Double-stranded Origin Research Articles

Characterization of a small erythromycin resistance plasmid pLFE1 from the food-isolate Lactobacillus plantarum M345

This paper reports the complete 4031 bp nucleotide sequence of the small erythromycin resistance plasmid pLFE1 isolated from the raw-milk cheese isolate Lactobacillus plantarum M345. Analysis of the sequence revealed the coding regions for the erythromycin resistance determinant Erm(B). A replication initiation protein RepB was identified belonging to the RepB proteins of the pMV158 family of rolling-circle replicating plasmids. The transcriptional repressor protein CopG and a small counter transcribed RNA, two elements typically involved in replication control within this family were also found. A putative replication initiation site including a single-strand origin ( sso) -like region succeeded by a characteristic pMV158 family double-strand origin ( dso) was located upstream of the replication region. An open reading frame following a typical origin of transfer ( oriT) site and coding for a putative truncated mobilization (Mob) protein with a size of 83 aa was detected. The product of the putative mob gene showed large similarity to the N-terminal region of the pMV158 family of Pre/Mob proteins, but was much smaller than other proteins of this family. We therefore suggest that the Mob function in pLFE1 is supplied in trans from another plasmid present in L. plantarum M345. Filter-mating experiments showed that pLFE1 has a broad host-range with transconjugants obtained from Lactobacillus rhamnosus, Lactococcus lactis, Listeria innocua, the opportunistic pathogen Enterococcus faecalis and the pathogen Listeria monocytogenes.

Analysis of pYC2, a cryptic plasmid in Lactobacillus sakei BM5 isolated from kimchi

Analysis of the structural properties of pYC2, a cryptic plasmid from Lactobacillus sakei BM5 isolated from kimchi, determined its length as 1,970 bp with a G + C content of 34%. The double-strand origin (dso) and single-strand origin (sso) of rolling-circle replicating plasmids were found in the nucleotide sequence of the pYC2 plasmid. Sequence analysis of pYC2 revealed that ORF 1 and ORF 2 showed high homology with the Cop and Rep proteins encoded by the pMV158 family of plasmids replicating via the rolling-circle mechanism. pYC2 also replicates by this mechanism, as confirmed by Southern hybridization analysis.

Characterization of a cryptic plasmid pM4 fromLactobacillus plantarumM4

A cryptic plasmid from Lactobacillus plantarum M4 isolated from fresh milk, designated as pM4, was sequenced and characterized. It was 3320 bp in length with a G+C content of 38.73 mol%. The plasmid pM4 was predicted to encode three putative ORFs, in which ORF1 shared 99% and 98% homology, respectively, with the Rep proteins of reported plasmids pWCFS101 and pF8801, members of the rolling circle replication (RCR) pC194 family. Sequence analysis revealed a typical pC194 family double strand origin (dso) and a putative single strand origin (sso) located upstream of the rep gene. Mung bean nuclease analysis and Southern hybridization confirmed the presence of single-stranded DNA (ssDNA) intermediates, suggesting that pM4 belongs to the RCR pC194 family. Accumulation of ssDNA in rifampicin-treated strains implied that the host-encoded RNA polymerase was involved in the conversion of ssDNA to double-stranded DNA. Furthermore, the relative copy number of pM4 was estimated to be about 25 in each cell by real-time PCR. The new RCR plasmid would be valuable in constructing cloning vectors for application in the food industry.

Precise Determination, Cross-Recognition, and Functional Analysis of the Double-Strand Origins of the Rolling-Circle Replication Plasmids in Haloarchaea

The precise nick site in the double-strand origin (DSO) of pZMX201, a 1,668-bp rolling-circle replication (RCR) plasmid from the haloarchaeon Natrinema sp. CX2021, was determined by electron microscopy and DSO mapping. In this plasmid, DSO nicking occurred between residues C404 and G405 within a heptanucleotide sequence (TCTC/GGC) located in the stem region of an imperfect hairpin structure. This nick site sequence was conserved among the haloarchaeal RCR plasmids, including pNB101, suggesting that the DSO nick site might be the same for all members of this plasmid family. Interestingly, the DSOs of pZMX201 and pNB101 were found to be cross-recognized in RCR initiation and termination in a hybrid plasmid system. Mutation analysis of the DSO from pZMX201 (DSO(Z)) in this hybrid plasmid system revealed that: (i) the nucleotides in the middle of the conserved TCTCGGC sequence play more-important roles in the initiation and termination process; (ii) the left half of the hairpin structure is required for initiation but not for termination; and (iii) a 36-bp sequence containing TCTCGGC and the downstream sequence is essential and sufficient for termination. In conclusion, these haloarchaeal plasmids, with novel features that are different from the characteristics of both single-stranded DNA phages and bacterial RCR plasmids, might serve as a good model for studying the evolution of RCR replicons.

