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A recently emerged plant disease, bacterial canker of kiwifruit (Actinidia deliciosa and A. chinensis), is caused by Pseudomonas syringae pv. actinidiae (PSA). The disease was first reported in China and Japan in the 1980s. A severe outbreak of PSA began in Italy in 2008 and has spread to other European countries. PSA was found in both New Zealand and Chile in 2010. To study the evolution of the pathogen and analyse the transmission of PSA between countries, genomes of strains from China and Japan (where the genus Actinidia is endemic), Italy, New Zealand and Chile were sequenced. The genomes of PSA strains are very similar. However, all strains from New Zealand share several single nucleotide polymorphisms (SNPs) that distinguish them from all other PSA strains. Similarly, all the PSA strains from the 2008 Italian outbreak form a distinct clonal group and those from Chile form a third group. In addition to the rare SNPs present in the core genomes, there is abundant genetic diversity in a genomic island that is part of the accessory genome. The island from several Chinese strains is almost identical to the island present in the New Zealand strains. The island from a different Chinese strain is identical to the island present in the strains from the recent Italian outbreak. The Chilean strains of PSA carry a third variant of this island. These genomic islands are integrative conjugative elements (ICEs). Sequencing of these ICEs provides evidence of three recent horizontal transmissions of ICE from other strains of Pseudomonas syringae to PSA. The analyses of the core genome SNPs and the ICEs, combined with disease history, all support the hypothesis of an independent Chinese origin for both the Italian and the New Zealand outbreaks and suggest the Chilean strains also originate from China. [ABSTRACT FROM AUTHOR]

Retrotransposons carrying tyrosine recombinases (YR) are widespread in eukaryotes. The first described tyrosine recombinase mobile element, DIRS1, is a retroelement from the slime mold Dictyostelium discoideum. The YR elements are bordered by terminal repeats related to their replication via free circular dsDNA intermediates. Site-specific recombination is believed to integrate the circle without creating duplications of the target sites. Recently a large number of YR retrotransposons have been described, including elements from fungi (mucorales and basidiomycetes), plants (green algae) and a wide range of animals including nematodes, insects, sea urchins, fish, amphibia and reptiles. YR retrotransposons can be divided into three major groups: the DIRS elements, PAT-like and the Ngaro elements. The three groups form distinct clades on phylogenetic trees based on alignments of reverse transcriptase/ribonuclease H (RT/RH) and YR sequences, and also having some structural distinctions. A group of eukaryote DNA transposons, cryptons, also carry tyrosine recombinases. These DNA transposons do not encode a reverse transcriptase. They have been detected in several pathogenic fungi and oomycetes. Sequence comparisons suggest that the crypton YRs are related to those of the YR retrotransposons. We suggest that the YR retrotransposons arose from the combination of a crypton-like YR DNA transposon and the RT/RH encoding sequence of a retrotransposon. This acquisition must have occurred at a very early point in the evolution of eukaryotes.

