Plasmid replication functions: two distinct segments of plasmid R1, RepA and RepD, express incompatibility and are capable of autonomous replication.

The genetic determinants for replication and incompatibility of plasmid R1 were investigated by gene cloning methods, and three types of R1 miniplasmid derivatives were generated. The first, exemplified by plasmid pKT300, consisted of a single BglII endonuclease-generated deoxyribonucleic acid fragment derived from the R1 region that is located between the determinants for conjugal transfer and antibiotic resistance. Two types of miniplasmids could be formed from PstI endonuclease-generated fragments of pKT300. One of these, which is equivalent to miniplasmids previously generated from plasmids R1-19 and R1-19B2, consisted of two adjacent PstI fragments that encode the RepA replication system of plasmid R1. The other type contained a segment of R1, designated the RepD replication region, that is adjacent to the RepA region and that has not been identified previously as having the capacity for autonomous replication. Plasmid R1, therefore, contained two distinct deoxyribonucleic acid segments capable of autonomous replication. The RepA-RepD miniplasmid pKT300 had a copy number about eightfold higher than that of R1 and hence lacked a determinant for the regulation of plasmid copy number. Like R1, it was maintained stably in dividing bacteria. RepA miniplasmids had copy numbers which were two- to fourfold higher than that of R1 (i.e., which were lower than that of pKT300) and were maintained slightly less stably than those of pKT300 and R1. The RepD miniplasmid was not maintained stably in dividing bacteria. Previous experiments have shown that incompatibility of IncFII group plasmids is specified by a plasmid copy control gene. Despite the fact that RepA miniplasmids of R1 were defective in copy control, they nevertheless expressed incompatibility. This suggests that two genes are responsible for plasmid copy control, one that specifies incompatibility and is located on RepA miniplasmids and another that is located outside of, but adjacent to, the RepA replication region. Hybrid plasmids composed of pBR322 and one PstI fragment from the RepA region, P-8, exhibited incompatibility towards R2 and RepA miniplasmids but not the RepD miniplasmid, whereas hybrids composed of pBR322 and the PstI fragment of the RepD region, P-3, exhibited incompatibility towards R1 and the RepD miniplasmid but not RepA miniplasmids. These results indicate that the two replication systems are functionally distinct and that, although the RepA system is the principal replication system of R1, the RepD system also plays a role in the maintenance of this plasmid.     

Molecular interactions of a replication initiator protein, RepA, with the replication origin of the enterococcal plasmid p703/5

We previously identified the origin of replication of p703/5, a small cryptic plasmid from the KBL703 strain of Enterococcus faecalis. The origin of replication contains putative regulatory cis-elements required for replication and a replication initiator (RepA) gene. The replicon of p703/5 is similar in its structural organization to theta-type plasmids, and RepA is homologous to a family of Rep proteins identified in several plasmids from Gram-positive bacteria. Here, we report molecular interactions between RepA and the replication origin of p703/5. DNase I footprinting using recombinant RepA together with electrophoretic mobility shift assays confirmed the binding of RepA to the replication origin of p703/5 via iterons and an inverted repeat. We also demonstrated the formation of RepA dimers and the different binding of RepA to the iteron and the inverted repeat using gel filtration chromatographic analysis, a chemical crosslinking assay, and electrophoretic mobility shift assays in the presence of guanidine hydrochloride. Our results suggest that RepA plays a regulatory role in the replication of the enterococcal plasmid p703/5 via mechanisms similar to those of typical iteron-carrying theta-type plasmids.     

Two replication determinants of an antibiotic-resistance plasmid, pTB19, from a thermophilic bacillus

Two different replication determinants were found on an antibiotic resistance plasmid, pTB19, from a thermophilic bacillus. One replication determinant (designated RepA) was functional only in Bacillus subtilis, whereas the other (designated RepB) functioned in both B. subtilis and Bacillus stearothermophilus. A deletion plasmid, pTB90, carrying the RepB derived from pTB19 coincidentally contained the specific 1.0 MDal EcoRI fragment of a cryptic plasmid pBSO2 from B. stearothermophilus. The presence of this 1.0 MDal EcoRI fragment in various deletion plasmids from pTB90 increased transformation frequencies for B. stearothermophilus 10(3) to 10(4) times and lowered plasmid copy numbers in the host strain to about one-tenth of those found for plasmids lacking this fragment.     

