Sequence Analysis of a Small Cryptic Plasmid Isolated from Streptococcus suis Serotype 2

A 3.6-kbp DNA fragment was cloned from the extrachromosomal DNA of a pathogenic plant mollicute, onion yellows phytoplasma (OY-W). Sequence analysis of the fragment revealed an open reading frame (ORF) encoding the replication (Rep) protein of rolling-circle replication (RCR)-type plasmids. This result suggests the existence of a plasmid (pOYW1) in OY-W that uses the RCR mechanism. This assumption was confirmed by detecting the single-stranded DNA (ssDNA) of a replication intermediate that is specifically produced by the RCR mechanism. This is the first report on the identification of the replication system of this plasmid and the genes encoded in it. With a DNA fragment including the Rep gene region of pOYW1 used as a probe, Southern and Northern (RNA) blot hybridizations were employed to examine the heterogeneity between the plasmids found in OY-W and a pathogenic mutant (OY-M) isolated from OY-W. Multiple bands were detected in the DNA and RNA extracted from both OY-W and OY-M infected plants, although the banding patterns were different. Moreover, the copy number of plasmids from OY-W was about 4.2 times greater than that from OY-M. These results indicate constructive heterogeneity between OY-W and OY-M plasmids, and the possibility of a relationship between the plasmid-encoded genes and the pathogenicity of the phytoplasma was suggested.

Novel circular DNA viruses identified in Procordulia grayi and Xanthocnemis zealandica larvae using metagenomic approaches

Identification of Plasmids in the GenusMarinococcusand Complete Nucleotide Sequence of Plasmid pPL1 fromMarinococcus halophilus

Complete DNA sequence and analysis of two cryptic plasmids isolated from Lactobacillus plantarum

We have analysed DNA from African cassava mosaic virus (ACMV)-infected Nicotiana benthamiana by two-dimensional agarose gel electrophoresis and detected ACMV-specific DNAs by blot-hybridisation. ACMV DNA forms including the previously characterised single-stranded, open-circular, linear and supercoiled DNAs along with five previously uncharacterised heterogeneous DNAs (H1-H5) were resolved. The heterogeneous DNAs were characterised by their chromatographic properties on BND-cellulose and their ability to hybridise to strand-specific and double-stranded probes. The data suggest a rolling circle mechanism of DNA replication, based on the sizes and strand specificity of the heterogeneous single-stranded DNA forms and their electrophoretic properties in relation to genome length single-stranded DNAs. Second-strand synthesis on a single-stranded virus-sense template is evident from the position of heterogeneous subgenomic complementary-sense DNA (H3) associated with genome-length virus-sense template (VT) DNA. The position of heterogeneous virus-sense DNA (H5), ranging in size from one to two genome lengths, is consistent with its association with genome-length complementary-sense template (CT) DNA, reflecting virus-sense strand displacement during replication from a double-stranded intermediate. The absence of subgenomic complementary-sense DNA associated with the displaced virus-sense strand suggests that replication proceeds via an obligate single-stranded intermediate. The other species of heterogeneous DNAs comprised concatemeric single-stranded virus-sense DNA (H4), and double-stranded or partially single-stranded DNA (H1 and H2).

Structural instability of small rolling circle replication plasmids from Selenomonas ruminantium

Rolling-circle plasmids from Bacillus subtilis: complete nucleotide sequences and analyses of genes of pTA1015, pTA1040, pTA1050 and pTA1060, and comparisons with related plasmids from gram-positive bacteria.