DNA sequence analysis of a putative primary replicon from a cryptic plasmid pNM214 of a hydrocarbon utilizing marine Bacillus strain NM21

Marine Bacillus strain NM21 isolated from hydrocarbon-contaminated site at Naval Harbour, Mumbai grows on high-speed diesel as a source of carbon and energy. This bacterium harbours four plasmids in it. The smallest plasmid, pNM214 was digested with EcoRI enzyme and cloned in pUC19 vector. The clone Om4 containing largest insert of >3.5 kb was sequenced by primer walking. DNA sequence analysis showed this fragment to be homologous to replication initiation protein (rep) gene and dso (double strand origin) of different plasmids from Bacillus subtilis and Bacillus pumilus species. The putative rep gene sequence of pNM214 showed 74.3-91.6% DNA identity to B. subtilis plasmids (pTA1015, pTA1060 and pTA1040) and 86.3% to 88.9% DNA identity to B. pumilus plasmids (pPL7065, pPL10 and pSH1452). The translated amino acid sequence of rep shows that it contains all the three conserved motifs present in the Rep protein of pC194 family of plasmids. DNA sequence comparison of putative dso of pNM214 with other bacillus plasmids belonging to pC194 group shows that it contains highly conserved nick site sequence 5'-TCTTTTCTTATCTTGATA-3' and surrounding inverted repeats. Thus, it indicates that pNM214 to be a rolling circle replicating plasmid belonging to the pC194 group. The presence of rep and dso like sequences in the sequenced EcoRI fragment indicate that the cloned fragment contain putative primary replicon of pNM214.

Analysis of pMA67, a predicted rolling-circle replicating, mobilizable, tetracycline-resistance plasmid from the honey bee pathogen, Paenibacillus larvae

This work characterizes a recently discovered natural tetracycline-resistance plasmid called pMA67 from Paenibacillus larvae—a Gram-positive bacterial pathogen of honey bees. We provide evidence that pMA67 replicates by the rolling-circle mechanism, and sequence comparisons place it in the pMV158 family of rolling-circle replicons. The plasmid contains predicted rep, cop, and rnaII genes for control of replication initiating at a predicted double-strand origin. The plasmid has an ssoT single-strand origin, which is efficient enough to allow only very small amounts of the single-stranded DNA intermediate to accumulate. The overall efficiency of replication is sufficient to render the plasmid segregationally stable without selection in P. larvae and in Bacillus megaterium, but not in Escherichia coli. The plasmid is expected to be mobilizable due to the presence of a mob gene and an oriT site. The plasmid contains a tetL gene, whose predicted amino acid sequence implies a relatively ancient divergence from all previously known plasmid-encoded tetL genes. We confirm that the tetL gene alone is sufficient for conferring resistance to tetracyclines. Sequence comparisons, mostly with the well-characterized pMV158, allow us to predict promoters, DNA and RNA secondary structures, DNA and protein motifs, and other elements.

Identification and characterization of nine novel types of small staphylococcal plasmids carrying the lincosamide nucleotidyltransferase gene lnu(A)

To date, very little is known about lincosamide resistance plasmids in staphylococci with only a single lnu(A)-carrying staphylococcal plasmid having been sequenced completely. The aim of this study was to characterize small lnu(A)-carrying plasmids isolated from bovine coagulase-negative staphylococci (CoNS). Nine CoNS isolates with MICs of the lincosamide pirlimycin of 1-4 mg/L were tested for the presence of the lnu(A) gene. Its location was determined by Southern-blot hybridization. The lnu(A)-carrying plasmids were transformed into Staphylococcus aureus RN4220 and compared by restriction mapping and subsequent sequencing. Selected plasmids were investigated for their copy number and their lnu(A) gene expression via RT real-time PCR. The lnu(A) gene was detected on plasmids in all isolates. Sequence analysis revealed that these plasmids carried a rep gene, coding for the replication initiator protein, and the resistance gene lnu(A), coding for a lincosamide nucleotidyltransferase. While the Lnu(A) proteins were closely related (91.3-100% amino acid identity), the Rep proteins differed distinctly (27.4-100% amino acid identity), but showed similarity (81.4-98.5%) to Rep proteins of other small staphylococcal resistance plasmids. Sequence features of rolling-circle plasmids, such as the single-strand (ssoA) and double-strand (dso) origins of replication, were identified. For two plasmid types detected, the lincosamide resistance level varied with regard to the amounts of lnu(A) transcripts detected. Structurally different lnu(A)-carrying plasmids were detected in various CoNS species. The detection of the same lnu(A) gene in different plasmid backbones suggested the exchange of the gene via interplasmid recombinational events.