Excess intracellular concentration of the pSC101 RepA protein interferes with both plasmid DNA replication and partitioning.

RepA, a plasmid-encoded gene product required for pSC101 replication in Escherichia coli, is shown here to inhibit the replication of pSC101 in vivo when overproduced 4- to 20-fold in trans. Unlike plasmids whose replication is prevented by mutations in the repA gene, plasmids prevented from replicating by overproduction of the RepA protein were lost rapidly from the cell population instead of being partitioned evenly between daughter cells. Removal of the partition (par) locus increased the inhibitory effect of excess RepA on replication, while host and plasmid mutations that compensate for the absence of par, or overproduction of the E. coli DnaA protein, diminished it. A repA mutation (repA46) that elevates pSC101 copy number almost entirely eliminated the inhibitory effect of RepA at high concentration and stimulated replication when the protein was moderately overproduced. As the RepA protein can exist in both monomer and dimer forms, we suggest that overproduction promotes RepA dimerization, reducing the formation of replication initiation complexes that require the RepA monomer and DnaA; we propose that the repA46 mutation alters the ability of the mutant protein to dimerize. Our discovery that an elevated intracellular concentration of RepA specifically impedes plasmid partitioning implies that the RepA-containing complexes initiating pSC101 DNA replication participate also in the distribution of plasmids at cell division.     

Analysis of the incompatibility determinants of I-complex plasmids.

The isolation and characterization of minireplicons corresponding to group B and I-complex plasmids are reported. The molecular structures of the small RNAs that may play a major role in the replication control and incompatibility reactions of the plasmids are compared. A mutant plasmid with changed copy number and incompatibility specificity is described. This mutant had a single-base-pair substitution in the DNA region that codes for the small RNA.     

A small plasmid, pCA2.4, from the cyanobacterium Synechocystis sp. strain PCC 6803 encodes a rep protein and replicates by a rolling circle mechanism.

Different cryptic plasmids are widely distributed in many strains of cyanobacteria. A small cryptic plasmid, pCA2.4, from Synechocystis strain PCC 6803 was completely sequenced, and its replication mode was determined. pCA2.4 contained 2,378 bp and encoded a replication (Rep) protein, designated RepA. An analysis of the deduced amino acid sequence revealed that RepA of pCA2.4 has significant homology with Rep proteins of pKYM from Shigella sonnei, a pUB110 plasmid family from gram-positive bacteria, and with a protein corresponding to an open reading frame in a Nostoc plasmid and open reading frame C of Plectonema plasmid pRF1. pKYM and pUB110 family plasmids replicate by a rolling circle mechanism in which a Rep protein nicks the origin of replication to allow the generation of a single-stranded plasmid as a replication intermediate. RepA encoded by pC2.4 was expressed in Escherichia coli cells harboring a vector, pCRP336, containing the entire repA gene. The observed molecular weight of RepA was consistent with the value of 39,200 calculated from its deduced amino acid sequence, as was the N-terminal sequence analysis done through the 12th residue. Single-stranded plasmid DNA of pCA2.4 that was specifically degraded by S1 nuclease was detected in Synechocystis cells by Southern hybridization. These observations suggest that pCA2.4 replicates by a rolling circle mechanism in Synechocystis cells.     

Protein domains and conformational changes in the activation of RepA, a DNA replication initiator.

RepA is the DNA replication initiator protein of the Pseudomonas plasmid pPS10. RepA has a dual function: as a dimer, it binds to an inversely-repeated sequence acting as a repressor of its own synthesis; as a monomer, RepA binds to four directly-repeated sequences to constitute a specialized nucleoprotein complex responsible for the initiation of DNA replication. We have previously shown that a Leucine Zipper-like motif (LZ) at the N-terminus of RepA is responsible for protein dimerization. In this paper we characterize the existence in RepA of two protein globular domains C-terminal to the LZ. We propose that dissociation of RepA dimers into monomers results in a conformational change from a compact arrangement of both domains, competent for binding to the operator, to an extended species that is suited for iteron binding. This model establishes the structural basis for the activation of DNA replication initiators in plasmids from Gram-negative bacteria.     

