Single-stranded Origin Of Replication Research Articles

Transfer of a mobile Staphylococcus saprophyticus plasmid isolated from fermented seafood that confers tetracycline resistance.

The complete nucleotide sequence of a tetracycline-resistance gene (tetK)-carrying plasmid from a Staphylococcus saprophyticus isolate from jeotgal, a Korean high-salt-fermented seafood, was determined. The plasmid, designated pSSTET1, was 4439 bp in length and encoded typical elements found in plasmids that replicate via a rolling-circle mechanism, including the replication protein gene (rep), a double-stranded origin of replication, a single-stranded origin of replication, and a counter-transcribed RNA sequence. Additionally, the plasmid recombination enzyme gene (pre), which may be involved in inter-plasmid recombination and conjugation, was found. Each gene exhibited >94% sequence identity with those harbored in other Staphylococcus species. pSSTET1 was conditionally transferred to Staphylococcus species in a host-dependent manner and transferred to an Enterococcus faecalis strain in vitro. Antibiotic susceptibility of the transconjugants was host-dependent and transconjugants maintained a tetracycline-resistant phenotype in the absence of selective pressure over 100 generations.

Characterization of Mobile Staphylococcus equorum Plasmids Isolated from Fermented Seafood That Confer Lincomycin Resistance.

The complete nucleotide sequences of lincomycin-resistance gene (lnuA)-containing plasmids in Staphylococcus equorum strains isolated from the high-salt-fermented seafood jeotgal were determined. These plasmids, designated pSELNU1–3, are 2638-bp long, have two polymorphic sites, and encode typical elements found in plasmids that replicate via a rolling-circle mechanism including the replication protein gene (rep), a double-stranded origin of replication, a single-stranded origin of replication, and counter-transcribed RNA sequence, as well as lnuA. Plasmid sequences exhibit over 83% identity to other Staphylococcus plasmids that harbor rep and lnuA genes. Further, three pairs of identified direct repeats may be involved in inter-plasmid recombination. One plasmid, pSELNU1, was successfully transferred to other Staphylococcus species, Enterococcus faecalis, and Tetragenococcus halophilus in vitro. Antibiotic susceptibility of the transconjugants was host-dependent, and transconjugants maintained a lincomycin resistance phenotype in the absence of selective pressure over 60 generations.

Identification of a Single Strand Origin of Replication in the Integrative and Conjugative Element ICEBs1 of Bacillus subtilis.

We identified a functional single strand origin of replication (sso) in the integrative and conjugative element ICEBs1 of Bacillus subtilis. Integrative and conjugative elements (ICEs, also known as conjugative transposons) are DNA elements typically found integrated into a bacterial chromosome where they are transmitted to daughter cells by chromosomal replication and cell division. Under certain conditions, ICEs become activated and excise from the host chromosome and can transfer to neighboring cells via the element-encoded conjugation machinery. Activated ICEBs1 undergoes autonomous rolling circle replication that is needed for the maintenance of the excised element in growing and dividing cells. Rolling circle replication, used by many plasmids and phages, generates single-stranded DNA (ssDNA). In many cases, the presence of an sso enhances the conversion of the ssDNA to double-stranded DNA (dsDNA) by enabling priming of synthesis of the second DNA strand. We initially identified sso1 in ICEBs1 based on sequence similarity to the sso of an RCR plasmid. Several functional assays confirmed Sso activity. Genetic analyses indicated that ICEBs1 uses sso1 and at least one other region for second strand DNA synthesis. We found that Sso activity was important for two key aspects of the ICEBs1 lifecycle: 1) maintenance of the plasmid form of ICEBs1 in cells after excision from the chromosome, and 2) stable acquisition of ICEBs1 following transfer to a new host. We identified sequences similar to known plasmid sso's in several other ICEs. Together, our results indicate that many other ICEs contain at least one single strand origin of replication, that these ICEs likely undergo autonomous replication, and that replication contributes to the stability and spread of these elements.

Characterization of a novel mosquitocidal strain of Bacillus thuringiensis serovar aizawai which harbors a rolling-circle replication plasmid, pBt1–3

Bacillus thuringiensis 1–3, isolated from a Korean soil sample, was determined to belong to ssp. aizawai (H7) type by an H antiserum agglutination test, and produced bipyramidal-shaped crystal proteins with a molecular weight of 130kDa. PCR analysis with specific cry gene primers showed that B. thuringiensis 1–3 contained cry1Aa, cry1Ab, cry1C, cry1D and cry2A genes, differing from that of serovar of aizawai (reference strain) which contains cry1Aa, cry1Ab, cry1C and cry1D genes. In contrast to the reference strain, B. thuringiensis aizawai showed insecticidal activity against Plutella xylostella larvae, the B. thuringiensis 1–3 showed insecticidal activity against not only P. xylostella, but also Aedes aegypti, owing to its Cry2A crystal protein. In this study, we modified the plasmid capture system (PCS) through in vitro transposition to clone small cryptic plasmids from B. thuringiensis 1–3. Fifty-three clones were acquired, and their sizes were approximately 10kb. Based on the sequence analysis, they were classified into four groups, showing similarities with four known B. thuringiensis plasmids, pGI3, pBMB175, pGI1 and pGI2, respectively. One of the pGI3-like clones, pBt1–3, was fully sequenced, and its putative open reading frames (ORFs), Rep-protein, double-strand origin of replication (dso), single-strand origin of replication (sso), have been identified. The structure of pBt1–3 showed high similarity with pGI3, which is of the rolling-circle replication (RCR) group VI family.

