Single-strand Origin Research Articles

Staphylococcus equorum plasmid pKS1030-3 encodes auxiliary biofilm formation and trans-acting gene mobilization systems

The foodborne bacterium Staphylococcus equorum strain KS1030 harbours plasmid pSELNU1, which encodes a lincomycin resistance gene. pSELNU1 undergoes horizontal transfer between bacterial strains, thus spreading antibiotic resistance. However, the genes required for horizontal plasmid transfer are not encoded in pSELNU1. Interestingly, a relaxase gene, a type of gene related to horizontal plasmid transfer, is encoded in another plasmid of S. equorum KS1030, pKS1030-3. The complete genome of pKS1030-3 is 13,583 bp long and encodes genes for plasmid replication, biofilm formation (the ica operon), and horizontal gene transfer. The replication system of pKS1030-3 possesses the replication protein-encoding gene repB, a double-stranded origin of replication, and two single-stranded origins of replication. The ica operon, relaxase gene, and a mobilization protein-encoding gene were detected in pKS1030-3 strain-specifically. When expressed in S. aureus RN4220, the ica operon and relaxase operon of pKS1030-3 conferred biofilm formation ability and horizontal gene transfer ability, respectively. The results of our analyses show that the horizontal transfer of pSELNU1 of S. equorum strain KS1030 depends on the relaxase encoded by pKS1030-3, which is therefore trans-acting. Genes encoded in pKS1030-3 contribute to important strain-specific properties of S. equorum KS1030. These results could contribute to preventing the horizontal transfer of antibiotic resistance genes in food.

Generating a Small Shuttle Vector for Effective Genetic Engineering of Methanosarcina mazei Allowed First Insights in Plasmid Replication Mechanism in the Methanoarchaeon.

Due to their role in methane production, methanoarchaea are of high ecological relevance and genetic systems have been ever more established in the last two decades. The system for protein expression in Methanosarcina using a comprehensive shuttle vector is established; however, details about its replication mechanism in methanoarchaea remain unknown. Here, we report on a significant optimisation of the rather large shuttle vector pWM321 (8.9 kbp) generated by Metcalf through a decrease in its size by about 35% by means of the deletion of several non-coding regions and the ssrA gene. The resulting plasmid (pRS1595) still stably replicates in M. mazei and—most likely due to its reduced size—shows a significantly higher transformation efficiency compared to pWM321. In addition, we investigate the essential gene repA, coding for a rep type protein. RepA was heterologously expressed in Escherichia coli, purified and characterised, demonstrating the significant binding and nicking activity of supercoiled plasmid DNA. Based on our findings we propose that the optimised shuttle vector replicates via a rolling circle mechanism with RepA as the initial replication protein in Methanosarcina. On the basis of bioinformatic comparisons, we propose the presence and location of a double-strand and a single-strand origin, which need to be further verified.

Unique properties of spacer acquisition by the type III-A CRISPR-Cas system.

Type III CRISPR-Cas systems have a unique mode of interference, involving crRNA-guided recognition of nascent RNA and leading to DNA and RNA degradation. How type III systems acquire new CRISPR spacers is currently not well understood. Here, we characterize CRISPR spacer uptake by a type III-A system within its native host, Streptococcus thermophilus. Adaptation by the type II-A system in the same host provided a basis for comparison. Cas1 and Cas2 proteins were critical for type III adaptation but deletion of genes responsible for crRNA biogenesis or interference did not detectably change spacer uptake patterns, except those related to host counter-selection. Unlike the type II-A system, type III spacers are acquired in a PAM- and orientation-independent manner. Interestingly, certain regions of plasmids and the host genome were particularly well-sampled during type III-A, but not type II-A, spacer uptake. These regions included the single-stranded origins of rolling-circle replicating plasmids, rRNA and tRNA encoding gene clusters, promoter regions of expressed genes and 5′ UTR regions involved in transcription attenuation. These features share the potential to form DNA secondary structures, suggesting a preferred substrate for type III adaptation. Lastly, the type III-A system adapted to and protected host cells from lytic phage infection.

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 a Cryptic Rolling-Circle Replication Plasmid pMK8 from Enterococcus durans 1-8.

