The region essential for efficient autonomous replication of pSa in Escherichia coli

Summary Comparative analyses were made between plasmid pSa17, a deletion derivative of pSa that is capable of replicating efficiently in Escherichia coli and plasmid pSa3, a derivative that is defective for replication. By comparing the restriction maps of these two derivatives, the regions essential for replication and for stable maintenance of the plasmid were determined. A 2.5 kb DNA segment bearing the origin of DNA replication of pSa17 was sequenced. A 36 kDa RepA protein was encoded in the region essential for replication. Downstream of the RepA coding region was a characteristic sequence including six 17 bp direct repeats, the possible binding sites of RepA protein, followed by AT-rich and GC-rich sequences. Furthermore, an 8 bp incomplete copy of the 17 bp repeat was found in the promoter region of the repA gene. Based on the hypothesis that RepA protein binds to this partial sequence as well as to intact 17 bp sequences, an autoregulatory system for the synthesis of RepA protein may be operative. Another open reading frame (ORF) was found in the region required for the stability of the plasmid. The putative protein encoded in this ORF showed significant homology to several site-specific recombination proteins. A possible role of this putative protein in stable maintenance of the plasmid is discussed.     

Isolation and sequencing of the replication region of plasmid pBFp1 isolated from a marine biofilm

A 24 kb plasmid, pBFp1, encoding mercury resistance was previously isolated from a marine biofilm. Isolation and sequencing of a 4280 bp DNA fragment containing the plasmid replicon (rep-pBFp1) revealed a putative open reading frame encoding a RepA protein and an oriV-like region containing an A+T rich sub-region, iterons, and DnaA boxes. Sequence comparisons showed significant similarities to the incW plasmid pSa both for the RepA amino acid sequence and in the iteron DNA sequence. Plasmid pBFp1 was also shown to be incompatible with pSa in standard incompatibility testing. A probe from the repA gene of pBFp1 was further made and tested on a collection of plasmids exogenously isolated from marine habitats in a previous study.     

Boundaries of the pSC101 minimal replicon are conditional.

The DNA segment essential for plasmid replication commonly is referred to as the core or minimal replicon. We report here that host and plasmid genes and sites external to the core replicon of plasmid pSC101 determine the boundaries and competence of the replicon and also the efficiency of partitioning. Missense mutations in the plasmid-encoded RepA protein or mutation of the Escherichia coli topoisomerase I gene enable autonomous replication of a 310-bp pSC101 DNA fragment that contains only the actual replication origin plus binding sites for RepA and the host-encoded DnaA protein. However, in the absence of a repA or topA mutation, the DNA-bending protein integration host factor (IHF) and either of two cis-acting elements are required. One of these, the partitioning (par) locus, is known to promote negative DNA supercoiling; our data suggest that the effects of the other element, the inverted repeat (IR) sequences that overlap the repA promoter, are mediated through the IR's ability to bind RepA. The concentrations of RepA and DnaA, which interact with each other and with plasmid DNA in the origin region (T. T. Stenzel, T. MacAllister, and D. Bastia, Genes Dev. 5:1453-1463, 1991), also affect both replication and partitioning. Our results, which indicate that the sequence requirements for replication of pSC101 are conditional rather than absolute, compel reassessment of the definition of a core replicon. Additionally, they provide further evidence that the origin region RepA-DnaA-DNA complex initiating replication of pSC101 also mediates the partitioning of pSC101 plasmids at cell division.     

Nucleotide sequence from the genetic left end of bacteriophage T7 DNA to the beginning of gene 4