The mobile elements termed inteins have a sporadic distribution in microorganisms. It is unclear how these elements are maintained. Inteins are intervening protein sequences that autocatalytically excise themselves from a precursor. Excision is a post-translational process referred to as 'protein splicing' in which the sequences flanking the intein are ligated, reforming the mature host protein. Some inteins contain a homing endonuclease domain (HEG) that is proposed to facilitate propagation of the intein element within a gene pool. We have previously demonstrated that the HEG of the PRP8 intein is highly active during meiosis in Botrytis cinerea. Here we analysed the Prp8 gene status in 21 additional Botrytis species to obtain insight into the mode of intein inheritance within the Botrytis lineage. Of the 21 species, 15 contained a PRP8 intein whereas six did not. The analysis was extended to closely related (Sclerotiniaceae) and distantly related (Ascomycota) taxa, focussing on evolutionary diversification of the PRP8 intein, including their possible acquisition by horizontal transfer and loss by deletion. Evidence was obtained for the occurrence of genetic footprints of previous intein occupation. There is no compelling evidence of horizontal transfer among species. Three distinct states of the Prp8 allele were identified, distributed over different orders within the Ascomycota: an occupied allele; an empty allele that was never occupied; an empty allele that was presumably previously occupied, from which the intein was precisely deleted. The presence of the genetic footprint identifies 20 species (including Neurospora crassa, Magnaporthe oryzae and Fusarium oxysporum) that previously contained the intein but have lost it entirely, while only 18 species (including Podospora anserina and Fusarium graminearum) appear never to have contained a PRP8 intein. The analysis indicates that inteins may be maintained in an equilibrium state., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The process of decomposition of bodies in the marine environment is poorly understood and almost nothing is currently known about the microorganisms involved. This study aimed to investigate the microbes involved in decomposition in the sea and to evaluate the potential use of marine bacterial succession for postmortem submersion interval (PMSI) estimation, for which there is currently no reliable method. Partial pig remains were completely submerged during autumn and winter and were regularly sampled to document marine bacterial colonisation and the changes in community composition over time. Five stages of decomposition were recognised, some of which exhibited characters specific for partial carrion. Marine bacteria rapidly colonised the submerged remains in a successional manner. Seasonal differences were observed for the rate of decomposition and also for several groups of colonising bacteria. Marine bacteria specific for particular PMSIs were identified. This study provides an insight into the involvement of saprophytic marine bacteria in the decomposition of mammalian remains in the sea and is the first to explore the use of marine bacterial colonisation and succession as a novel tool for PMSI estimation. We propose that with further study, marine bacterial succession will prove useful for determination of the length of time a body may have been immersed in a marine environment., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Strains of Botrytis cinerea are polymorphic for the presence of an intein in the Prp8 gene (intein +/-). The intein encodes a homing endonuclease (HEG). During meiosis in an intein +/- heterozygote, the homing endonuclease initiates intein 'homing' by inducing gene conversion. In such meioses, the homing endonuclease triggers gene conversion of the intein together with its flanking sequences into the empty allele. The efficiency of gene conversion of the intein was found to be 100%. The extent of flanking sequence affected by the gene conversion varied in different meioses. A survey of the inteins and flanking sequences of a group B. cinerea isolates indicates that there are two distinct variants of the intein both of which have active HEGs. The survey also suggests that the intein has been actively homing during the evolution of the species and that the PRP8 intein may have entered the species by horizontal transfer.

Background: Target-primed (non-LTR) retrotransposons, such as the human L1 element, are mobile genetic elements found in many eukaryotic genomes. They are often present in large numbers and their retrotransposition can cause mutations and genomic rearrangements. Despite their importance, many aspects of their replication are not well understood., Results: We have developed a yeast model system for studying target-primed retrotransposons. This system uses the Zorro3 element from Candida albicans. A cloned copy of Zorro3, tagged with a retrotransposition indicator gene, retrotransposes at a high frequency when introduced into an appropriate C. albicans host strain. Retrotransposed copies of the tagged element exhibit similar features to the native copies, indicating that the natural retrotransposition pathway is being used. Retrotransposition is dependent on the products of the tagged element's own genes and is highly temperature-regulated. The new assay permits the analysis of the effects of specific mutations introduced into the cloned element., Conclusion: This Zorro3 retrotransposition assay system complements previously available target-primed retrotransposition assays. Due to the relative simplicity of the growth, manipulation and analysis of yeast cells, the system should advance our understanding of target-primed retrotransposition.

