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.     

Replication Protein RepN Encoded by the RC Plasmid of Thermophilic Bacterium Thermoanaerobacterium saccharolyticum: Mutation Analysis and Deletion Mapping of Domains Responsible for Its Lethal Effect

Amino acid sequence analysis of the product encoded by repN of Thermoanaerobacterium saccharolyticum (Clostridium thermosaccharolyticum) pNB2, which is capable of rolling-circle (RC) replication, revealed all known motifs conserved among replication (Rep) proteins that initiate RC replication of plasmids related to pC194/pUB110. Using the T7 expression system in Escherichia coli, RepN was identified as a 35K protein. Its lethal effect on bacterial cells was unusually high for a protein of the kind. Mutation analysis of the potential active centers (Y85F and Y211F) showed that the lethal effect of RepN is not associated with its putative topoisomerase (relaxase) activity. On evidence of deletion mapping, the lethal effect was attributed to the N- and C-terminal domains, each accounting for about 30% of the total protein. The RepN fragments essential for the lethal effect were found to share a motif without appreciable homology to known conserved motifs. The high lethal effect of RepN was assumed to result from duplication of the motif and to play an adaptive role, providing for stable maintenance of the AT-rich plasmid in thermophilic bacterial cells.     

Replication protein RepN encoded by the RC plasmid of thermophilic bacterium Thermoanaerobacterium saccharolyticum: mutational analysis and deletion mapping of domains responsible for its lethal effect

Amino acid sequence analysis of the product encoded by repN of Thermoanaerobacterium saccharolyticum (Clostridium thermosaccharolyticum) pNB2, which is capable of rolling-circle (RC) replication, revealed all known motifs conserved among replication (Rep) proteins that initiate RC replication of plasmids related to pC194/pUB110. Using the T7 expression system in Escherichia coli, RepN was identified as a 35K protein. Its lethal effect on bacterial cells was unusually high for a protein of the kind. Mutation analysis of the potential active centers (Y85F and Y211F) showed that the lethal effect of RepN is not associated with its putative topoisomerase (relaxase) activity. On evidence of deletion mapping, the lethal effect was attributed to the N- and C-terminal domains, each accounting for about 30% of the total protein. The RepN fragments essential for the lethal effect were found to share a motif, which showed no appreciable homology to known conserved motifs. The high lethal effect of RepN was assumed to result from duplication of the motif and to play an adaptive role, providing for the stable maintenance of the AT-rich plasmid in thermophilic bacterial cells.     

Molecular analysis of pDL10 from Acidianus ambivalens reveals a family of related plasmids from extremely thermophilic and acidophilic archaea.

The 7598-bp plasmid pDL10 from the extremely thermophilic, acidophilic, and chemolithoautotrophic Archaeon Acidianus ambivalens was sequenced. It contains 10 open reading frames (ORFs) organized in five putative operons. The deduced amino acid sequence of the largest ORF (909 aa) showed similarity to bacterial Rep proteins known from phages and plasmids with rolling-circle (RC) replication. From the comparison of the amino acid sequences, a novel family of RC Rep proteins was defined. The pDL10 Rep protein shared 45-80% identical residues with homologous protein genes encoded by the Sulfolobus islandicus plasmids pRN1 and pRN2. Two DNA regions capable of forming extended stem-loop structures were also conserved in the three plasmids (48-69% sequence identity). In addition, a putative plasmid regulatory protein gene (plrA) was found, which was conserved among the three plasmids and the conjugative Sulfolobus plasmid pNOB8. A homolog of this gene was also found in the chromosome of S. solfataricus. Single-stranded DNA of both pDL10 strands was detected with a mung bean nuclease protection assay using PCR detection of protected fragments, giving additional evidence for an RC mechanism of replication.     