The RepA protein of plasmid pSC101 controls Escherichia coli cell division through the SOS response

Although plasmid copy number varies widely among different plasmid species, normally copy number is maintained within a narrow range for any given plasmid. Such copy number control has been shown to occur by regulation of the rate of plasmid DNA replication. Here we report a novel mechanism by which the pSC101 plasmid also can detect an imbalance between the cellular level of its replication protein, RepA, and plasmid-borne RepA binding sites to inhibit bacterial DNA replication and delay host cell division when RepA is in relative excess. We show that delayed cell division occurs by RepA-mediated induction of the SOS response and can be reversed by over-expression of the host DNA primase, DnaG. The effects of RepA excess are prevented by introducing a surfeit of RepA binding sites. The mechanism reported here may help to limit variation in plasmid copy number and allow repopulation of cells with plasmids when copy number falls--potentially pre-empting plasmid loss in cultures of dividing cells.     

A single amino acid difference between Rep proteins of P1 and P7 affects plasmid copy number

P1 and P7 are closely related plasmid prophages which are members of the same incompatibility group. We report the complete DNA sequence of the replication region of P7 and compare it to that of P1. The sequence predicts a single amino acid difference between the RepA proteins of these two plasmids, no differences in methylation sites or regions where dnaA protein is expected to bind, and no difference in the spacing of the major features of the two replicons. A P1 replicon with a mutation in repA, the gene that encodes an essential replication protein, is complemented for replication by providing either the P1 RepA protein (RepA1) or the P7 RepA protein (RepA7) in trans. Furthermore, when either of these proteins is supplied in trans, the plasmid copy number of P1 cop mutants drops to that of P1 cop+. However, when RepA7 is supplied, the copy number of P1 cop and P1 cop+ is higher than that when RepA1 is supplied. This indicates that the single amino acid difference between the two versions of the RepA protein plays an important role in determining the plasmid copy number.     

Effects of mutations in the repA gene of plasmid Rts1 on plasmid replication and autorepressor function.

We constructed a system in which wild-type RepA or RepAcop1 protein was supplied in trans in various amounts to coexisting mini-Rts1 plasmids by clones of the repA or repAcop1 gene under the control of the native promoter with or without its operator sequence. RepAcop1 protein which contains a single amino acid substitution (Arg-142 to Lys) within its 288 amino acids could initiate the replication of the mini-Rts1 plasmid efficiently at both 37 and 42 degrees C even if it was supplied in excess. In contrast, excess wild-type RepA inhibited plasmid replication at 37 degrees C but supported replication at 42 degrees C. Therefore, it appears that the initiator activity of RepA is not related to the incompatibility phenotype associated with an excess of RepA protein. An immunoblot analysis revealed that neither RepA nor RepAcop1 synthesis was temperature sensitive and that both were autogenously regulated to a similar extent because of the presence of an operator located immediately upstream of the promoter. Two mutant RepA proteins, each of which contains a 4-amino-acid insertion in the middle of the protein, maintained the autorepressor and incompatibility activities but lost the ori(Rts1)-activating function.     

Isolation of a new broad-host-range IncQ-like plasmid, pTC-F14, from the acidophilic bacterium Acidithiobacillus caldus and analysis of the plasmid replicon

A moderately thermophilic (45 to 50 degrees C), highly acidophilic (pH 1.5 to 2.5), chemolithotrophic Acidithiobacillus caldus strain, f, was isolated from a biooxidation process used to treat nickel ore. Trans-alternating field electrophoresis analysis of total DNA from the A. caldus cells revealed two plasmids of approximately 14 and 45 kb. The 14-kb plasmid, designated pTC-F14, was cloned and shown by replacement of the cloning vector with a kanamycin resistance gene to be capable of autonomous replication in Escherichia coli. Autonomous replication was also demonstrated in Pseudomonas putida and Agrobacterium tumefaciens LBA 4404, which suggested that pTC-F14 is a broad-host-range plasmid. Sequence analysis of the pTC-F14 replicon region revealed five open reading frames and a replicon organization like that of the broad-host-range IncQ plasmids. Three of the open reading frames encoded replication proteins which were most closely related to those of IncQ-like plasmid pTF-FC2 (amino acid sequence identities: RepA, 81%; RepB, 78%; RepC, 74%). However, the two plasmids were fully compatible and pTC-F14 represents a new IncQ-like plasmid replicon. Surprisingly, asymmetrical incompatibility was found with the less closely related IncQ plasmid R300B derivative pKE462 and the IncQ-like plasmid derivative pIE1108. Analysis of the pTC-F14 oriV region revealed five direct repeats consisting of three perfectly conserved 22-bp iterons flanked by iterons of 23 and 21 bp. Plasmid pTC-F14 had a copy number of 12 to 16 copies per chromosome in both E. coli, and A. caldus. The rep gene products of pTC-F14 and pTF-FC2 were unable to functionally complement each other's oriV regions, but replication occurred when the genes for each plasmid's own RepA, RepB, and RepC proteins were provided in trans. Two smaller open reading frames were found between the repB and repA genes of pTC-F14, which encode proteins with high amino acid sequence identity (PasA, 81%; PasB, 72%) to the plasmid addiction system of pTF-FC2. This is the second time a plasmid stability system of this type has been found on an IncQ-like plasmid.     