A novel cryptic plasmid from Enterococcus durans 1-8, designated as pMK8, was sequenced and analyzed in this study. It consists of 3337bp with a G + C content of 33.11%. Sequence analysis of pMK8 revealed three putative open reading frames (ORFs). Based on homology, two of them were identified as genes encoding replication initiation (RepC) and mobilization (Mob) protein, respectively. Sequence analysis revealed a pT181 family double-strand origin (dso) and a putative single-strand origin (sso) located upstream of the repC gene. Sequence homology analysis indicated that the sso belongs to the ssoW family. Southern hybridization confirmed the presence of single-strand DNA (ssDNA) intermediates, suggesting that pMK8 replicates via the RCR mechanism. Furthermore, the relative copy number of pMK8 was estimated by real-time PCR to be 175 ± 14 copies in each cell.

Autonomous Replication of the Conjugative Transposon Tn916.

Integrative and conjugative elements (ICEs) drive horizontal gene transfer and the spread of antibiotic resistances in bacteria. ICEs reside integrated in a host genome but can excise to create a plasmid that is the substrate for transfer to other cells. Here we show that Tn916, an ICE with broad host range, undergoes autonomous rolling-circle replication when in the plasmid form. We found that the origin of transfer functions as a double-stranded origin of replication and identified a single-stranded origin of replication. It was long thought that ICEs do not undergo autonomous replication. Our work adds to the evidence that ICEs replicate autonomously as part of their normal life cycle and indicates that diverse ICEs use the same replicative mechanism.

Generation of a Stable Plasmid for In Vitro and In Vivo Studies of Staphylococcus Species.

A major shortcoming to plasmid-based genetic tools is the necessity of using antibiotics to ensure plasmid maintenance. While selectable markers are very powerful, their use is not always practical, such as during in vivo models of bacterial infection. During previous studies, it was noted that the uncharacterized LAC-p01 plasmid in Staphylococcus aureus USA300 isolates was stable in the absence of a known selection and therefore could serve as a platform for new genetic tools for Staphylococcus species. LAC-p01 was genetically manipulated into an Escherichia coli-S. aureus shuttle vector that remained stable for at least 100 generations without antibiotic selection. The double- and single-stranded (dso and sso) origins were identified and found to be essential for plasmid replication and maintenance, respectively. In contrast, deletion analyses revealed that none of the four LAC-p01 predicted open reading frames were necessary for stability. Subsequent to this, the shuttle vector was used as a platform to generate two plasmids. The first plasmid, pKK22, contains all genes native to the plasmid for use in S. aureus USA300 strains, while the second, pKK30, lacks the four predicted open reading frames for use in non-USA300 isolates. pKK30 was also determined to be stable in Staphylococcus epidermidis Moreover, pKK22 was maintained for 7 days postinoculation during a murine model of S. aureus systemic infection and successfully complemented an hla mutant in a dermonecrosis model. These plasmids that eliminate the need for antibiotics during both in vitro and in vivo experiments are powerful new tools for studies of StaphylococcusIMPORTANCE Plasmid stability has been problematic in bacterial studies, and historically antibiotics have been used to ensure plasmid maintenance. This has been a major limitation during in vivo studies, where providing antibiotics for plasmid maintenance is difficult and has confounding effects. Here, we have utilized the naturally occurring plasmid LAC-p01 from an S. aureus USA300 strain to construct stable plasmids that obviate antibiotic usage. These newly modified plasmids retain stability over a multitude of generations in vitro and in vivo without antibiotic selection. With these plasmids, studies requiring genetic complementation, protein expression, or genetic reporter systems would not only overcome the burden of antibiotic usage but also eliminate the side effects of these antibiotics. Thus, our plasmids can be used as a powerful genetic tool for studies of Staphylococcus species.

Characterization of a Rolling-Circle Replication Plasmid pM411 from Lactobacillus plantarum 1–3

A cryptic plasmid pM411 isolated from Lactobacillus plantarum 1-3 consisted of a 2303-bp circular molecule with a G+C content 32.96%. Sequence analysis of pM411 revealed four putative open reading frames (ORFs). ORF1 shared 99 and 94% similarities, respectively, with the Rep proteins of plasmids pLC2 and pYC2, which belong to the rolling-circle replication pMV158 family. A typical pMV158 family double-strand origin (dso) and a putative single-strand origin (sso) located upstream of the rep gene. Southern hybridization confirmed the presence of single-strand DNA (ssDNA) intermediates, suggesting that pM411 belongs to the RCR pMV158 family. Sequence homology analysis indicated that the sso belongs to the ssoW family. Furthermore, the relative copy number of pM411 was about 88 copies in each cell by real-time PCR.