Specificity of the interactions between the Rep proteins and the origins of replication of Staphylococcus aureus plasmids pT181 and pC221

Summary pT181 and pC221 are closely relatedStaphylococcus aureus plasmids with the same genome organization, which is characterized by the overlapping of the origin of replication with the sequence encoding a protein, Rep, essential for plasmid replication. Former results have shown the lack of in vivo cross-complementation between these two plasmids, while in vitro studies have revealed the ability of both Rep proteins to act on either origin. One possible explanation for this difference was based on a previous analysis of the incompatibility expressed by the origin of replication of these plasmids, showing that the origin embedded in therep gene competes for Rep utilization with the origin of a test plasmid and that changes in the sequence of the origin reduce its ability to compete. To avoid this problem, in the present work special hybrids were constructed in which the origin of replication overlapping therep gene was mutationally inactivated, without changing the amino acid sequence of the encoded protein. The level of Rep expression by these hybrids could be varied by taking advantage of what is presently known about the control of Rep synthesis in plasmid pT181. The results of complenentation studies conducted using these hybrids have shown that: (i) at the usual level of expression for a wild-type plasmid each Rep protein can initiate replication strictly from its corresponding origin; (ii) when overproduced, the pT181 RepC protein could also act efficiently on the pC221 origin; a functional pT181 origin present in the same host completely prevented this complementation; (iii) in excess, the RepD protein encoded by pC221 could replicate a plasmid carrying the pT181 origin but could not ensure the hereditary stability of such a plasmid in the absence of another active replication system; (iv) when overproduced both RepC and RepD could act on the origin of replication of three other related plasmids pS194, pC223 and pUB112.     

DNA sequence analysis of a putative primary replicon from a cryptic plasmid pNM214 of a hydrocarbon utilizing marine <Emphasis Type="Italic">Bacillus</Emphasis> 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.     

Lactobacillus hilgardii plasmid pLAB1000 consists of two functional cassettes commonly found in other gram-positive organisms.

A Lactobacillus hilgardii plasmid, pLAB1000, was studied to understand the organization of autonomous replicons from lactobacilli. Two cassettes could be identified. First, the replication region consisted of a sequence coding for a replication protein (Rep) and its corresponding target site, similar to those from plasmids pUB110, pC194 (Staphylococcus aureus), pFTB14, pBAA1 (Bacillus sp.), and pLP1 (Lactobacillus sp.). Sequence analysis indicated the possible synthesis of an antisense RNA that might regulate Rep production. The results also suggested that pLAB1000 replicates via a single-stranded DNA intermediate, and a putative lagging-strand initiation site was found that had similarities to those of alpha 3, St-1, and G4 isometric bacteriophages. The second cassette of pLAB1000 consisted of a sequence coding for a putative mobilization protein (Mob) and its corresponding RSA site. This cassette was similar to those found in pT181, pUB110, pE194 (S. aureus), and pG12 (Bacillus sp.), and it was found to be conserved among different Lactobacillus plasmid replicons. The origin and evolution of these functional cassettes are also discussed.     

Characterization of a novel Streptococcus thermophilus rolling-circle plasmid used for vector construction

The complete nucleotide sequence of pER371, a native plasmid in Streptococcus thermophilus ST137, was determined. A putative open reading frame coding for a replication protein, Rep371, was identified. A characteristic promoter sequence and ribosome-binding site were found upstream of rep371. Rep371 (247 amino acid residues) does not show homology with RepA and RepS of the small S. thermophilus cryptic plasmids pST1-No.29 and pST1 respectively. The plus-origin sequence and Rep371 are highly homologous to the corresponding elements of the Staphylococcus aureus plasmids pC194 and pSK89. A novel 140-nucleotide palindromic minus-origin sequence, which is structurally similar but does not show sequence homology to the palA region of pC194, was identified in pER371. A palindromic sequence capable of forming a putative hairpin structure was identified and subsequently recognized as being highly conserved among several lactococcal rolling-circle plasmids. Cloning vectors derived from pER371 should provide valuable gene-delivery vehicles for the genetic engineering of lactic acid bacteria. Received: 25 November 1997 / Received last revision: 13 April 1998 / Accepted: 19 April 1998     

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.     

