Identification and nucleotide sequence of the minimal replicon of the low-copy-number plasmid pBS2

The plasmid pBS2 has a low copy number and is endogenous to Bacillus subtilis. The replication of this plasmid depends on the function of most of the host's dna genes including dnaB, which is unique to B. subtilis and is required for both the initiation of chromosome replication and the DNA-membrane association. We have identified the region that is essential for the replication of pBS2 and determined the complete 2279-bp nucleotide sequence of this region. In this region, there are two stretches of sequence homologous to the 18-bp consensus sequence which commonly appears at the origin of replication of plasmids pUB110 and pC194. The entire region contains six sizable open reading frames. Two of them are probably translated. One open reading frame, designated ORF A, coding for 269 amino acids, has significant homology, in terms of amino acid sequence, with the open reading frame of the gene for the Rep U protein of plasmid pUB110. The similarities between pBS2 and other plasmids suggest that the pBS2 may also replicate as a rolling circle, which appears to be the salient feature of a mechanism of replication that is common to small plasmids in gram-positive bacteria.     

Characterization,sequence and mode of replication of plasmid pNB2 from the thermophilic bacterium Clostridium thermosaccharolyticum

The 1882-bp nucleotide sequence of the cryptic plasmid pNB2 isolated from the thermophilic bacterium Clostridium thermosaccharolyticum was determined. pNB2 DNA has very low GC content (27%) and may serve as a model for studying the modes of maintenance and replication of AT-rich DNA under conditions of thermophilic growth. The plasmid sequence revealed three open reading frames (ORFs) which would encode polypeptides of 289, 68 and 59 amino acids, respectively, and these proteins were synthesized in E. coli extracts primed with the plasmid. We found that the product of ORF289 may be initiated at the non-ATG start codon, TTG, and has similarities with the conserved motifs of Rep proteins encoded by rolling circle (RC) plasmids of the pC194/pUB110 family. Southern hybridization analysis of lysates of C. thermosaccharolyticum cells harboring pNB2 revealed single-stranded intermediates, suggesting that this plasmid is able to replicate in clostridial cells via the RC mechanism. The most significant similarities are found between pNB2 Rep protein and the Rep proteins of three RC plasmids of the pC194 family (pTB913, pBC1 and pST1) isolated from thermophilic bacteria. Comparative analysis of these Rep proteins showed that despite the significant level of divergence, these Rep proteins share a high degree of similarity in the regions of five well-known conserved domains of RC Rep proteins and fall into two groups in accordance with the similarities found in their active sites.     

Characterization, sequence and mode of replication of plasmid pNB2 from the thermophilic bacterium Clostridium thermosaccharolyticum

 The 1882-bp nucleotide sequence of the cryptic plasmid pNB2 isolated from the thermophilic bacterium Clostridium thermosaccharolyticum was determined. pNB2 DNA has very low GC content (27%) and may serve as a model for studying the modes of maintenance and replication of AT-rich DNA under conditions of thermophilic growth. The plasmid sequence revealed three open reading frames (ORFs) which would encode polypeptides of 289, 68 and 59 amino acids, respectively, and these proteins were synthesized in E. coli extracts primed with the plasmid. We found that the product of ORF289 may be initiated at the non-ATG start codon, TTG, and has similarities with the conserved motifs of Rep proteins encoded by rolling circle (RC) plasmids of the pC194/pUB110 family. Southern hybridization analysis of lysates of C. thermosaccharolyticum cells harboring pNB2 revealed single-stranded intermediates, suggesting that this plasmid is able to replicate in clostridial cells via the RC mechanism. The most significant similarities are found between pNB2 Rep protein and the Rep proteins of three RC plasmids of the pC194 family (pTB913, pBC1 and pST1) isolated from thermophilic bacteria. Comparative analysis of these Rep proteins showed that despite the significant level of divergence, these Rep proteins share a high degree of similarity in the regions of five well-known conserved domains of RC Rep proteins and fall into two groups in accordance with the similarities found in their active sites. Received: 25 April 1996 / Accepted: 20 June 1996     