The nucleotide sequence running from the genetic left end of bacteriophage T7 DNA to within the coding sequence of gene 4 is given, except for the internal coding sequence for the gene 1 protein, which has been determined elsewhere. The sequence presented contains nucleotides 1 to 3342 and 5654 to 12,100 of the approximately 40,000 base-pairs of T7 DNA. This sequence includes: the three strong early promoters and the termination site for Escherichia coli RNA polymerase: eight promoter sites for T7 RNA polymerase; six RNAase III cleavage sites; the primary origin of replication of T7 DNA; the complete coding sequences for 13 previously known T7 proteins, including the anti-restriction protein, protein kinase, DNA ligase, the gene 2 inhibitor of E. coli RNA polymerase, single-strand DNA binding protein, the gene 3 endonuclease, and lysozyme (which is actually an N-acetylmuramyl-l-alanine amidase); the complete coding sequences for eight potential new T7-coded proteins; and two apparently independent initiation sites that produce overlapping polypeptide chains of gene 4 primase. More than 86% of the first 12,100 base-pairs of T7 DNA appear to be devoted to specifying amino acid sequences for T7 proteins, and the arrangement of coding sequences and other genetic elements is very efficient. There is little overlap between coding sequences for different proteins, but junctions between adjacent coding sequences are typically close, the termination codon for one protein often overlapping the initiation codon for the next. For almost half of the potential T7 proteins, the sequence in the messenger RNA that can interact with 16 S ribosomal RNA in initiation of protein synthesis is part of the coding sequence for the preceding protein. The longest non-coding region, about 900 base-pairs, is at the left end of the DNA. The right half of this region contains the strong early promoters for E. coli RNA polymerase and the first RNAase III cleavage site. The left end contains the terminal repetition (nucleotides 1 to 160), followed by a striking array of repeated sequences (nucleotides 175 to 340) that might have some role in packaging the DNA into phage particles, and an A · T-rich region (nucleotides 356 to 492) that contains a promoter for T7 RNA polymerase, and which might function as a replication origin.     

Genes and sites involved in replication and incompatibility of an R100 plasmid derivative based on nucleotide sequence analysis

Summary The nucleotide sequence of the entire region required for autonomous replication and incompatibility of an R100 plasmid derivative, pSM1, has been determined. This region includes the replication region and all plasmid encoded information required for replication. Numerous reading frames for possible proteins can be found in this region. The existence of one of these proteins called RepA1 (285 amino acids; 33,000 daltons) which is encoded within the region known by cloning analysis to be required for replication is supported by several lines of evidence. These include an examination of the characteristic sequences on the proximal and distal ends of the coding region, a comparison of the sequence of the replication regions of pSM1 and the highly related R1 plasmid derivative Rsc13 as well as other biochemical and genetic evidence. The existence of two other proteins, RepA3 (64 amino acids; 7000 daltons) and RepA2 (103 amino acids; 11,400 daltons) is also consistent with most of the criteria mentioned above. However, the region encoding RepA3, which by cloning analysis is within the region responsible for both replication and incompatibility, has never been demonstrated to produce a 7,000 dalton polypeptide. Since a large secondary structure can be constructed in this region, it is possible that the region contains structure or other information that is responsible for incompatibility. RepA2, encoded entirely within the region identified by cloning analysis to be responsible for incompatibility but not for replication can be visualized in vivo and in vitro. However, the nucleotide sequence of the region encoding RepA2 is completely different in mutually incompatible plasmid derivatives of R1 and R100. It is therefore unlikely that RepA2 plays a major role in incompatibility. Thus, we predict that RepA1 is required to initiate DNA synthesis at the replication origin and that the region proximal to RepA1 either encodes a gene product or structure information that is responsible for incompatibility.     

Molecular analysis of the replication region of the theta-replicating plasmid pUCL287 from Tetragenococcus (Pediococcus) halophilus ATCC33315

The complete nucleotide sequence of the 8.7-kb theta-replicating plasmid pUCL287 from Tetragenococcus halophilus (formerly Pediococcus halophilus) ATCC33315 has been determined. The replication region was identified and analyzed. Its nucleotide sequence contains an untranslated region, the replication origin, followed by two open reading frames (ORFs) encoding two proteins of 311 (RepA287) and 168 (RepB287) amino acids, respectively. Evidence is presented to show that RepA287 represents the plasmid replication protein. RepB287, which is non-essential for replication, is involved in the plasmid copy-number control and segregational stability. The roles of lactococcal proteins homologous to RepB287 have not been defined so far. Nevertheless, the structural organization of the pUCL287 replication region is remarkably similar to those of well known theta-replicating lactococcal plasmids despite the absence of homology of the replication origin and of the replication protein, and this suggests that pUCL287 uses the same mechanism of replication. Nucleotide sequence comparisons show that pSMB74, a pediococcal plasmid encoding bacteriocin production, is a member of the pUCL287 replicon family.     