As the Cne PRP8 intein is active and exists in an essential gene of an important fungal pathogen, inhibitors of splicing and assays for intein activity are of interest. The self-splicing activity of Cne PRP8, the intein from the Prp8 gene of Cryptococcus neoformans, was assessed in different heterologous fusion proteins expressed in Escherichia coli. Placement of a putatively inactive variant of the intein adjacent to the alpha-complementation peptide abolished the peptide's ability to restore beta-galactosidase activity, while an active variant allowed complementation. This alpha-complementation peptide therefore provides a facile assay of splicing which can be used to test potential inhibitors. When placed between two heterologous protein domains, splicing was impaired by a beta-branched amino acid immediately preceding the intein, while splicing occurred only with a hydroxyl or thiol immediately following the intein. Both these assays sensitively report impairment of splicing and provide information on how context constrains the splicing ability of Cne PRP8.

The dependence of protein splicing on conserved residues of the Cne PRP8 intein was assessed by alanine scanning mutagenesis in a foreign protein context. Corroboration was obtained for the involvement of residues at the splice junctions and of the conserved threonine and histidine of motif B. Five additional residues were identified as absolutely required for splicing. Variant W151A displayed premature C-terminal cleavage, not seen with other Cne PRP8 mutants. We propose a model whereby W151 acts to prevent premature C-terminal cleavage, favoring complete splicing as opposed to two disjointed cleavage events.

An intein is a protein sequence embedded within a precursor protein that is excised during protein maturation. Inteins were first found encoded in the VMA gene of Saccharomyces cerevisiae. Subsequently, they have been found in diverse organisms (eukaryotes, archaea, eubacteria and viruses). The VMA intein has been found in various saccharomycete yeasts but not in other fungi. Different inteins have now been found widely in the fungi (ascomycetes, basidiomycetes, zygomycetes and chytrids) and in diverse proteins. A protein distantly related to inteins, but closely related to metazoan hedgehog proteins, has been described from Glomeromycota. Many of the newly described inteins contain homing endonucleases and some of these are apparently active. The enlarged fungal intein data set permits insight into the evolution of inteins, including the role of horizontal transfer in their persistence. The diverse fungal inteins provide a resource for biotechnology using their protein splicing or homing endonuclease capabilities.

Background: Inteins are self-splicing protein elements. They are translated as inserts within host proteins that excise themselves and ligate the flanking portions of the host protein (exteins) with a peptide bond. They are encoded as in-frame insertions within the genes for the host proteins. Inteins are found in all three domains of life and in viruses, but have a very sporadic distribution. Only a small number of intein coding sequences have been identified in eukaryotic nuclear genes, and all of these are from ascomycete or basidiomycete fungi., Results: We identified seven intein coding sequences within nuclear genes coding for the second largest subunits of RNA polymerase. These sequences were found in diverse eukaryotes: one is in the second largest subunit of RNA polymerase I (RPA2) from the ascomycete fungus Phaeosphaeria nodorum, one is in the RNA polymerase III (RPC2) of the slime mould Dictyostelium discoideum and four intein coding sequences are in RNA polymerase II genes (RPB2), one each from the green alga Chlamydomonas reinhardtii, the zygomycete fungus Spiromyces aspiralis and the chytrid fungi Batrachochytrium dendrobatidis and Coelomomyces stegomyiae. The remaining intein coding sequence is in a viral relic embedded within the genome of the oomycete Phytophthora ramorum. The Chlamydomonas and Dictyostelium inteins are the first nuclear-encoded inteins found outside of the fungi. These new inteins represent a unique dataset: they are found in homologous proteins that form a paralogous group. Although these paralogues diverged early in eukaryotic evolution, their sequences can be aligned over most of their length. The inteins are inserted at multiple distinct sites, each of which corresponds to a highly conserved region of RNA polymerase. This dataset supports earlier work suggesting that inteins preferentially occur in highly conserved regions of their host proteins., Conclusion: The identification of these new inteins increases the known host range of intein sequences in eukaryotes, and provides fresh insights into their origins and evolution. We conclude that inteins are ancient eukaryote elements once found widely among microbial eukaryotes. They persist as rarities in the genomes of a sporadic array of microorganisms, occupying highly conserved sites in diverse proteins.