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 the Lactobacillus plantarum plasmid pCD033 and generation of the plasmid free strain L. plantarum 3NSH

Lactobacillus plantarum CD033, a strain isolated from grass silage in Austria, harbors a 7.9kb plasmid designated pCD033. Sequence analysis identified 14 open reading frames and 8 of these were supposed to be putative coding sequences. Gene annotation revealed no putative essential genes being plasmid encoded, but a plasmid addiction system based on a PemI/PemK-like toxin–antitoxin system, able to stabilize plasmid maintenance. Absence of a replication initiation protein, a double strand origin as well as a single strand origin on plasmid pCD033 suggests replication via a new type of theta mechanism, whereby plasmid replication is potentially initiated and regulated by non-coding RNA. Detailed examination of segregational stability of plasmid vectors consisting of pCD033-fragments, combined with a selection marker, resulted in definition of a stably maintained minimal replicon. A gene encoding a RepB/OrfX-like protein was found to be not essential for plasmid replication. Alignment of the amino acid sequence of this protein with related proteins unveiled a highly conserved amino acid motif (LLDQQQ). L. plantarum CD033 was cured of pCD033 resulting in the novel plasmid free strain L. plantarum 3NSH. Plasmid curing demonstrated that no essential features are provided by pCD033 under laboratory conditions.

Completing the specificity swap: Single-stranded DNA recognition by F and R100 TraI relaxase domains

During conjugative plasmid transfer, one plasmid strand is cleaved and transported to the recipient bacterium. For F and related plasmids, TraI contains the relaxase or nickase activity that cleaves the plasmid DNA strand. F TraI36, the F TraI relaxase domain, binds a single-stranded origin of transfer (oriT) DNA sequence with high affinity and sequence specificity. The TraI36 domain from plasmid R100 shares 91% amino acid sequence identity with F TraI36, but its oriT DNA binding site differs by two of eleven bases. Both proteins readily distinguish between F and R100 binding sites. In earlier work, two amino acid substitutions in the DNA binding cleft were shown to be sufficient to change the R100 TraI36 DNA-binding specificity to that of F TraI36. In contrast, three substitutions could make F TraI36 more “R100-like”, but failed to completely alter the specificity. Here we identify one additional amino acid substitution that completes the specificity swap from F to R100. To our surprise, adding further substitutions from R100 to the F background were detrimental to binding instead of being neutral, indicating that their effects were influenced by their structural context. These results underscore the complex and subtle nature of DNA recognition by relaxases and have implications for the evolution of relaxase binding sites and oriT sequences.

New shuttle vector-based expression system to generate polyhistidine-tagged fusion proteins in Staphylococcus aureus and Escherichia coli.

Four Staphylococcus aureus-Escherichia coli shuttle vectors were constructed for gene expression and production of tagged fusion proteins. Vectors pBUS1-HC and pTSSCm have no promoter upstream of the multiple cloning site (MCS), and this allows study of genes under the control of their native promoters, and pBUS1-Pcap-HC and pTSSCm-Pcap contain the strong constitutive promoter of S. aureus type 1 capsule gene 1A (Pcap) upstream of a novel MCS harboring codons for the peptide tag Arg-Gly-Ser-hexa-His (rgs-his6). All plasmids contained the backbone derived from pBUS1, including the E. coli origin ColE1, five copies of terminator rrnB T1, and tetracycline resistance marker tet(L) for S. aureus and E. coli. The minimum pAMα1 replicon from pBUS1 was improved through either complementation with the single-strand origin oriL from pUB110 (pBUS1-HC and pBUS1-Pcap-HC) or substitution with a pT181-family replicon (pTSSCm and pTSSCm-Pcap). The new constructs displayed increased plasmid yield and segregational stability in S. aureus. Furthermore, pBUS1-Pcap-HC and pTSSCm-Pcap offer the potential to generate C-terminal RGS-His6 translational fusions of cloned genes using simple molecular manipulation. BcgI-induced DNA excision followed by religation converts the TGA stop codon of the MCS into a TGC codon and links the rgs-his6 codons to the 3' end of the target gene. The generation of the rgs-his6 codon-fusion, gene expression, and protein purification were demonstrated in both S. aureus and E. coli using the macrolide-lincosamide-streptogramin B resistance gene erm(44) inserted downstream of Pcap. The new His tag expression system represents a helpful tool for the direct analysis of target gene function in staphylococcal cells.