Structural organization of pLP1, a cryptic plasmid from Lactobacillus plantarum CCM 1904

To construct shuttle vectors based on an endogenous replicon, we isolated a small cryptic plasmid (pLP1) from Lactobacillus plantarum CCM 1904. The nucleotide sequence (2093 bp, 38.25 GC mol%) revealed one major open reading frame encoding for a 317 amino acid protein (Rep). Comparisons with proteins encoded by other Gram-positive bacteria plasmids strongly suggest that the protein encoded by pLP1 has a replicative role. The presence of a consensus sequence including a tyrosine residue known to be the replication protein binding site to the DNA (in phage phi X174) strengthens this hypothesis. The DNA sequence contains also a sequence similar to the pC194 origin nick sequence, which initiates the plasmid replication at the plus origin, characteristic of plasmids which replicate following a rolling circle mechanism via single-stranded DNA intermediates. A set of 13 direct repeats of 17 bp could be involved in the expression of the incompatibility or in the copy number control as in the other plasmids. A promoter sequence located at the rep 5' region has been identified and is functional in Bacillus subtilis.     

Structural organization of pLP1, a cryptic plasmid from Lactobacillus plantarum CCM 1904

To construct shuttle vectors based on an endogenous replicon, we isolated a small cryptic plasmid (pLP1) from Lactobacillus plantarum CCM 1904. The nucleotide sequence (2093 bp, 38.25 GC mol%) revealed one major open reading frame encoding for a 317 amino acid protein (Rep). Comparisons with proteins encoded by other Gram-positive bacteria plasmids strongly suggest that the protein encoded by pLP1 has a replicative role. The presence of a consensus sequence including a tyrosine residue known to be the replication protein binding site to the DNA (in phage φX174) strengthens this hypothesis. The DNA sequence contains also a sequence similar to the pC194 origin nick sequence, which initiates the plasmid replication at the plus origin, characteristic of plasmids which replicate following a rolling circle mechanism via single-stranded DNA intermediates. A set of 13 direct repeats of 17 bp could be involved in the expression of the incompatibility or in the copy number control as in the other plasmids. A promoter sequence located at the rep 5′ region has been identified and is functional in Bacillus subtilis.     

Nucleotide sequence analysis of pRS1, a cryptic plasmid from Oenococcus oeni

A new cryptic plasmid, pRS1, from an Oenococcus oeni strain isolated from Spanish wines is reported. Nucleotide sequence analysis (2523 bp) revealed the presence of three major open reading frames (ORFs) whose nucleotide sequence and encoded proteins exhibit high homology with those of pOg32, a previously described plasmid of O. oeni. Common features in other plasmids from O. oeni (i.e., pLo13 and pOg32) have been found in pRS1. They have three major ORFs in the same strand; the putative encoded proteins by two of these ORFs exhibit homology with the replication (Rep) and the recombination (Pre) proteins, respectively, of the pT181 plasmid family and related gram-positive bacteria plasmids; these plasmids contain the DNA sequences required for plasmid replication by the rolling circle mechanism and for recombination (i.e., double-strand origin, DSO; single-strand origin, SSO; recombination-specific sites, RSA and RSB); and finally, all these plasmids have a third ORF of unknown function. These features suggest that pRS1 could constitute together with pLo13 and pOg32 a family of small cryptic plasmids of O. oeni.     

pIH01, a small cryptic plasmid from Leuconostoc citreum IH3

A small cryptic plasmid pIH01 from Leuconostoc citreum IH3 was characterized. This 1.8-kb sized plasmid contains single open reading frame that encodes a RepC class protein (342 amino acids) and a conserved pT181-type double strand origin, suggesting a rolling circle replication mode. This putative replicase protein shows the highest similarity to a replicase from pFR18 plasmid of Leuconostoc mesenteroides FR52 (64% identity), one of the pT181-type rolling circle plasmid family and contains a strictly conserved RepC-type active site sequence of pT181 family. A shuttle vector that was developed on the basis of this cryptic plasmid by insertion of both erythromycin resistance gene (ermC) from pE194 and Escherichia coli ColE1 origin was able to transform Leuconostoc strains, Lactobacillus plantarum, and Lactococcus lactis. Therefore, pIH01 derivative plasmids might be useful for the manipulation of Leuconostoc strains.     

