Molecular description of Holospora caryophila, the highly infectious macronuclear endosymbiont of Paramecium spp.

In questo studio, viene presentata la prima descrizione molecolare di Holospora caryophila, un simbionte macronucleare infettivo del Paramecium. In accordo con la descrizione originale, H. caryophila è affiliata al genere Holospora sulla base di morfologia, peculiarità del ciclo vitale e specificità d’ospite. Otto ceppi di Paramecium sono stati sottoposti ad analisi comparativa del marcatore molecolare 16S rRNA dei simbionti ospitati. Tutti i ceppi di H. caryophila investigati hanno mostrato valori di similarità del gene 16S rRNA pari od inferiori all’ 87,8 % con i membri del genere Holospora finora descritti a livello molecolare, tra cui la “type species” Holospora undulata. Grazie al recente aumento di risoluzione nella filogenesi degli Holospora-like bacteria (HLBs), siamo adesso in grado di ricostruire un modello evolutivo del meccanismo di trasmissione orizzontale di questi parassiti. Viene infatti qui dimostrato, su base filogenetica, che la capacità delle forme infettive (IFs) di accumularsi nel “connecting piece” durante la divisione nucleare dell’ospite è un apomorfia delle olospore maggiormente specializzate nello stile di vita parassitario. Vista l'incapacità di H. caryophila di indurre il “connecting piece” durante il ciclo infettivo, e dati i modesti valori di similarità del 16S rRNA rispetto agl’altri HLBs, viene qui proposta la sua rimozione dal genere Holospora, il trasferimento ad un nuovo taxon Preeria gen. nov. e la combinazione Preeria caryophila comb. nov.. Infine, in un sottogruppo dei ceppi di H. caryophila investigati, è stato caratterizzato l’ “Internal Transcribed Spacer” (ITS), e l'estremità 5' del gene 23S rRNA. L'analisi di questa regione ha rivelato una sorprendente “etereogeneità molecolare” tra i ceppi ospitati dalle due morfospecie di Paramecium naturalmente infettate da H. caryophila; il Paramecium aurelia complex ed il Paramecium caudatum. Anche se apparentemente identici, i ceppi di H. caryophila infettanti le due morfospecie ospite potrebbero essere geneticamente distinti. Protists belonging to phylum Ciliophora are frequently colonized by intracellular bacteria traditionally referred to as “endosymbionts”. Among ciliates, Paramecium (Eukaryota, Alveolata, Ciliophora, Intramacronucleata) represents a suitable model for studying the biodiversity and phylogeny of these bacteria, as many as 60 prokaryotic organisms have been found to inhabit virtually all of its subcellular compartments. In this study is presented the first molecular description of Holospora caryophila, a highly infectious macronuclear symbiont of Paramecium. In agreement with the original description, H. caryophila is affiliated with the genus Holospora on the basis of morphology, life cycle peculiarities, and host specificity. Eight Paramecium strains (naturally infected with H. caryophila) were examined by comparative analysis of the SSU 16S rRNA of the infecting symbionts and fluorescence in situ hybridization (FISH) experiments. Each investigated H. caryophila strain, including the type strain from P. biaurelia stock 562, showed 16S rRNA similarity values equal or lower than 87.8 % with the members of the genus Holospora accounted for in the analysis, including the type species Holospora undulata. As the phylogeny of the Holospora-like bacteria (HLBs) is increasing in resolution, the evolutionary pattern of the horizontal-transmission mechanism is revealed. Here, is shown on a molecular basis, that the ability of the infectious forms (IFs) to assemble in the connecting piece during the host nuclear division, is an apomorphy of the most specialized Holosporas, and has to be regarded as a highly advanced trait. According to the inability of H. caryophila to “induce” the connecting piece during the infective cycle, and the modest 16S rRNA similarity shared with others HLBs we propose its removal from the genus Holospora, its transfer to the new taxon Preeria, gen. nov., and the new combination Preeria caryophila comb. nov.. Furthermore, in a subgroup of the investigated H. caryophila strains, the Internal Transcribed Spacer (ITS) and the 5’ end of the 23S rRNA gene have been characterized. The analysis of this region revealed an unexpected heterogeneous molecular composition between strains harbored by the two natural host morphospecies, namely the Paramecium aurelia complex and Paramecium caudatum