A novel cryptic plasmid pBMB175 from Bacillus thuringiensis subsp. tenebrionis YBT-1765

A new cryptic plasmid pBMB175 from Bacillus thuringiensis subsp. tenebrionis YBT-1765 was isolated and characterized. Sequence analysis showed that pBMB175 (14,841 bp and 31% GC content) contained at least eighteen putative open reading frames (ORFs), among which nine ORFs displayed the homology with the hypothetical proteins in rolling-circle replication plasmid pGI3. Deletion analysis revealed that the pBMB175 minireplicon located in a novel 1,151 bp fragment. This fragment contains ORF7 coding sequence, which encodes a protein (Rep175, 149 amino acids [aa]) indispensable for plasmid replication. Rep175 has no significant homology with known function proteins. Furthermore, a putative double-strand origin (dso), having no DNA similarity with characterized dso of other replicon so far, was identified in this minireplicon fragment. These features showed that pBMB175 could be placed into a new plasmid family.

Isolation and characterization of a new plasmid pSpnP1 from a multidrug-resistant clone of Streptococcus pneumoniae

A novel Streptococcus pneumoniae plasmid (pSpnP1; 5413 bp) has been isolated from the multidrug-resistant clone Poland 23F-16, and its complete nucleotide sequence has been determined. Sequence analysis predicted seven co-directional open reading frames and comparative analyses revealed that plasmid pSpnP1 is different to pDP1, the only previously described pneumococcal plasmid, whereas it is highly similar to pSt08, a plasmid from Streptococcus thermophilus. A double-stranded origin for replication similar to the replication origin of the pC194/pUB110 family was located upstream of the putative rep gene ( orf2). It also contained a 144-bp region with over 60% identity to the single-stranded origin type A of the Streptococcus agalactiae plasmid pMV158/pLS1. Detection of single-stranded DNA by Southern blot analysis indicated that pSpnP1 replicates via a rolling circle mechanism. Interestingly, the product of orf1 has a putative Zonular occludens toxin conserved domain present in toxigenic strains of Vibrio cholerae. Real-time PCR assays revealed that this ORF was expressed. Hybridization experiments showed that the pSpnP1 replicon was unusual among other examined antibiotic-resistant pneumococcal clones, although the recombinant plasmids based on pSpnP1 were able to replicate in Bacillus subtilis and Lactococcus lactis.

Characterization of a cryptic plasmid from Bacillus sphaericus strain LP1-G

A cryptic plasmid from Bacillus sphaericus strain LP1-G, designated as pLG, was sequenced and characterized. It was an 11,066 bp circular molecule, with G + C content of 37%. The plasmid pLG was predicted to encode 23 putative ORFs, and ORF 21 shared the highest identity with Rep of pGI1 and pBMB9741, members of rolling-circle replication (RCR) pC194-family. Sequence analysis revealed a pC194-type double strand origin ( dso) and a single strand origin ( sso) like sequence located upstream and downstream of ORF 21, respectively. Moreover, Mung bean nuclease analysis and Southern hybridization confirmed the existence of single stranded DNA (ssDNA) intermediates, indicating that pLG belongs to the RCR pC194-family. Accumulation of multiple ssDNA intermediates in native strain LP1-G and decline of ssDNA and supercoiled DNA in rifampicin-treated strain implied that a special mechanism might be employed by pLG. Furthermore, the copy number of pLG in its original host was determined and about 58 copies of the plasmid exist in each cell. Subcloning and transformation experiments proved that the minimal replicon of pLG was within a 1.6-kb fragment, which was composed of rep gene and dso. These data are a good basis for the understanding of replication mechanisms and genetics of this B. sphaericus plasmid.

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.

Binding of the Universal Minicircle Sequence Binding Protein at the Kinetoplast DNA Replication Origin

Kinetoplast DNA, the mitochondrial DNA of trypanosomatids, is a remarkable DNA structure that contains, in the species Crithidia fasciculata, 5000 topologically linked duplex DNA minicircles. Their replication initiates at two conserved sequences, a dodecamer, known as the universal minicircle sequence (UMS), and a hexamer, which are located at the replication origins of the minicircle L and H strands, respectively. A UMS-binding protein (UMSBP) binds specifically the 12-mer UMS sequence and a 14-mer sequence that contains the conserved hexamer in their single-stranded DNA conformation. In vivo cross-linking analyses reveal the binding of UMSBP to kinetoplast DNA networks in the cell. Furthermore, UMSBP binds in vitro to native minicircle origin fragments, carrying the UMSBP recognition sequences. UMSBP binding at the replication origin induces conformational changes in the bound DNA through its folding, aggregation and condensation.