Cloning and molecular characterization of a novel rolling-circle replicating plasmid, pK1S-1, from Bacillus thuringiensis subsp. kurstaki K1

Bacillus thuringiensis, an entomopathogenic bacterium belonging to the B. cereus group, harbors numerous extra-chromosomal DNA molecules whose sizes range from 2 to 250 kb. In this study, we used a plasmid capture system (PCS) to clone three small plasmids from B. thuringiensis subsp. kurstaki Kl which were not found in B. thuringiensis subsp. kurstaki HD-1, and determined the complete nucleotide sequence of plasmid pKlS-1 (5.5 kb). Of the six putative open reading frames (ORF2-ORF7) in pKlS-1, ORF2 (MobKl) showed approximately 90% aa identity with the Mob-proteins of pGI2 and pTX14-2, which are rolling circle replicating group VII (RCR group VII) plasmids from B. thuringiensis. In addition, a putative origin of transfer (oriT) showed 95.8% identity with those of pGI2 and pTX14-2. ORF3 (RepK1) showed relatively low aa identity (17.8-25.2%) with the Rep protein coded by RCR plasmids, however. The putative double-strand origin of replication (dso) and single-strand origin of replication (sso) of pKlS-1 exhibited approximately 70% and 64% identities with those of pGI2 and pTX14-2. ORF6 and 7 showed greater than 50% similarities with alkaline serine protease, which belongs to the subtilase family. The other 2 ORFs were identified as hypothetical proteins. To determine the replicon of pKlS-1, seven subclones were contructed in the B. thuringiensis ori-negative pHTIK vector and were electroporated into a plasmid cured B. thuringiensis strain. The 1.6 kb region that included the putative ORF3 (ReplK), dso and ORF4, exhibited replication ability. These findings identified pKlS-1 as a new RCR group VII plasmid, and determined its replication region.

Characterization and application of a rolling-circle-type plasmid from Cupriavidus taiwanensis

Two small cryptic plasmids, pTJ86-1 and pTJ86-2, identified in Cupriavidus taiwanensis strain TJ86, were detected and characterized. Complete sequencing of pTJ86-1 and pTJ86-2 revealed these plasmids to be 2221 and 2229 bp in length with a GC content of 61.7% and 61.6%, respectively. Both plasmids harbored four open reading frames (ORF1, 2, 3 and 4). Only the predicted ORF1 gene product of both plasmids (436 amino acids) was homologous to Rep proteins previously identified on plasmids replicated using a rolling-circle replication (RCR). A double-stranded origin (DSO) of replication, highly conserved in the group III (cluster III) RCR plasmids, was identified and located immediately upstream of this putative Rep gene. In addition, both plasmids contained a putative single-stranded origin of replication (SSO) exhibiting similarity to the ssoA-type. Detection of single-stranded plasmid DNA by Southern analysis and S1 nuclease digestion confirmed that the cryptic plasmid replicated via an RCR mechanism. A potential shuttle vector, pS4-tet R, was constructed by ligation of pTJ86-1 to the cloning vector pBluescript II SK(+) along with the insertion of a tetracycline-resistance (tet R) gene. It was successfully used for the transformation of genera Burkholderia and Cupriavidus.

The patchwork nature of rolling-circle plasmids: comparison of six plasmids from two distinct Bacillus thuringiensis serotypes

Bacillus thuringiensis, the entomopathogenic bacteria from the Bacillus cereus group, harbors numerous extrachromosomal molecules whose sizes vary from 2 to more than 200 kb. Apart from the genes coding for the biopesticide δ-endotoxins located on large plasmids, little information has been obtained on these plasmids and their contribution to the biology of their host. In this paper, we embarked on a detailed comparison of six small rolling-circle replicating (RCR) plasmids originating from two major B. thuringiensis strains. The complete nucleotide sequences of plasmid pGI1, pGI2, pGI3, pTX14-1, pTX14-2, and pTX14-3 have been obtained and compared. Replication functions, comprising, for each plasmid, the gene encoding the Rep-protein, double-strand origin of replication ( dso), single-strand origin of replication ( sso), have been identified and analyzed. Two new families, or homology groups, of RCR plasmids originated from the studies of these plasmids (Group VI based on pGI3 and Group VII based on pTX14-3). On five of the six plasmids, loci involved in conjugative mobilization (Mob-genes and origin of transfer ( oriT)) were identified. Plasmids pTX14-1, pTX14-2, and pTX14-3 each harbor an ORF encoding a polypeptide containing a central domain with repetitive elements similar to eukaryotic collagen (Gly–X–Y triplets). These genes were termed bcol for Bacillus-collagen-like genes.