Replication origins of single-stranded-DNA plasmid pUB110.

The two replication origins of plasmid pUB110 have been characterized. The site of initiation of DNA replication at the plus origin was mapped to within an 8-base-pair sequence. DNA synthesis initiated at the origin was made to terminate precociously in an inserted sequence of 18 base pairs that is homologous to a sequence in the origin. This suggests that pUB110 replicates as a rolling circle. The minus origin of plasmid pUB110 has been characterized, and the minimal sequence required for function has been determined. As with other minus origins, activity is orientation specific with respect to the direction of replication. Its activity is sensitive to rifampin in vivo, suggesting that RNA polymerase catalyzes single-strand to double-strand conversion. Unlike all other plasmids of gram-positive bacteria thus far described, the pUB110 minus origin is functional in more than one host.     

Characterization of a region of plasmid pBL1 of Brevibacterium lactofermentum involved in replication via the rolling circle model.

The minimal region for autonomous replication of pBL1, a 4.5-kb cryptic plasmid of Brevibacterium lactofermentum ATCC 13869 that has been used to construct a variety of corynebacterium vectors, was shown to be contained on a 1.8-kb HindII-SphI DNA fragment. This region contains two open reading frames (ORFs) (ORF1 and ORF5) which are essential for pBL1 replication in B. lactofermentum. Accumulation of single-strand intermediates in some of the constructions indicates that plasmid pBL1 replicates via the rolling circle replication model; its plus strand and minus strand were identified by hybridization with two synthetic oligonucleotide probes complementary to each pBL1 strand. ORF1 seems to encode the Rep protein and showed partial homology with sequences for Rep proteins from Streptomyces plasmids which replicate via rolling circle replication such as pIJ101, pSB24, and pJV1.     

Molecular, genetic, and functional analysis of the basic replicon of pVA380-1, a plasmid of oral streptococcal origin.

The 4.2-kb cryptic plasmid pVA380-1 has been used as a vector for the cloning of antibiotic resistance genes directly in streptococci, and in the construction of Escherichia coli/Streptococcus shuttle vectors. The results of subcloning experiments located the basic replicon of pVA380-1 within a 2.5-kb region. The nucleotide base sequence of this region was determined and contained a single complete open reading frame (ORF) encoding a 237-amino-acid peptide with a predicted size of 29 kDa. This peptide and a region of the DNA molecule 5' to the ORF encoding it shared homology with the replication protein and plus origin, respectively, of the Staphylococcus aureus plasmid pUB110. Data from Tn5 mutagenesis and complementation studies indicated that the protein product of the ORF was required for pVA380-1 replication in streptococci. Deletion of a region of the basic replicon distal to the plus origin and ORF produced an unstable derivative, and resulted in the accumulation of single-stranded replicative intermediates, consistent with the loss of a minus origin. All of these results suggest that pVA380-1 replicates by a rolling circle mode, and is most closely related to the pC194 family of single-stranded DNA plasmids.     