Bringing them together: Plasmid pMV158 rolling circle replication and conjugation under an evolutionary perspective

Rolling circle-replicating plasmids constitute a vast family that is particularly abundant in, but not exclusive of, Gram-positive bacteria. These plasmids are constructed as cassettes that harbor genes involved in replication and its control, mobilization, resistance determinants and one or two origins of lagging strand synthesis. Any given plasmid may contain all, some, or just only the replication cassette. We discuss here the family of the promiscuous streptococcal plasmid pMV158, with emphasis on its mobilization functions: the product of the mobM gene, prototype of the MOBV relaxase family, and its cognate origin of transfer, oriT. Amongst the subfamily of MOBV1 plasmids, three groups of oriT sequences, represented by plasmids pMV158, pT181, and p1414 were identified. In the same subfamily, we found four types of single-strand origins, namely ssoA, ssoU, ssoW, and ssoT. We found that plasmids of the rolling-circle Rep_2 family (to which pMV158 belongs) are more frequently found in Lactobacillales than in any other bacterial order, whereas Rep_1 initiators seemed to prefer hosts included in the Bacillales order. In parallel, MOBV1 relaxases associated with Rep_2 initiators tended to cluster separately from those linked to Rep_1 plasmids. The updated inventory of MOBV1 plasmids still contains exclusively mobilizable elements, since no genes associated with conjugative transfer (other than the relaxase) were detected. These plasmids proved to have a great plasticity at using a wide variety of conjugative apparatuses. The promiscuous recognition of non-cognate oriT sequences and the role of replication origins for lagging-strand origin in the host range of these plasmids are also discussed.

Phenotypic and molecular characteristics of an Aeromonas hydrophila strain isolated from the River Nile

Aeromonas hydrophila, an inhabitant of aquatic ecosystems found in most parts of the world, has considerable virulence potential. The polymerase chain reaction technique was used to assay for the presence of five virulence factor genes: haemolytic toxins aerA and ahh1, elastase ahyB, the enterotoxin act, and the polar flagella flaA/flaB in the A. hydrophila strain isolated from the River Nile. Drug screening showed high levels of resistance to β-lactam antibiotics and tetracycline. Slime production was determined by the Congo red agar plate test. The isolate produced two restriction enzymes named AehI and AehII which are isoschizomers of XhoI and StuI respectively. The complete nucleotide sequence of the cryptic plasmid pAhy2.5 (2524bp) from this strain was determined. Sequence analysis revealed the presence of two open reading frames (ORFs) encoding putative proteins. The protein coded by ORF1 is homologous with Rep proteins of plasmids belonging to the pC194 family, which are known to replicate by the rolling-circle mechanism. The putative double-strand origin of replication and a region with palindromic sequences that could function as a single-strand origin were detected in pAhy2.5.

Chromosomal integration of the novel plasmid pUR3912 from methicillin-susceptible Staphylococcus aureus ST398 of human origin

AbstractThe novel erm(T)-cadDX-carrying plasmid pUR3912 has recently been described in the methicillin-susceptible Staphylococcus aureus ST398-t571 strain C3912 from a healthy human in Spain. Structural analysis revealed that pUR3912 belongs to the pC194 replicon family, replicates via a rolling circle mechanism and harbours putative double-strand (dso) and single-strand (sso) origins of replication. Besides its plasmid location, a copy of pUR3912 was also found in the chromosomal DNA of strain C3912. Two IS431 copies, which flank the plasmid, most probably mediated its chromosomal integration. Its ability to not only exist extrachromosomally, but also to integrate into the chromosomal DNA ensures persistence and dissemination of pUR3912.