Sequence and analysis of pBM02, a novel RCR cryptic plasmid from Lactococcus lactis subsp. cremoris P8-2-47

This paper reports the complete nucleotide sequence of the 3.85 kbp plasmid pBM02 from Lactococcus lactis subsp. cremoris P8-2-47. Analysis of the sequence predicted six ORFs larger than 25 amino acids. They all were transcribed from the same strand and organized in two functional cassettes: the replication region and a putative mobilization region. In the replication region, two ORFs specifying proteins homologous to others found in some classes of rolling circle-replicating plasmids were encountered ( copG and repB). In fact, single-stranded DNA was detected as a replication intermediate of pBM02. copG and repB, together with some upstream sequences, formed part of the minimal replication unit of the plasmid. Interestingly, pBM02 shared a 212 bp stretch with plasmids of the pWV01 type, in which the whole single-strand origin of replication is included. In the mobilization region, an ORF coding for a mobilization-like protein was present, preceded by a putative oriT sequence homologous to that of plasmid pMV158. The replicon of pBM02 is of the wide-host range type, and functions in both Gram-positive and Gram-negative bacteria, including Lactobacillus casei, Lactobacillus plantarum, Bacillus subtilis, and Escherichia coli.

Nucleotide Sequence and Analysis of pWC1, A pC194-Type Rolling Circle Replicon inLactococcus lactis

A 2.8-kb cryptic plasmid showing no homology to either pFX3 (rolling circle, pE194-type) or pCI305 (theta-type) lactococcal replicons was identified inLactococcus lactissubsp.cremoris2204. The plasmid, pWC1, was compatible with both pCI3340 (a pCI305 derivative) and pFX3 inL. lactissubsp.cremoris2204. Sequence analysis of pWC1 showed one major ORF encoding a protein with a deduced size of 316 amino acids (aa). Database comparisons showed that the protein was distinct from the pFX- and pCI-type replication proteins (less than 21% aa identity), but shared significant homology (up to 57% aa identity) with the replication proteins from a different group of rolling circle plasmids (pC194-type) commonly found in gram-positive bacteria. A pC194-type rolling circle plasmid has not been previously described inL. lactis.Further sequence analysis showed a conserved double-stranded origin of replication in pWC1 preceded by a large (118-bp) direct repeat. The chloramphenicol-resistance gene from pC194 was inserted into a nonessential region of pWC1 to give pCP12. The host range of pCP12 includedStreptococcus thermophilus, Enterococcus faecalis,andStaphylococcus aureus,but notEscherichia coli.Both pCP12 and to a lesser extent pWC1 generated single-stranded DNA (ssDNA) inL. lactis.A possible single-stranded origin of replication was identified by sequence analysis of pWC1 and by comparing levels of ssDNA produced by pCP12 deletion derivatives. The pWC1 replicon may be a useful addition to other replicons currently available for vector construction.

Complete Nucleotide Sequence of the Bacillus thuringiensis subsp. israelensis Plasmid pTX14-3 and Its Correlation with Biological Properties

The complete nucleotide sequence of the plasmid pTX14-3 from Bacillus thuringiensis subsp. israelensis has been determined. The circular DNA molecule was 7649 bp and had a G + C content of 35.1%. Twenty-two open reading frames larger than 50 codons were identified. Ten of these open reading frames are suggested to be protein coding regions. The existence of the polypeptides encoded by the mob14-3 and rep14-3 genes were verified by maxi-cells analysis in Escherichia coli. Even though the rep14-3 gene was expressed in E. coli the plasmid pTX14-3 was unable to replicate in this bacterium. The minimal region of the plasmid pTX14-3 required for replication in B. thuringiensis was identified. Potential secondary structures upstream of the rep14-3 gene indicated regulation by antisense RNA and transcription attenuation. Extensive sequence homology with the B. thuringiensis subsp. thuringiensis plasmid pG12 was found in the last part of the mob14-3 gene, downstream of the rep14-3 gene, and in the region containing the single-strand origin of replication (i.e., the minus origin) of pTX14-3. A sequence of 700 bp containing multiple direct repeats was found in an ORF encoding a glycine and proline rich protein of 35.9 kDa. 1.2 kbp upstream and 0.1 kbp downstream of this ORF was found a large direct repeat of 230 bp (87% identity). The region between this direct repeat was often spontaneously deleted from plasmid derivatives containing the entire pTX14-3.