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.     

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     

Replication Mechanism and Sequence Analysis of the Replicon of pAW63, a Conjugative Plasmid from Bacillus thuringiensis

A 5.8-kb fragment of the large conjugative plasmid pAW63 from Bacillus thuringiensis subsp. kurstaki HD73 containing all the information for autonomous replication was cloned and sequenced. By deletion analysis, the pAW63 replicon was reduced to a 4.1-kb fragment harboring four open reading frames (ORFs). Rep63A (513 amino acids [aa]), encoded by the largest ORF, displayed strong similarity (40% identity) to the replication proteins from plasmids pAMbeta1, pIP501, and pSM19035, indicating that the pAW63 replicon belongs to the pAMbeta1 family of gram-positive theta-replicating plasmids. This was confirmed by the facts that no single-stranded DNA replication intermediates could be detected and that replication was found to be dependent on host-gene-encoded DNA polymerase I. An 85-bp region downstream of Rep63A was also shown to have strong similarity to the origins of replication of pAMbeta1 and pIP501, and it is suggested that this region contains the bona fide pAW63 ori. The protein encoded by the second large ORF, Rep63B (308 aa), was shown to display similarity to RepB (34% identity over 281 aa) and PrgP (32% identity over 310 aa), involved in copy control of the Enterococcus faecalis plasmids pAD1 and pCF10, respectively. No significant similarity to known proteins or DNA sequences could be detected for the two smallest ORFs. However, the location, size, hydrophilicity, and orientation of ORF6 (107 codons) were analogous to those features of the putative genes repC and prgO, which encode stability functions on plasmids pAD1 and pCF10, respectively. The cloned replicon of plasmid pAW63 was stably maintained in Bacillus subtilis and B. thuringiensis and displayed incompatibility with the native pAW63. Hybridization experiments using the cloned replicon as a probe showed that pAW63 has similarity to large plasmids from other B. thuringiensis subsp. kurstaki strains and to a strain of B. thuringiensis subsp. alesti.     

Temperature-sensitive cloning vector for Corynebacterium glutamicum

We constructed a temperature-sensitive form of the Corynebacterium glutamicum ATCC13869 cryptic plasmid, pBL1. The C. glutamicum/Escherichia coli shuttle vector pSFK6, which is composed of pBL1 and the E. coli cloning vector pK1, was mutagenized in vitro by treatment with hydroxylamine, and introduced into C. glutamicum cells. A mutant plasmid, which was stably maintained at 25 degrees C but not at 34 degrees C, was isolated from the cells. Sequencing the plasmid, which was named p48K, revealed four substitutions in the Rep protein coding region. Moreover, site-directed single-nucleotide substitutions showed that a G to A transition at position 2,920, which resulted in a Pro-47 to Ser substitution in the Rep protein, was responsible for its temperature-sensitive replication. Pro-47 is conserved among the Rep proteins of the pIJ101/pJV1 family of plasmids. This temperature-sensitive cloning vector will be useful for disrupting genes in this industrially important bacterium.     

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.     

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 11,066bp 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.     

Sequence analysis of the cryptic plasmid pMG101 from Rhodopseudomonas palustris and construction of stable cloning vectors

A 15-kb cryptic plasmid was obtained from a natural isolate of Rhodopseudomonas palustris. The plasmid, designated pMG101, was able to replicate in R. palustris and in closely related strains of Bradyrhizobium japonicum and phototrophic Bradyrhizobium species. However, it was unable to replicate in the purple nonsulfur bacterium Rhodobacter sphaeroides and in Rhizobium species. The replication region of pMG101 was localized to a 3.0-kb SalI-XhoI fragment, and this fragment was stably maintained in R. palustris for over 100 generations in the absence of selection. The complete nucleotide sequence of this fragment revealed two open reading frames (ORFs), ORF1 and ORF2. The deduced amino acid sequence of ORF1 is similar to sequences of Par proteins, which mediate plasmid stability from certain plasmids, while ORF2 was identified as a putative rep gene, coding for an initiator of plasmid replication, based on homology with the Rep proteins of several other plasmids. The function of these sequences was studied by deletion mapping and gene disruptions of ORF1 and ORF2. pMG101-based Escherichia coli-R. palustris shuttle cloning vectors pMG103 and pMG105 were constructed and were stably maintained in R. palustris growing under nonselective conditions. The ability of plasmid pMG101 to replicate in R. palustris and its close phylogenetic relatives should enable broad application of these vectors within this group of alpha-proteobacteria.     