Molecular analysis of the Lactococcus lactis subspecies lactis CNRZ270 bidirectional theta replicating lactose plasmid pUCL22

pUCL22 is the lactose protease plasmid of Lactococcus lactis ssp. lactis CNRZ270. The nucleotide sequence of its replication region Rep22 contains a non-transcribed region, the replication origin, followed by a gene encoding a putative 388-amino-acid protein named Rep22A. The promoter regions of the rep22A and pC194 cat genes share strong similarities and the pUCL22 replicon exerted trans or cis negative control on the pC194 cat gene expression in L. lactis. We suggest that Rep22A binds to its own promoter as well as to the pC194 cat promoter and thus is autoregulated. We show that pUCL22 replicates mainly by a bidirectional theta mechanism in L. lactis, and is representative of a widely distributed replicon family, members of which could be co-resident. We propose that compatibility between these closely related replicons results from minor replication protein modifications coupled with base changes in their respective binding sites, supporting the co-existence of numerous related replicons in lactococcal strains.     

Active site of the replication protein of the rolling circle plasmid pC194.

Mutation analysis of the rolling circle (RC) replication initiator protein RepA of plasmid pC194 was targeted to tyrosine and acidic amino acids (glutamate and aspartate) which are well conserved among numerous related plasmids. The effect of mutations was examined by an in vivo activity test. Mutations of one tyrosine and two glutamate residues were found to greatly impair or abolish activity, without affecting affinity for the origin, as deduced from in vitro gel mobility assays. We conclude that all three amino acids have a catalytic role. Tyrosine residues were found previously in active sites of different RC plasmid Rep proteins and topoisomerases, but not in association with acidic residues, which are a hallmark of the active sites of DNA hydrolyzing enzymes, such as the exo- and endonucleases. We propose that the active site of RepA contains two different catalytic centers, corresponding to a tyrosine and a glutamate. The former may be involved in the formation of the covalent DNA-protein intermediate at the initiation step of RC replication, and the latter may catalyze the release of the protein from the intermediate at the termination step.     

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.     

Structural relationships among chloramphenicol-resistance plasmids of Staphylococcus aureus

Abstract Twelve different chloramphenicol-resistance (Cm^r) plasmids detected in Staphylococcus aureus strains isolated between 1952 and 1981 were characterized by restriction endonuclease, DNA hybridization and heteroduplex analyses. These studies revealed three families of Cm^r plasmids which were distinguished by their chloramphenicol acetyltransferase sequences; the prototype plasmids of the families were pC221, pC223 and pC194. The cat and replication ( rep ) genes of the plasmid pC221 were highly conserved in other pC221 family members and were related to their homologs in the pC223 family plasmids; however, the cat and rep genes of the pC194 family plasmids were distinct.     

The active site of the rolling circle replication protein Rep75 is involved in site-specific nuclease, ligase and nucleotidyl transferase activities

The plasmid pGT5 from the hyperthermophilic archaeon Pyrococcus abyssi replicates via a rolling circle mechanism. The protein Rep75, encoded by this plasmid, exhibits a nicking-closing (NC) activity in vitro on single-stranded oligonucleotides containing the pGT5 double-stranded origin sequence. In addition, Rep75 catalyses a site-specific nucleotidyl terminal transferase (NTT) activity, e.g. it can transfer one AMP or dAMP (from ATP or dATP) to the 3'-OH of an oligonucleotide corresponding to the left part of the nicking site. The Rep75 sequence contains a motif similar to the active-site motifs of Rep proteins from the PhiX174/pC194 superfamily. We show here that the tyrosine present in this motif is indeed essential for DNA cleavage by Rep75, but is dispensable for its NTT activity. However, a nearby arginine, which is not required for DNA cleavage, is involved in both NTT and closing, indicating that the same active site is involved in the NC and NTT activities of Rep75. For both NTT and NC, the G residue in 3' of the nicking site is essential, whereas the A residue in 5' is dispensable for NC, despite its conservation in RC plasmids of the PhiX174/pC194 superfamily. The NTT and closing activities have an optimal temperature lower than the nicking activity. These data indicate that the three reactions catalysed by Rep75 can be uncoupled, although they share part of their mechanisms. Finally, we show that NC is inhibited by ATP or dATP at concentrations that promote NTT. We propose a model in which the NTT activity of Rep75 plays a role in the regulation of pGT5 replication in vivo.     

