Genomic Sequencing and Biological Characteristics of a Novel Escherichia Coli Bacteriophage 9g, a Putative Representative of a New Siphoviridae Genus

Bacteriophage 9g was isolated from horse feces using Escherichia coli C600 as a host strain. Phage 9g has a slightly elongated capsid 62 × 76 nm in diameter and a non-contractile tail about 185 nm long. The complete genome sequence of this bacteriophage consists of 56,703 bp encoding 70 predicted open reading frames. The closest relative of phage 9g is phage PhiJL001 infecting marine alpha-proteobacterium associated with Ircinia strobilina sponge, sharing with phage 9g 51% of amino acid identity in the main capsid protein sequence. The DNA of 9g is resistant to most restriction endonucleases tested, indicating the presence of hypermodified bases. The gene cluster encoding a biosynthesis pathway similar to biosynthesis of the unusual nucleoside queuosine was detected in the phage 9g genome. The genomic map organization is somewhat similar to the typical temperate phage gene layout but no integrase gene was detected. Phage 9g efficiently forms stable associations with its host that continues to produce the phage over multiple passages, but the phage can be easily eliminated via viricide treatment indicating that no true lysogens are formed. Since the sequence, genomic organization and biological properties of bacteriophage 9g are clearly distinct from other known Enterobacteriaceae phages, we propose to consider it as the representative of a novel genus of the Siphoviridae family

Hybridization of the RT-CR probes on chromosomes of <i>D</i>. <i>villosum</i>, <i>P</i>. <i>spicata</i>, <i>Th</i>. <i>bessarabicum</i>, and <i>Th</i>. <i>intermedium</i>.

<p><b>a-c</b> FISH analysis using RT-CR probes on the root-tip cells at mitotic metaphase in a) <i>D</i>. <i>villosum</i>, b) <i>P</i>. <i>spicata</i>, c) <i>Th</i>. <i>bessarabicum</i>; the metaphase plates are probed with <i>Davi</i> (a, green), <i>Pssp</i> (b, pink) and <i>Thbe</i> (c, pink). Bar = 5 μm. <b>d-f</b> Sequential multicolor FISH and multicolor GISH on the root-tip cells at mitotic metaphase in <i>Th</i>. <i>intermedium</i>: d) the mcFISH results using the <i>Thbe</i> probe (red); e) the same cell in (d) with <i>Pssp</i> probe (green); f) GISH analysis for the same cell in (d) and (e) with the labeled genomic DNA of <i>P</i>. <i>spicata</i> (green) and <i>D</i>. <i>villosum</i> (pink) as probes and <i>T</i>. <i>aestivum</i> genomic DNA (ABD genome) as a block. Bar = 10 μm. <b>g-i</b> Karyotype of chromosomes of <i>Th</i>. <i>intermedium</i> from (d-f) with the results of sequential multicolor FISH and multicolor GISH. Chromosomes organized into genomes J^r, J^vs and St according to St- and V-genome DNA labeling and FISH signal intensity in centromeric region. The J^vs chromosomes grouped into J^v chromosomes (in frame) and J^s chromosomes. The translocated chromosome marked with asterisk. g) Chromosomes with <i>Thbe</i> probe (red); h) The same chromosomes with <i>Pssp</i> probe (green); i) The same chromosomes labeled with genomic DNA of <i>P</i>. <i>spicata</i> (St genome, green) and <i>D</i>. <i>villosum</i> (V genome, pink) as probes and <i>T</i>. <i>aestivum</i> genomic DNA (ABD genome) as a block. Chromosomes counterstained with DAPI (blue).</p

The dendrogram of the deduced RT-CR amino acid sequences and their homologs from the NCBI Nucleotide collection search database.

<p>The dendrogram was constructed using the Maximum Likelihood method based on the JTT matrix-based model. The bootstrap consensus tree inferred from 1000 replicates. The colored bullets indicate the RT-CRs obtained in the present study.</p

Variation in Copy Number of Ty3/Gypsy Centromeric Retrotransposons in the Genomes of <i>Thinopyrum intermedium</i> and Its Diploid Progenitors

<div><p>Speciation and allopolyploidization in cereals may be accompanied by dramatic changes in abundance of centromeric repeated transposable elements. Here we demonstrate that the reverse transcriptase part of Ty3/gypsy centromeric retrotransposon (RT-CR) is highly conservative in the segmental hexaploid <i>Thinopyrum intermedium</i> (J^rJ^vsSt) and its possible diploid progenitors <i>Th</i>. <i>bessarabicum</i> (J^b), <i>Pseudoroegneria spicata</i> (St) and <i>Dasypyrum villosum</i> (V) but the abundance of the repeats varied to a large extent. Fluorescence <i>in situ</i> hybridization (FISH) showed hybridization signals in centromeric region of all chromosomes in the studied species, although the intensity of the signals drastically differed. In <i>Th</i>. <i>intermedium</i>, the strongest signal of RT-CR probe was detected on the chromosomes of J^v, intermediate on J^r and faint on J^s and St subgenome suggesting different abundance of RT-CR on the individual chromosomes rather than the sequence specificity of RT-CRs of the subgenomes. RT-CR quantification using real-time PCR revealed that its content per genome in <i>Th</i>. <i>bessarabicum</i> is ~ 2 times and <i>P</i>. <i>spicata</i> is ~ 1,5 times higher than in genome of <i>D</i>. <i>villosum</i>. The possible burst of Ty3/gypsy centromeric retrotransposon in <i>Th</i>. <i>intermedium</i> during allopolyploidization and its role in proper mitotic and meiotic chromosome behavior in a nascent allopolyploid is discussed.</p></div