The smr gene resides on a novel plasmid pSP187 identified in a Staphylococcus pasteuri isolate recovered from unpasteurized milk

This work describes a novel plasmid pSP187 (5550 bp) carrying the small multidrug resistance determinant smr encoding resistance to quaternary ammonium compounds (QACs). pSP187 was identified in a Staphylococcus pasteuri isolate recovered from bulk milk in a dairy cattle herd in Norway. Sequence analysis revealed 6 putative ORFs in addition to the smr gene within a cassette with identical genetic organization to that found in the pSK41-like Staphylococcus aureus plasmid pTZ22. A protein homology search suggested the gene product of ORF7 to be a putative replication initiation protein, while ORF2 was predicted to encode a protein homologous to members of FtsK/SpoIIIE cell division-DNA segregation protein families. Sequence similarities to some initiator proteins of rolling circle replicons (RCR) indicated that pSP187 uses a RCR mode of replication, supported by the detection of intermediate ssDNA using S1 nuclease treatment and hybridization analysis. Interestingly, a 30-bp sequence found upstream from ORF7 showed high similarity to other dyad symmetry motifs proposed as putative double-strand origins of replication in the plasmids pGI3 ( Bacillus thuringiensis), pSTK1 ( Bacillus stearothermophilus), and pER1-2 ( Streptococcus thermophilus). In conclusion: The novel smr-containing plasmid pSP187 is the first member of RCR group VI to be identified in a Staphylococcus sp.

Characterization of <italic>Nocardia</italic> Plasmid pXT 107

Nocardia, Rhodococcus and Streptomyces, all members of the actinomycetes family, are Gram-positive eubacteria with high G+C content and able to form mycelium. We report here a newly identified plasmid pXT107 of Nocardia sp. 107, one of the smallest circular plasmids found in Nocardia. The complete nucleotide sequence of pXT107 consisted of 4335 bp with 65% G+C content, and encoded one replication extragenic palindromic (Rep) and six hypothetical proteins. The Rep, double-strand origin and single-strand origin of pXT107 resembled those of typical rolling-circle-replication plasmids, such as pNI100 of Nocardia, pRE8424 of Rhodococcus and pIJ101 of Streptomyces. The Escherichia coli-Nocardia shuttle plasmid pHAQ22, containing the rep gene of pXT107, is able to propagate in Nocardia but not in Streptomyces.

DNA sequence analysis of a small cryptic plasmid from Lactococcus lactis subsp. lactis M14

A small plasmid designated pAR141 was isolated from Lactococcus lactis subsp. lactis M14 and its complete 1594 base pair nucleotide sequence was determined. Analysis of the sequence indicated that this plasmid does not carry any industrially important determinants besides the elements involved in plasmid replication and control. The transcriptional repressor CopG and replication initiation protein RepB appeared as a single operon. A small countertranscribed RNA (ctRNA) coding region was found between the copG and repB genes. The double strand origin ( dso) and single strand origin ( sso) of rolling circle replicating (RCR) plasmids were also identified in pAR141, suggesting that this plasmid replicates by rolling circle (RC) mode. This observation was supported by S1 nuclease and Southern hybridization analyses.

Characterization of a rolling-circle replication plasmid from Thermus aquaticus NTU103

The thermophilic bacterium Thermus aquaticus NTU103 harbors a 1965-bp plasmid, pTA103. Sequencing analysis revealed that pTA103 contains two open reading frames. One of the open reading frames ( orf2) shares no significant homology with protein in the data bank. The other one has 50% similarity and 34% identity with RepA-like protein of pRm1132f, which is a rolling-circle replication (RCR) plasmid isolated from Sinorhizobium meliloti. S1 nuclease analysis demonstrated that pTA103 contains a single-stranded intermediate, confirming that pTA103 replicates via RCR mechanism. Sequence data also revealed putative double-stranded origin and single-stranded origin sites, indicating the importance of these cis elements in pTA103 replication.

Double-stranded origin nicking and replication initiation are coupled in the replication of a rolling circle plasmid, pT181.

The Staphylococcus aureus rolling circle plasmid pT181 initiator RepC is modified by the addition of an oligodeoxynucleotide, giving rise to a new form, RepC*. RepC/RepC* heterodimer is an inhibitor of replication. However, in order to act effectively, the initiator/inhibitor protein must be stable. We show here that RepC is stable for at least 90 min, which enables it to function effectively as an inhibitor of replication. This finding also allowed us to carry out the two stages in pT181 replication sequentially: first, binding/nicking of the double-strand origin (DSO) by the pT181-encoded RepC, followed by initiation/elongation by the host cell's DNA replication apparatus. The results demonstrate that these two stages in pT181 replication are functionally coupled and that interruptions in this continuous process generate relaxed pT181 DNA that cannot be used as a template for replication.