Structural organization of the Corynebacterium glutamicum plasmid pCG100.

pCG100, a 3 kb cryptic plasmid of Corynebacterium glutamicum ATCC 13058, probably identical with pSR1 from C. glutamicum ATCC 19223, was characterized. The minimum region for autonomous replication was shown to be contained on a 1.9 kb BglII-NcoI fragment; a 380 bp HindIII-SphI fragment can replicate in the presence of the parental plasmid, which presumably provides a trans-acting replication factor. Derivatives of pCG100 are able to replicate in several Corynebacterium, Brevibacterium and Arthrobacter strains. pCG100 is compatible with pBL1, a cryptic plasmid of Brevibacterium lactofermentum. Shuttle plasmid vectors, containing the kanamycin-resistance gene from Tn903 or from Streptococcus faecalis as selectable markers and the AmpR, TetR or lacZ alpha genes for insertional inactivation, were constructed using the minimum replication fragment of pCG100.     

Properties and construction of plasmid pFW213, a shuttle vector with the oral Streptococcus origin of replication

Streptococcus parasanguinis is among the most successful colonizers of the human body. Strain FW213 harbors a 7.0-kb cryptic plasmid, pFW213, with a copy number at 5 to 10 per chromosome. Sequence and functional analyses of pFW213 revealed that the open reading frame (ORF) encoding the replication protein (Rep) is essential for the replication of pFW213, and the putative plasmid addiction system (RelB and RelE) and an ORF (ORF6) with no known function are required for its stability. The minimal replicon of pFW213 contains the rep gene and its 5'-flanking 390-bp region. Within the minimal replicon, an A/T-rich region followed by 5 contiguous 22-bp repeats was located 5' of the ATG of rep. No single-stranded replication intermediates were detected in the derivatives of pFW213, suggesting that pFW213 replicates via the theta replication mechanism. The minimal replicon was unstable in streptococcal hosts without selection, but the stability was greatly enhanced in derivatives containing the intact relBE genes. A Streptococcus-Escherichia coli shuttle vector, pCG1, was constructed with the pFW213 replicon. Plasmid pCG1 features a multiple cloning region and a spectinomycin resistance determinant that is expressed in both Streptococcus spp. and E. coli. Various streptococcal DNA fragments were cloned in pCG1, and the recombinant constructs were stably maintained in the streptococcal hosts. Since pCG1 is compatible with the most widely used streptococcal replicon, pVA380-1, pCG1 will provide a much needed tool allowing the cloning of two genes that work in concert in the same host.     

Characterization of the cryptic plasmid pCC1 from Corynebacterium callunae and its use for vector construction

The complete nucleotide sequence of the cryptic plasmid pCC1 from Corynebacterium callunae (4109 bp) was determined. DNA sequence analysis revealed five open reading frames longer than 200 bp. One of the deduced polypeptides showed homology with the Rep proteins encoded by plasmids of the pIJ101/pJV1 family of plasmids replicating by the rolling-circle (RC) mechanism. Within this plasmid family, the Rep protein of pCC1 showed the highest degree of similarity to the Rep proteins of corynebacterial plasmids pAG3 and pBL1. These data suggest that the plasmid pCC1 replicates by the RC mechanism. The Escherichia coli/Corynebacterium glutamicum shuttle cloning vector pSCCD1, carrying the pCC1 rep gene on the 2.1-kb DNA fragment and the streptomycin/spectinomycin resistance determinant, was constructed. This vector is stably maintained in population of C. glutamicum cells grown in the absence of selection pressure and it is compatible with plasmid vectors based on corynebacterial plasmids pBL1 and pSR1.     