Background: We recently described a mini-intein in the PRP8 gene of a strain of the basidiomycete Cryptococcus neoformans, an important fungal pathogen of humans. This was the second described intein in the nuclear genome of any eukaryote; the first nuclear encoded intein was found in the VMA gene of several saccharomycete yeasts. The evolution of eukaryote inteins is not well understood. In this report we describe additional PRP8 inteins (bringing the total of these to over 20). We compare and contrast the phylogenetic distribution and evolutionary history of the PRP8 intein and the saccharomycete VMA intein, in order to derive a broader understanding of eukaryote intein evolution. It has been suggested that eukaryote inteins undergo horizontal transfer and the present analysis explores this proposal., Results: In total, 22 PRP8 inteins have been detected in species from three different orders of euascomycetes, including Aspergillus nidulans and Aspergillus fumigatus (Eurotiales), Paracoccidiodes brasiliensis, Uncinocarpus reesii and Histoplasma capsulatum (Onygales) and Botrytis cinerea (Helotiales). These inteins are all at the same site in the PRP8 sequence as the original Cryptococcus neoformans intein. Some of the PRP8 inteins contain apparently intact homing endonuclease domains and are thus potentially mobile, while some lack the region corresponding to the homing endonuclease and are thus mini-inteins. In contrast, no mini-inteins have been reported in the VMA gene of yeast. There are several examples of pairs of closely related species where one species carries the PRP8 intein while the intein is absent from the other species. Bio-informatic and phylogenetic analyses suggest that many of the ascomycete PRP8 homing endonucleases are active. This contrasts with the VMA homing endonucleases, most of which are inactive., Conclusion: PRP8 inteins are widespread in the euascomycetes (Pezizomycota) and apparently their homing endonucleases are active. There is no evidence for horizontal transfer within the euascomycetes. This suggests that the intein is of ancient origin and has been vertically transmitted amongst the euascomycetes. It is possible that horizontal transfer has occurred between the euascomycetes and members of the basidiomycete genus Cryptococcus.

Only two nuclear encoded inteins have been described. The first, SceVMA, was found in a vacuolar ATPase gene of Saccharomyces cerevisiae and related yeasts. The second, CnePRP8, was found in the PRP8 gene of Cryptococcus neoformans. CnePRP8 contains protein sequences associated with intein splicing but no endonuclease domain. We compared allelic mini-inteins in both varieties of C. neoformans (var. neoformans and var. grubii) and in the related primary pathogen C. gattii to study the evolution of both the mini-intein and the host. We also describe a full-length, endonuclease-containing intein in Cryptococcus laurentii, a moderately distant relation of C. neoformans. We did not detect an intein in the PRP8 gene of other species of Cryptococcus including species closely related to the C. neoformans/C. gattii group. It is therefore probable that the C. neoformans/C. gattii mini-intein was derived from horizontal transfer in which C. laurentii or another intein-containing species was the source.

Helitrons, a novel class of eukaryote mobile genetic elements, are distinguished from other transposable elements by encoding a 'rolling circle' replication (RCR) protein (Rep) and a helicase. Helitrons have recently been described from Arabidopsis, rice and the nematode Caenorhabditis. We now report the discovery of Helitron-like elements in vertebrates, specifically in the genomes of the fish Danio rerio and Sphoeroides nephelus. We also describe Helitrons from the white rot fungus Phanerochaete chrysosporium and from the Anopheles genome. Many of the fish Helitrons have an uncorrupted open reading frame encoding both the RCR Rep protein and a helicase. These fish elements are of particular interest because they also encode, within the single open reading frame, an apurinic-apyrimidinic (AP) endonuclease most closely related to those of certain non-long terminal repeat retrotransposons. As they invariably carry an endonuclease and also form a very distinct clade, we have named these vertebrate elements 'helentrons'. It is likely that these helentrons are still active.