Characterization of the Campylobacter jejuni cryptic plasmid pTIW94 recovered from wild birds in the southeastern United States

Development of a host-vector system for Lactobacillus plantarum L137 isolated from a traditional fermented food produced in the Philippines

Rolling circle replication (RCR) is ubiquitously used by cellular and viral systems for genome and plasmid replication. While the molecular mechanism of RCR has been described, the structural mechanism is desperately lacking. Circular-rep encoded single stranded DNA (CRESS-DNA) viruses employ a viral encoded replicase (Rep) to initiate RCR. The recently identified prokaryotic homologues of Reps may also be responsible for initiating RCR. Reps are composed of an endonuclease, oligomerization, and ATPase domain. Recent structural studies have provided structures for all these domains such that an overall mechanism of RCR initiation can begin to be synthesized. However, structures of Rep in complex with its various DNA substrates and/or ligands are lacking. Here we provide a 3D bioinformatic review of the current structural information available for Reps. We combine an excess of 1590 sequences with experimental and predicted structural data from 22 CRESS-DNA groups to identify similarities and differences between Reps that lead to potentially important functional sites. Experimental studies of these sites may shed light on how Reps execute their functions. Furthermore, we identify Rep-substrate or Rep-ligand structures that are urgently needed to better understand the structural mechanism of RCR.

Characterization of a cryptic plasmid pPZZ84 from Bacillus pumilus

Nucleotide Sequence of a Plasmid pCL2.1 fromLactococcus lactisssp.lactisML8

Rubus yellow net virus (RYNV) is a member of the genus Badnavirus (family: Caulimoviridae). RYNV infects Rubus species causing chlorosis of the tissue along the leaf veins, giving an unevenly distributed netted symptom in some cultivars of red and black raspberry. Recently, a strain of RYNV was sequenced from a Rubus idaeus plant in Alberta, Canada, exhibiting such symptoms. The viral genome contained seven open reading frames (ORFs) with five of them in the sense-strand, including a large polyprotein. Here we describe a graft-transmissible strain of RYNV from Europe infecting cultivar 'Baumforth's Seedling A' (named RYNV-BS), which was sequenced using rolling circle amplification, enzymatic digestion, cloning and primer walking, and it was resequenced at a 5X coverage. This sequence was then compared with the RYNV-Ca genome and significant differences were observed. Genomic analysis identified differences in the arrangement of coding regions, promoter elements, and presence of motifs. The genomic organization of RYNV-BS consisted of five ORFs (four ORFs in the sense-strand and one ORF in the antisense-strand). ORFs 1, 2, and 3 showed a high degree of homology to RYNV-Ca, while ORFs 4 and 6 of RYNV-BS were quite distinct. Also, the predicted ORFs 5 and 7 in the RYNV-Ca were absent in the RYNV-BS sequence. These differences may account for the lack of aphid transmissibility of RYNV-BS.

Electron microscopy and sequencing of reverse transcriptase and ribonuclease H (RT/RNase H) region of Badnavirus genome from two banana cultivars: Poovan (triploid: AAB) and Safed velchi (diploid: AB), exhibiting leaf streak symptoms, confirmed the association of Banana streak OL virus (BSOLV). As per ICTV species demarcation threshold of 80 % identity in RT/RNase H region, both the isolates were identified as BSOLV. Rolling circle and end-to-end amplification showed the association of two short episomal BSOLV variants: BSOLV-IN1 and BSOLV-IN2 from Poovan and Safed velchi banana, respectively. The genome sizes of both isolates were 6,950 nucleotides long, but shorter than the typical BSOLV genome of 7,389 bp. Open reading frames (ORFs) 1 and 2 of shorter BSOLV isolates shared almost complete nucleotide identity (>99 %) to that of BSOLV. However, the ORF 3 (5,130 bp) and intergenic region (IGR), 886 bp, showed deletions compared with ORF 3 (5,499 bp) and IGR (956 bp) of BSOLV. In phylogenetic analysis for ORF 3 polyprotein, both the isolates clustered with BSOLV, Banana streak CA virus (BSCAV), and Sugarcane bacilliform GA virus (SCBGAV). Identical ORF 1, ORF 2, and the presence of all the conserved domains in short ORF 3 and promoter elements in IGR indicated that these isolates represent replicationally competent shorter variants of BSOLV. These two shorter-than-BSOLV genome sequences and two other identical banana streak virus sequences in GenBank (BSV-TRY; DQ859899 and BSV-GD; DQ451009) might have evolved due to error-prone reverse transcription and splicing or excision from the integrated sequences by homologous recombination in natural banana hybrids under field conditions.

Novel divergent rhabdovirus in feces of red fox, Spain.