Cloning of a functional replication origin of phage G4 into the genome of phage M13.

A chimeric single-stranded DNA phage, M13Gori1, has been formed as a result of the in vitro insertion of a 2216 base-pair HaeII fragment of bacteriophage G4 replicative form DNA into the replicative form DNA of bacteriophage M13. The inserted G4 DNA carries the dnaG-dependent origin for G4 complementary strand synthesis. The cloned G4 origin functions both in vivo and in vitro in the conversion of M13Gori1 single-stranded viral DNA to the duplex replicative form by a rifampicin-resistant mechanism. Labelling of the 3′ terminus of the single discontinuity in M13Gori1 replicative form II molecules synthesized in crude extracts and subsequent restriction analysis indicate that M13Gori1 complementary strand synthesis can be initiated at either the RNA polymeraseprimed M13 origin or at the dnaG-primed G4 origin. The M13Gori1 complementary strand initiated at the G4 origin terminates in the vicinity of the G4 origin after progressing around the circular template and traversing the M13 origin region, indicating the absence of a specific nucleotide sequence in the M13 origin for termination of the newly formed complementary strand. The ability of this chimeric phage to utilize the cloned G4 origin in vivo even in the presence of the presumed M13 pilot protein (gene 3 protein) indicate that the nucleotide sequence of the replication origin is sufficient for recognizing the appropriate initiation enzymes. Since decapsidation of M13 is tightly coupled to replicative form formation, initiation at the G4 origin, located over 1000 nucleotides from the M13 complementary strand origin, indicates that widely separated nucleotide sequences contained in the filamentous virion can be exposed to the cell cytoplasm during eclipse.     

Induced bending of plasmid pLS1 DNA by the plasmid-encoded protein RepA

The broad host range streptococcal plasmid pLS1 encodes for a 5.1-kDa repressor protein, RepA. This protein has affinity for DNA (linear or supercoiled) and is translated from the same mRNA as the replication initiator protein RepB. By gel retardation assays, we observed that RepA shows specificity for binding to the plasmid HinfID fragment, which includes the target of the protein. The target of RepA within the plasmid DNA molecule has been located around the plasmid single site ApaLI. This site is included in a region that contains the promoter for the repA and repB genes and is contiguous to the plasmid ori(+). A complex sequence-directed DNA curvature is observed in this region of pLS1. Upon addition of RepA to plasmid linear DNA or to circularly permuted restriction fragments, this intrinsic curvature was greatly enhanced. Thus, a strong RepA-induced bending could be located in the vicinity of the ApaLI site. Visualization of the bent DNA was achieved by electron microscopy of complexes between RepA and plasmid DNA fragments containing the RepA target.     

Deletion analysis of the mini-P1 plasmid origin of replication and the role of Escherichia coli DnaA protein

The mini-P1 plasmid origin of replication is contained on a 246 base pair (bp) piece of DNA. At one end there are five 19-bp binding sites for the P1 initiator protein, RepA, and near the other end there are two 9-bp DnaA protein-binding sites. To further define the limits of the origin, we cloned the origin region in M13 and constructed deletions of either end. We sequenced the DNA and tested the replicative form I DNA of the deletion phages for their ability to support RepA-dependent DNA replication in an in vitro system. The origin that is functional in vitro could be reduced to 202 bp. It includes three intact and one incomplete RepA-binding sites at one end and the two DnaA-binding sites at the other end. When the two naturally occurring DnaA-binding sites were replaced with one or two synthetic sites, only the construction containing two sites was active in vitro. We found that the minimal origin that is functional in vivo contains all of the five RepA and the two DnaA-binding sites. Mini-P1 plasmid replication both in vivo and in vitro requires two initiator proteins, the Escherichia coli DnaA protein and the P1 RepA protein. We have found that the ADP form of DnaA is as active as the ATP form of the protein in the in vitro replication of mini-P1. In contrast, only the ATP form is active for in vitro replication of plasmids carrying the E. coli origin (Bramhill, D., and Kornberg, A. (1988) Cell 52, 743-755).     