Complete nucleotide sequence and characterization of pUA140, a cryptic plasmid from Streptococcus mutans

Approximately 5% of strains of Streptococcus mutans contain plasmid DNA. Strain UA140 harbors a 5.6-kb cryptic plasmid, pUA140, with an overall G+C content of 32.7%. Five open reading frames (ORF), encoding peptides of larger than 100 amino acid residues, were initially designated as ORF1 to ORF5. These five ORFs were located on the same strand of pUA140. ORF1 (258 amino acids) resembled a replication protein, Rep. Upstream of the putative Rep gene, a double-stranded origin for plasmid replication that showed strong similarity to those of a number of plasmids in the pT181 family was identified. Further upstream was a region constituting the single-stranded origin of replication. A single-stranded DNA intermediate was detected during plasmid replication. Taken together, these results suggest that pUA140 replicated by the rolling circle replication mechanism but exhibited several characteristics that differ from those of other members of the pT181 plasmid family.     

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

The complete nucleotide sequence of two cryptic plasmids isolated from Lactobacillus plantarum strain AS1.2986 has been determined. The smaller plasmid, designated pLP2000, encodes a 37.0kDa Rep protein and has a 17bp sequence repeated 10 times. Sequence analysis of the larger plasmid, designated pLP9000, revealed nine putative open reading frames (ORFs). Based on sequence similarity, ORF1 codes for a putative magnesium transporter protein that shows similarities to CorA from plasmid pCIS3 (Lactococcus lactis). None of the nine ORFs shows similarity to any known Rep protein. Southern blot analysis indicates these two plasmids both replicate via a rolling circle (RC) mechanism.     

Complete nucleotide sequence and structural organization of pPB1, a small Lactobacillus plantarum cryptic plasmid that originated by modular exchange

A small cryptic plasmid designated pPB1 was isolated from Lactobacillus plantarum BIFI-38 and its complete 2899 bp nucleotide sequence was determined. Sequence analysis revealed four putative open reading frames. Based on sequence analysis two modules could be identified. First, the replication module consisted of a sequence coding for a replication protein (RepB) and its corresponding target site, and two putative repressor proteins (RepA and RepC). Sequence analysis indicated the possible synthesis of an antisense RNA that might regulate RepB production. A putative lagging-strand initiation site was also found, suggesting that pPB1 replicates via a rolling circle mechanism. The second module of pPB1 consisted of a sequence coding for a putative mobilization protein and its corresponding oriT site. Since the nucleotide sequence of the replication module showed 94.5% identity to the similar region on the Leuconostoc lactis plasmid pCI411, and the nucleotide sequence of the mobilization module had 97.5% identity to L. plantarum plasmid pLB4, it is concluded that pPB1 originated by modular exchange between two such plasmids by homologous recombination. Putative recombination sites where crossover might have taken place were also identified.     

Molecular analysis and characterization of a broad-host-range plasmid, pEP2.

Plasmid pEP2 was found to encode a protein, RepA, which is essential and rate limiting for its replication in Escherichia coli and Corynebacterium pseudotuberculosis. Mutations which altered the rate of synthesis of this protein in E. coli affected the copy number and segregational stability of pEP2 in the two hosts. RepA contains 483 amino acid residues and has the calculated molecular weight of 53,925. It shows 45% amino acid residue identity with open reading frame ORF2 of pSR1, a plasmid isolated from Corynebacterium glutamicum (J. A. C. Archer and A. J. Sinskey, J. Gen. Microbiol. 139:1753-1759, 1993). Plasmid pEP2 was shown to accumulate single-stranded DNA corresponding to the RepA coding strand during its replication in E. coli and C. pseudotuberculosis, suggesting that it may replicate by a rolling circle mechanism. However, RepA has no significant sequence homology with the replication initiator proteins of plasmids known to use this mode of replication.     

Molecular structure of the replication origin of a Bacillus subtilis (natto) plasmid, pUH1.

The structure of a 2.0-kb BstEII DNA sequence necessary and sufficient for the replication of a 5.7-kb Natto plasmid, pUH1, which is responsible for gamma-polyglutamate production by Bacillus subtilis (natto), has been characterized by using a trimethoprim resistance gene derived from B. subtilis chromosomal DNA as a selective marker. The 2.0-kb DNA sequence contains an open reading frame, rep, stretching for 999 bp; a promoter region for rep expression; and a possible replication origin for the plasmid upstream of the promotor. The predicted Rep protein has highly homologous amino acid sequences with rep14 of pFTB14 in B. amyloliquefaciens, RepB of pUB110, and protein A, which is necessary for pC194 replication in staphylococci throughout the protein molecule, but is not homologous with RepC of staphylococcal plasmid pT181.     