Determination of specific DNA strand discontinuities with nucleotide resolution in exponentionally growing bacteria harboring rolling circle-replicating plasmids.

Plasmid replication by the rolling circle mechanism and conjugative transfer of plasmids require the generation of a specific strand discontinuity in the DNA. In both processes cleavage at the so-called nic site is catalyzed by plasmid-encoded proteins. The strand discontinuities at the conjugative origins of transfer of plasmid pE194 and pMV158 were determined in Bacillus subtilis and Streptococcus pneumoniae, respectively, with a recently developed runoff DNA synthesis assay. The positions of intracellular cleavage within the respective transfer origins were shown to coincide with the site predicted for pE194 and with the nic site determined in vitro for pMV158. For pMV158, the influence of a mutation in the S. pneumoniae polA gene on the efficiency of replication was investigated. In addition, the nic site within the double-stranded origin of the-rolling circle-replicating plasmid pMV158 in S. pneumoniae as well as that of pFX2 in Escherichia coli was mapped with nucleotide resolution.

Role of RepB in the replication of plasmid pJB01 isolated from Enterococcus faecium JC1

The plasmid pJB01 (GenBank Accession No. AY425961) isolated from the pathogenic bacterium, Enterococcus faecium JC1, is 2235 base pairs in length and consists of a putative double-strand origin ( dso), a single-strand origin, a counter-transcribed RNA, and three open reading frames. A comparison of a few replication factors and motifs, bind and nic regions, for replication initiation on the nucleotide sequence level revealed that it belongs to the pMV158 family among RC-replicating plasmids. A runoff DNA synthesis assay demonstrated that nicking occurred between G525 and A526, which is located on the internal loop of a putative secondary structure in the dso. Unlike all the other plasmids of the pMV158 family having two or three direct repeats, pJB01 has three non-tandem direct repeats of 5′-CAACAAA-3′ separated by four nucleotides, as the RepB-binding site in the dso. Moreover, the nick site on the internal loop is located at 77 nucleotides upstream from the RepB-binding region. Irrespective of the structural difference of direct repeats from other members of the pMV158 family, we think, it is still a new member of this plasmid family. The introduction of mutations in conserved regions of RepB confirmed that RepB N-moiety is important for nicking/nick-closing activity. Within N-moiety, especially all of the motif R-III, the Y100 in R-IV and Y116 in R-V residues, played particularly critical roles in this activity, however, for its binding, both of the N- and C-moieties of RepB were needed.

Nucleotide sequence, structural organization and host range of pRS4, a small cryptic Pediococcus pentosaceus plasmid that contains two cassettes commonly found in other lactic acid bacteria

The complete nucleotide sequence of the cryptic plasmid pRS4 (3550 bp) from Pediococcus pentosaceus RS4 was determined. Sequence analysis revealed the presence of three open reading frames (ORFs). The putative protein coded by ORF 1 showed 93% identity with the mobilization protein of Lactobacillus casei plasmid pLC88 and 94% identity with a sequenced fragment of the mobilization protein of P. damnosus plasmid pF8801, suggesting a common origin for these three mobilization proteins. The putative protein coded by ORF 2 showed 92% identity with the replication protein of L. plantarum plasmid pWCFS101, a plasmid that replicates via the rolling circle (RC) mechanism, suggesting a similar replication mechanism for pRS4. Supporting this hypothesis, a putative double strand origin ( dso) and a region with palindromic sequences that could function as single strand origin ( sso), were detected in pRS4. A function could not be assigned to ORF 3. Since ORF 1 exhibits high identity with L. casei plasmid pLC88 but lower identity (58%) with other Lactobacillus plasmids, and ORF 2 exhibits high identity with the L. plantarum plasmid pWCFS101 but lower identity (55–58%) with other Lactobacillus plasmids (including pLC88), two independent cassettes, from different sources, seem to be involved in the structure of pRS4. Plasmids derived from pRS4 containing the chloramphenicol resistance gene were successfully electrotransformed in L. plantarum, L. casei, P. pentosaceus, and Pediococcus acidilactici, suggesting that pRS4 could be used as a cloning vector for lactic acid bacteria. To our knowledge pRS4 is the first RC-replicating plasmid of P. pentosaceus that has been completely sequenced and used as cloning vector.