Genetic and functional analysis of the basic replicon of pPS10, a plasmid specific for Pseudomonas isolated from Pseudomonas syringae patovar savastanoi.

The sequence of a 1823 base-pair region containing the replication functions of pPS10, a narrow host-range plasmid isolated from a strain of Pseudomonas savastanoi, is reported. The origin of replication, oriV, or pPS10 is contained in a 535 base-pair fragment of this sequence that can replicate in the presence of trans-acting function(s) of the plasmid. oriV contains four iterons of 22 base-pairs that are preceded by G+C-rich and A+T-rich regions. A dnaA box located adjacent to the repeats of the origin is dispensable but required for efficient replication of pPS10; A and T are equivalent bases at the 5' end of the box. repA, the gene of a trans-acting replication protein of 26,700 Mr has been identified by genetic and functional analysis. repA is adjacent to the origin of replication and is preceded by the consensus sequences of a typical sigma 70 promoter of Escherichia coli. The RepA protein has been identified, using the minicell system of E. coli, as a polypeptide with an apparent molecular mass of 26,000. A minimal pPS10 replicon has been defined to a continuous 1267 base-pair region of pPS10 that includes the oriV and repA sequences.     

Functional analysis of ori1 and repA of the R-plasmid pSJ5.6 from Neisseria gonorrhoeae

The functional ori1 of the 5.6kb gonococcal R-plasmid pSJ5.6 contains an A-T rich region followed by four 22bp direct repeats and one 19bp inverted repeat. The replication region of the plasmid also contains a gene encoding for a 39kD RepA protein. We have further assessed the functionality of the replication region in pSJ5.6, an-iteron type plasmid, using in vivo complementation assays in Escherichia coli. A 2.1kb PstI-RsaI fragment containing the ori1 and repA gene of pSJ5.6 was cloned into vector pZErO -2 to obtain pZA-MRR. The pUC origin in pZA-MRR was deleted to render the plasmid dependable on the cis-acting ori1 for replication. The resulting plasmid, pMRR, was capable of replication and maintenance in E. coli. We also cloned the ori1 and repA gene separately to obtain pA-Ori and pZG-Rep, respectively. Using in vivo complementation assays, we demonstrated that the ori1(+) plasmid (pA-Ori) was maintained only when the RepA protein was supplied in trans by the high copy number plasmid pZG-Rep.     

Role of RepA and DnaA proteins in the opening of the origin of DNA replication of an IncB plasmid

The replication initiator protein RepA of the IncB plasmid pMU720 was shown to induce localized unwinding of its cognate origin of replication in vitro. DnaA, the initiator protein of Escherichia coli, was unable to induce localized unwinding of this origin of replication on its own but enhanced the opening generated by RepA. The opened region lies immediately downstream of the last of the three binding sites for RepA (RepA boxes) and covers one turn of DNA helix. A 6-mer sequence, 5'-TCTTAA-3', which lies within the opened region, was essential for the localized unwinding of the origin in vitro and origin activity in vivo. In addition, efficient unwinding of the origin of replication of pMU720 in vitro required the native positioning of the binding sites for the initiator proteins. Interestingly, binding of RepA to RepA box 1, which is essential for origin activity, was not required for the localized opening of the origin in vitro.     

Interaction of the initiator protein of an IncB plasmid with its origin of DNA replication

The replication initiator protein RepA of the IncB plasmid pMU720 was purified and used in DNase I protection assays in vitro. RepA protected a 68-bp region of the origin of replication of pMU720. This region, which lies immediately downstream of the DnaA box, contains four copies of the sequence motif 5'AANCNGCAA3'. Mutational analyses identified this sequence as the binding site specifically recognized by RepA (the RepA box). Binding of RepA to the RepA boxes was ordered and sequential, with the box closest to the DnaA binding site (box 1) occupied first and the most distant boxes (boxes 3 and 4) occupied last. However, only boxes 1, 2, and 4 were essential for origin activity, with box 3 playing a lesser role. Changing the spacing between box 1 and the other three boxes affected binding of RepA in vitro and origin activity in vivo, indicating that the RepA molecules bound to ori(B) interact with one another.