DNA sequence and analysis of a cryptic 4.2-kb plasmid from the filamentous cyanobacterium, Plectonema sp. strain PCC 6402.

The 4194-bp plasmid, pRF1, from Plectonema sp. Strain PCC 6402 was completely sequenced and analyzed. Seven potential open reading frames were identified. The predicted amino acid sequence of open reading frame C (ORF C) had identities of 34, 29, and 25% with Rep B from the Staphylococcus aureus plasmid, pUB110; Rep from the Bacillus amyloliquefaciens plasmid, pFTB14; and protein A from the S. aureus plasmid, pC194, respectively. A 75-amino-acid region conserved in these proteins (Rep B, Rep, and protein A) also was highly conserved in ORF C with identities of 45, 37, and 40%, respectively. Significantly, 16 of the 21 amino acids conserved in Rep B, Rep, and protein A were found at the same positions in ORF C. This ORF may encode a replication protein that includes a region conserved in some eubacteria. Additional structural features include a 425-bp region that contains palindromes, tandem repeats, and short direct repeats which may correspond to the origin of replication. An 18-bp inverted repeat was located between two open reading frames, A and G.     

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

A small cryptic plasmid designated pSSU1 was isolated from Streptococcus suis serotype 2 strain DAT1. The complete sequence of pSSU1 was 4975 bp and contained six major open reading frames (ORFs). ORF1 and ORF2 encode for proteins highly homologous to CopG and RepB of the pMV158 family, respectively. ORF5 encodes for a protein highly homologous to Mob of pMV158. ORF4 encodes for a protein highly homologous to orf3 of pVA380-1 of S. ferus, but its function is unknown. There was no similarity between ORF3 and ORF6 and other protein sequences. In this plasmid, the ORF1 (CopG protein) was preceded by two multiples of direct repeat and the conserved nucleotides that could be the double-strand origin (DSO) of rolling circle replication (RCR) mechanism. The ORF5 (Mob protein) was followed by a potential hairpin loop that could be the single-strand origin (SSO) of RCR mechanism. The sequence, which was complementary to the leader region of Rep mRNA, was homologous to the countertranscribed RNA (ctRNA) of pLS1. Moreover, a 5-amino acid conserved sequence was found in C terminal of Rep and putative Rep proteins of several pMV158 family plasmids. These observations suggest that this plasmid replicates by use of the rolling circle mechanism. Received: 26 June 1999 / Accepted: 10 August 1999     

Complete nucleotide sequence of a cryptic plasmid, pbaw301, from the ruminai anaerobe Ruminococcus flavefaciens R13e2

Abstract The complete nucleotide sequence of a cryptic plasmid designated pBAW301, from the Gram-positive ruminai bacterium Ruminococcus flavefaciens R13e2, has been determined. This plasmid is 1768 bp in size and has an overall G+C content of 43.5%. Computer analysis of the sequence data revealed an open reading frame, ORF1 (256 amino acids), which is similar to the Rep protein of the Bacillus borstelensis plasmid pHT926. ORF1 is preceded by Shine-Dalgarno and Escherichia coli —10 and —35 like sequences. Nine smaller open reading frames showed no significant homologies to known protein sequences. Analysis of replication intermediates and the nucleotide sequence indicate that the plasmid does not replicate by a rolling-circle mode of replication similar to other plasmids from Gram-positive bacteria. Moreover, sequences typical of theta replication origins were not found in the nucleotide sequence of pBAW301. These data suggest that this plasmid either replicates by an as yet undescribed mechanism, or represents a new class of theta replicating plasmids.