Taxonomic classification of metagenomic sequences

Gerlach W. Taxonomic classification of metagenomic sequences. Bielefeld: Universität; 2012.Bacteria, archaea and microeukaryotes can be found in almost every habitat present in nature, in particular in soil, sediments and sea water. They typically live in complex communities with different kinds of symbiotic associations which include relationships with larger organisms like animals or plants. Examples are microbial communities in the gut or on the skin of animals and humans, or bacteria that live in symbiosis with plants. The vast majority of such microbes are unculturable and thus cannot be sequenced by means of traditional methods. The recently upcoming discipline of metagenomics provides various in vivo- and in silico-tools to overcome this limitation. In particular, high-throughput sequencing techniques like 454 or Solexa-Illumina make it possible to explore those microbes by studying whole natural microbial communities and analysing their biological diversity as well as the underlying metabolic pathways. A current limitation of theses technologies is that they can sequence only DNA fragments of a limited length. With this limitation it is usually not possible to recover complete microbial genomes. In addition, the DNA fragments are drawn randomly from the microbial communities and the exact species of origin is unknown. Over the past few years, different methods have been developed for the taxonomic and functional characterization of metagenomic shotgun sequences. However, the taxonomic classification of metagenomic sequences from novel species without close homologues in the biological sequence databases poses a challenge due to the high number of wrong taxonomic predictions on lower taxonomic ranks. In this thesis we present CARMA3, a novel method for the taxonomic classification of assembled and unassembled metagenomic sequences that has been adapted to work with both BLAST and HMMER3 homology searches. CARMA3 accepts protein-encoding DNA sequences, protein sequences, and 16S-rDNA sequences as input. In addition, we present WebCARMA, a web application for the analysis of protein-encoding DNA sequences with CARMA3 without the need for a local installation. We evaluate our novel method in different experiments using simulated and real shotgun metagenomes and show that CARMA3 makes fewer wrong taxonomic predictions (at the same sensitivity) than other BLAST-based methods. In the last experiment we show that also very short reads can, in principle, be used to describe the taxonomic content of a metagenome

Virus found in a boreal lake links ssDNA and dsDNA viruses

Viruses have impacted the biosphere in numerous ways since the dawn of life. However, the evolution, genetic, structural, and taxonomic diversity of viruses remain poorly understood, in part because sparse sampling of the virosphere has concentrated mostly on exploring the abundance and diversity of dsDNA viruses. Furthermore, viral genomes are highly diverse, and using only the current sequence-based methods for classifying viruses and studying their phylogeny is complicated. Here we describe a virus, FLiP (Flavobacterium-infecting, lipid-containing phage), with a circular ssDNA genome and an internal lipid membrane enclosed in the icosahedral capsid. The 9,174-nt-long genome showed limited sequence similarity to other known viruses. The genetic data imply that this virus might use replication mechanisms similar to those found in other ssDNA replicons. However, the structure of the viral major capsid protein, elucidated at near-atomic resolution using cryo-electron microscopy, is strikingly similar to that observed in dsDNA viruses of the PRD1-adenovirus lineage, characterized by a major capsid protein bearing two beta-barrels. The strong similarity between FLiP and another member of the structural lineage, bacteriophage PM2, extends to the capsid organization (pseudo T = 21 dextro) despite the difference in the genetic material packaged and the lack of significant sequence similarity.Peer reviewe

Idendification and characterisation of novel small RNAs from repetitve elements in mammals

Transposable elements account for the almost half of the sequence encoded by mammalian genomes, which become silenced during early embryonic development. This thesis sought to explore the hypothesis of the involvement of the RNAi pathway in the silencing of transposable elements in mammals, predominantly through the identification of transposon-associated RNA of -20-25nt using a gel blotting technique. Initially cell lines of embryonic and tumour origin were analysed. This lead to the identification of several previously unreported transposon-associated RNA ranging from 70-90nt. However, it was not until a more detailed analysis of the embryonic cell lines, with the induction of differentiation in cell culture that several discrete RNA of -20nt were detected for the mouse transposons L1 and B2. The differentiation of embryonic cell lines in culture also serendipitously lead to the detection of several short 55 rRNA of -22-26nt, these were also later detected in several human cell lines of breast cancer origin and healthy breast tissue. Intriguingly the -20-26nt repeat-associated identified were predominantly observed after two-days of differentiation in cell culture in several cell lines and often coincided with an ethidium bromide stainable band of -19nt. The latter may indicate a large proportion of these RNAs. Further analysis of the B2 and 55 rRNA repeat-associated short RNA revealed both to have reduce accumulation in Dicer-null embryonic stem cells, implicating a possible association with a known component of the RNAi pathway. Dicer was also observed to process the longer -50-80nt 55rRNA to -20-26nt in vitro. BLAST was also used to identify possible mRNA targets for the short B2 and 55 rRNA. One of these, the mRNA encoding sialic acid acetylesterase (SIAE) was consistently observed to be reduced with the accumulation of the short RNA using end-point RT-PCR, consistent with targeting through the RNAi or a similar pathway. However, no further links with the RNAi pathway were established, with no targeting detected for the short B2 or 55 rRNA using dual luciferase sensor assays. The repeat-associated RNA identified in this thesis are among the first of their type and further work will be required to establish what relevance they have to the RNAi pathway and transposon regulation