Characterization of the replication region of the Lactobacillus reuteri plasmid pTC82 potentially used in the construction of cloning vector

A 3.2 kb DNA fragment containing the replication region (RR) from pTC82 was cloned, sequenced, and found to contain elements typical of plasmids that replicate via a rolling-circle mechanism of replication (RCR), including double-strand origin (DSO), replication protein gene (rep), and single-strand origin (SSO). The DSO of pTC82 contains two domains showing 55.5% and 84.6% similarities in nucleotide (nt) sequence to the conserved functional elements bind and nic, respectively, which are required for the initiation of the leading strand typical of the pC194-RCR family. Although the predicted rep gene product of pTC82 (Rep82) shares little identity (less than 24%) with other known Reps, a region containing three motifs, characteristic of the pC194-family Reps, was identified, indicating the Rep82 as a novel Rep protein of this family. Downstream of the rep82 gene, strong similarity to the typical palT type-SSO could be detected. This is the first palT type-SSO to be identified from Lactobacillus. Through a series of deletion studies, the minimal replicon of the cloned RR was found to be 2.66 kb in size including the DSO region and rep gene. This RR was further identified as being highly stable in L. reuteri and also bearing a very narrow host-range property, suggesting it to be a good replicon potentially useful in vector construction for developing L. reuteri as a vaccine carrier.     

Characterization of the Replication Region of the Lactobacillus reuteri Plasmid pTC82 Potentially Used in the Construction of Cloning Vector

A 3.2 kb DNA fragment containing the replication region (RR) from pTC82 was cloned, sequenced, and found to contain elements typical of plasmids that replicate via a rolling-circle mechanism of replication (RCR), including double-strand origin (DSO), replication protein gene (rep), and single-strand origin (SSO). The DSO of pTC82 contains two domains showing 55.5% and 84.6% similarities in nucleotide (nt) sequence to the conserved functional elements bind and nic, respectively, which are required for the initiation of the leading strand typical of the pC194-RCR family. Although the predicted rep gene product of pTC82 (Rep82) shares little identity (less than 24%) with other known Reps, a region containing three motifs, characteristic of the pC194-family Reps, was identified, indicating the Rep82 as a novel Rep protein of this family. Downstream of the rep82 gene, strong similarity to the typical palT type-SSO could be detected. This is the first palT type-SSO to be identified from Lactobacillus. Through a series of deletion studies, the minimal replicon of the cloned RR was found to be 2.66 kb in size including the DSO region and rep gene. This RR was further identified as being highly stable in L. reuteri and also bearing a very narrow host-range property, suggesting it to be a good replicon potentially useful in vector construction for developing L. reuteri as a vaccine carrier.     

Characterization of a cryptic plasmid pM4 from Lactobacillus plantarum M4

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.     

Identification and Characterization of a Shuttle Plasmid with Antibiotic Resistance Gene from <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

While studying antibiotic-resistant plasmids from multi-drug-resistant nosocomial Staphylococcus aureus strains, we isolated a small (2.889 kb) chloramphenicol-resistant (Cm^r) plasmid, which was designated as pMC524/MBM. The molecular size of pMC524/MBM was close to that of pC194 (2.910 kb), a well-known Cm^r staphylococcal plasmid. Unlike pC194, this plasmid can replicate and express itself efficiently and stably in Escherichia coli. However, Cm is needed for stable maintenance of pMC524/MBM in different hosts. In this study, the nucleotide sequences of these two plasmids were compared after sequencing of pMC524/MBM [EMBL Accession No. AJ312056 SAU312056]. Although these two plasmids have striking nucleotide sequence homology, the Plus Origin, Minus Origin, the replication protein (Rep), and the chloramphenicol acetyl transferase (Cat) have considerable variations. Possibly, these changes have modulated pMC524/MBM into an efficient shuttle-plasmid. Received: 8 April 2002 / Accepted: 27 July 2002     

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.