Characterization of the cryptic plasmid pWCZ from Lactobacillus paracasei WCZ isolated from silage

A cryptic plasmid from Lactobacillus paracasei WCZ, designated as pWCZ, was sequenced and analyzed. It consisted of 3,078 bp with a G+C content of 42.6 %. The plasmid was predicted to encode three putative coding sequences. Based on homology, the three coding sequences were identified as genes encoding transcriptional repressor (CopG), replication initiation (Rep) and mobilization (Mob) protein, respectively. Various regulatory regions like promoters and ribosome binding site (RBS) were then deduced from the sequences of copG and mob. A double-strand origin (dso), which contained the conserved nick sequence of the rolling-circle replication (RCR) pMV158 family was identified. A putative single-strand origin (sso) consisting of successive inverted repeats was also detected. Detection of single-stranded DNA (ssDNA) intermediates by Southern hybridization and S1 nuclease treatment confirmed that pWCZ replicated via the RCR mechanism. Furthermore, the relative copy number of pWCZ was estimated by real-time PCR to be about 99 copies in each cell.

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.

Characterization of pMRI 5.2, a rolling-circle-type plasmid from Lactobacillus plantarum BFE 5092 which harbours two different replication initiation genes

Plasmid pMRI 5.2 from Lactobacillus plantarum BFE 5092 was sequenced and analysed. The sequence consists of 5206bp with a mol% G+C content of 35.8%. Nine putative open reading frames were identified. A typical pC194 family double strand origin (dso) and a putative single strand origin (sso) were predicted upstream of a rep gene. This rep gene encoded a replication protein of 314 amino acids exhibiting 98% amino acid sequence identity to the Rep protein of plasmid pLAB1000 from Lactobacillus hilgardii. A mob gene encoding a mobilization protein was also identified and this protein showed high amino acid similarity to Mob proteins from various L. plantarum plasmids. Downstream of the mob gene, a second putative replication region was identified that is similar to the pMV158 family of plasmids. It contains a dso as well as a putative sso, and encodes the 52 amino acid repressor-like protein RepA, the replication initiation protein RepB of 215 amino acids, and the 48 amino acid RepC that is similar to ORFD of the lactococcal plasmid pWVO1. RT-PCR and qRT-PCR expression analyses of the rep and repB genes showed that the repB gene was expressed at a higher level. To confirm that the plasmid replicated by the rolling-circle-type mechanism, the presence of a characteristic single strand intermediate DNA was shown to be produced during replication. Plasmid copy number was ca. 30 per equivalent chromosome copy number based on qRT-PCR analyses. The plasmid also encodes four additional putative proteins of unknown function. The unusual feature of a rolling-circle plasmid having two different plasmid-encoded replication initiation proteins from different replicon families suggests that the genes for these may have originated from different plasmids.

Characterization of a small cryptic plasmid pK50-2 isolated from Lactobacillus reuteri K50

The complete nucleotide sequence of a cryptic plasmid, pK50-2, from Lactobacillus reuteri K50 had been determined. It consisted of an 1866bp circular molecule with a G+C content of 35%, from which two putative open reading frames (orfs) could be predicted. Based on sequence similarity, the orf1 was not homologous to any known protein, while the N-terminus of the orf2 shared 56% and 64% identities with RepB proteins of plasmid pAR141 and an unnamed plasmid in L. reuteri strain PA-16, members of the rolling-circle replication (RCR) pMV158 family, respectively. Downstream of orf2, a sequence containing two conserved regions (i.e., bind and nick), possibly involved in the binding and nicking of Rep protein, similar to the dso (double strand origin) of RCR-pMV158 family was also identified. Furthermore, a sequence capable of forming the characteristic secondary structure of ssoT (single-strand origin type T) was subsequently determined upstream of the orf2 gene. Thus, the three elements essential for a RCR plasmid (i.e., dso, sso, and rep gene) were all deducible in the pK50-2. Noteworthy was that a conserved alpha helix-turn-alpha helix (HTH) motif, thus far only seen in theta-type plasmids, was for the first time identified in Rep protein of RCR plasmid, pK50-2. To estimate the pK50-2 could be an expression vector to deliver exogenous antigens, a shuttle vector pK50-S containing both pK50-2 and pUE80 (−) was used to analyze the segregational stability and copy-number, which were shown that pK50-S in L. reuteri DSM 20016 were estimated to be 98%, 77%, and 75% after 36, 72, and 100 generations and about 50 copies per chromosome equivalent by real-time PCR.