extreme genome variability was confirmed by whole-genome microarray analysis; it may reflect the organism's niche in the gut and should provide information on the evolution of virulence in this organism.

Genetic analysis of the capsular biosynthetic locus from all 90 pneumococcal serotypes.

Several major invasive bacterial pathogens are encapsulated. Expression of a polysaccharide capsule is essential for survival in the blood, and thus for virulence, but also is a target for host antibodies and the basis for effective vaccines. Encapsulated species typically exhibit antigenic variation and express one of a number of immunochemically distinct capsular polysaccharides that define serotypes. We provide the sequences of the capsular biosynthetic genes of all 90 serotypes of Streptococcus pneumoniae and relate these to the known polysaccharide structures and patterns of immunological reactivity of typing sera, thereby providing the most complete understanding of the genetics and origins of bacterial polysaccharide diversity, laying the foundations for molecular serotyping. This is the first time, to our knowledge, that a complete repertoire of capsular biosynthetic genes has been available, enabling a holistic analysis of a bacterial polysaccharide biosynthesis system. Remarkably, the total size of alternative coding DNA at this one locus exceeds 1.8 Mbp, almost equivalent to the entire S. pneumoniae chromosomal complement.

Complete genome sequence and lytic phase transcription profile of a Coccolithovirus.

The genus Coccolithovirus is a recently discovered group of viruses that infect the globally important marine calcifying microalga Emiliania huxleyi. Among the 472 predicted genes of the 407,339-base pair genome are a variety of unexpected genes, most notably those involved in biosynthesis of ceramide, a sphingolipid known to induce apoptosis. Uniquely for algal viruses, it also contains six RNA polymerase subunits and a novel promoter, suggesting this virus encodes its own transcription machinery. Microarray transcriptomic analysis reveals that 65% of the predicted virus-encoded genes are expressed during lytic infection of E. huxleyi.

Genome of the host-cell transforming parasite Theileria annulata compared with T. parva.

Theileria annulata and T. parva are closely related protozoan parasites that cause lymphoproliferative diseases of cattle. We sequenced the genome of T. annulata and compared it with that of T. parva to understand the mechanisms underlying transformation and tropism. Despite high conservation of gene sequences and synteny, the analysis reveals unequally expanded gene families and species-specific genes. We also identify divergent families of putative secreted polypeptides that may reduce immune recognition, candidate regulators of host-cell transformation, and a Theileria-specific protein domain [frequently associated in Theileria (FAINT)] present in a large number of secreted proteins.

Integration of tools and resources for display and analysis of genomic data for protozoan parasites.

Centralisation of tools for analysis of genomic data is paramount in ensuring that research is always carried out on the latest currently available data. As such, World Wide Web sites providing a range of online analyses and displays of data can play a crucial role in guaranteeing consistency of in silico work. In this respect, the protozoan parasite research community is served by several resources, either focussing on data and tools for one species or taking a broader view and providing tools for analysis of data from many species, thereby facilitating comparative studies. In this paper, we give a broad overview of the online resources available. We then focus on the GeneDB project, detailing the features and tools currently available through it. Finally, we discuss data curation and its importance in keeping genomic data 'relevant' to the research community.

The Chlamydophila abortus genome sequence reveals an array of variable proteins that contribute to interspecies variation.

The obligate intracellular bacterial pathogen Chlamydophila abortus strain S26/3 (formerly the abortion subtype of Chlamydia psittaci) is an important cause of late gestation abortions in ruminants and pigs. Furthermore, although relatively rare, zoonotic infection can result in acute illness and miscarriage in pregnant women. The complete genome sequence was determined and shows a high level of conservation in both sequence and overall gene content in comparison to other Chlamydiaceae. The 1,144,377-bp genome contains 961 predicted coding sequences, 842 of which are conserved with those of Chlamydophila caviae and Chlamydophila pneumoniae. Within this conserved Cp. abortus core genome we have identified the major regions of variation and have focused our analysis on these loci, several of which were found to encode highly variable protein families, such as TMH/Inc and Pmp families, which are strong candidates for the source of diversity in host tropism and disease causation in this group of organisms. Significantly, Cp. abortus lacks any toxin genes, and also lacks genes involved in tryptophan metabolism and nucleotide salvaging (guaB is present as a pseudogene), suggesting that the genetic basis of niche adaptation of this species is distinct from those previously proposed for other chlamydial species.

Extensive DNA inversions in the B. fragilis genome control variable gene expression.

The obligately anaerobic bacterium Bacteroides fragilis, an opportunistic pathogen and inhabitant of the normal human colonic microbiota, exhibits considerable within-strain phase and antigenic variation of surface components. The complete genome sequence has revealed an unusual breadth (in number and in effect) of DNA inversion events that potentially control expression of many different components, including surface and secreted components, regulatory molecules, and restriction-modification proteins. Invertible promoters of two different types (12 group 1 and 11 group 2) were identified. One group has inversion crossover (fix) sites similar to the hix sites of Salmonella typhimurium. There are also four independent intergenic shufflons that potentially alter the expression and function of varied genes. The composition of the 10 different polysaccharide biosynthesis gene clusters identified (7 with associated invertible promoters) suggests a mechanism of synthesis similar to the O-antigen capsules of Escherichia coli.