Complete Sequence of Plasmid pMP1 from the Marine Environmental Vibrio vulnificus and Location of Its Replication Origin

A novel cryptic plasmid, pMP1, from an environmental Vibrio vulnificus MP-4 isolated from Mai Po Nature Reserve in Hong Kong, has been characterized. The 7.6-kb plasmid had guanine–cytosine content of 40.03% and encoded four open reading frames (ORFs) with >100 amino acids. The predicted protein of ORF1 contained 478 amino acids showing 29% identity and 50% similarity over 309 amino acids to the integrase of Vibrio cholerae phage VP2. ORF2 encoded a putative protein of 596 amino acids, which were 23% identity and 42% similarity over 455 amino acids to the tail tape measure protein TP901 of Chromohalobacter salexigens phage. ORF3 and ORF4 encoded putative proteins of 103 and 287 amino acids, respectively, but showed no homologies to any known proteins. Further experiments indicated that a 3.2-kb fragment from EcoRI digestion could self-replicate. Analysis indicated that a sequence upstream of ORF4 had the features characteristic of theta-type replicons: AT-rich region, six potential direct repeats (iterons) spaced approximately two DNA helical turn apart (about 23 bp), two copies of 9 bp dnaA boxes, three Dam methylation sites, and five inverted repeats. Complementation experiments confirmed that the protein encoded by ORF4 was required for plasmid replication. We propose that ORF4 encode a new type of Rep protein and pMP1 is a new type of theta plasmid.     

A rolling circle replication initiator protein with a nucleotidyl-transferase activity encoded by the plasmid pGT5 from the hyperthermophilic archaeon Pyrococcus abyssi

The plasmid pGT5 from the hyperthermophilic archaeon Pyrococcus abyssi presents similarities to plasmids from the pC194 family that replicate by the rolling circle mechanism. These plasmids encode a replication initiator protein, which activates the replication origin by nicking one of the two DNA strands. The gene encoding the putative Rep protein of pGT5 (Rep75) has been cloned and overexpressed in Escherichia coli , and the recombinant protein has been purified to homogeneity. Rep75 exhibits a highly thermophilic nicking-closing activity in vitro on single-stranded oligonucleotides containing the putative double-stranded replication origin sequence of pGT5. Gel shift analyses on single-stranded oligonucleotides indicate that Rep75 recognizes the single-stranded DNA region upstream of the nicking site via non-covalent interaction and remains covalently linked to the 5'-phosphate of the downstream fragment after nicking. Besides these expected activities, Rep75 contains a dATP (and ATP) terminal transferase activity at the 3'-OH extremity of the nicking site, which had not been reported previously for proteins of this type. Rep75, which is the first replication initiator protein characterized in an archaeon, offers an attractive new model for the study of rolling circle replication.     

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.     

Characterization of broad host range cryptic plasmid pCR1 from Corynebacterium renale

Plasmid pCR1 is a cryptic plasmid harboured by Corynebacterium renale. It is the smallest corynebacterial plasmid known to date. Although its natural host is animal corynebacteria, it can replicate in several strains of soil corynebacteria. It can also replicate in Escherichia coli, in which it is stably maintained. The copy number of pCR1 in this host is higher than that of pUC19, with which it shows unidirectional incompatibility. It is also incompatible with pBK2, a plasmid bearing the common corynebacterial replicon pBL1. Its size is 1488bp, as revealed by DNA sequencing. A total of eight open reading frames (ORF) were detected in this plasmid, the largest of which codes for a putative Rep protein of predicted molecular mass of 21kDa. The plasmid pCR1 can be mobilized by the plasmid R6K from E. coli to other corynebacteria. Sequence analysis revealed the presence of an oriT homologous to that of R64. An E. coli plasmid pKL1 shows more than 90% identity with pCR1. Like many coryenbacterial plasmids, pCR1 also replicates by rolling circle mode.     

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.