Characterization and comparative sequence analysis of replication origins from three large Bacillus thuringiensis plasmids.

The replication origins of three large Bacillus thuringiensis plasmids, derived from B. thuringiensis HD263 subsp. kurstaki, have been cloned in Escherichia coli and sequenced. The replication origins, designated ori 43, ori 44, and ori 60, were isolated from plasmids of 43, 44, and 60 MDa, respectively. Each cloned replication origin exhibits incompatibility with the resident B. thuringiensis plasmid from which it was derived. Recombinant plasmids containing the three replication origins varied in their ability to transform strains of B. thuringiensis, Bacillus megaterium, and Bacillus subtilis. Analysis of the derived nucleotide and amino acid sequences indicates that the replication origins are nonhomologous, implying independent derivations. No significant homology was found to published sequences of replication origins derived from the single-stranded DNA plasmids of gram-positive bacteria, and shuttle vectors containing the three replication origins do not appear to generate single-stranded DNA intermediates in B. thuringiensis. The replication origin regions of the large plasmids are each characterized by a single open reading frame whose product is essential for replication in B. thuringiensis. The putative replication protein of ori 60 exhibits partial homology to the RepA protein of the Bacillus stearothermophilus plasmid pTB19. The putative replication protein of ori 43 exhibits weak but extensive homology to the replication proteins of several streptococcal plasmids, including the open reading frame E replication protein of the conjugative plasmid pAM beta 1. The nucleotide sequence of ori 44 and the amino acid sequence of its putative replication protein appear to be nonhomologous to other published replication origin sequences.     

Novel RepA-MCM proteins encoded in plasmids pTAU4, pORA1 and pTIK4 from Sulfolobus neozealandicus

Three plasmids isolated from the crenarchaeal thermoacidophile Sulfolobus neozealandicus were characterized. Plasmids pTAU4 (7,192 bp), pORA1 (9,689 bp) and pTIK4 (13,638 bp) show unusual properties that distinguish them from previously characterized cryptic plasmids of the genus Sulfolobus. Plasmids pORA1 and pTIK4 encode RepA proteins, only the former of which carries the novel polymerase-primase domain of other known Sulfolobus plasmids. Plasmid pTAU4 encodes a mini-chromosome maintenance protein homolog and no RepA protein; the implications for DNA replication are considered. Plasmid pORA1 is the first Sulfolobus plasmid to be characterized that does not encode the otherwise highly conserved DNA-binding PlrA protein. Another encoded protein appears to be specific for the New Zealand plasmids. The three plasmids should provide useful model systems for functional studies of these important crenarchaeal proteins.     

Control of replication in I-complex plasmids

The closely related plasmids that make up the I-complex group and the more distantly related IncL/M plasmids regulate the frequency of initiation of their replication by controlling the efficiency of translation of the rate limiting replication initiator protein, RepA. Translation initiation of repA is dependent on the formation of a pseudoknot immediately upstream of its Shine-Dalgarno sequence. Formation of this pseudoknot involves base pairing between two complementary sequences in the repA mRNA and requires that the secondary structure sequestering the distal sequence be disrupted by movement of the ribosome translating and terminating a leader peptide, whose coding sequence precedes and overlaps that of repA. Expression of repA is controlled by a small antisense RNA, RNAI, which on binding to its complementary target in the repA mRNA not only pre-empts formation of the pseudoknot, but also inhibits translation of the leader peptide. The requirement that translation of the leader peptide be completed for the pseudoknot to form increases the time available for the inhibitory interaction of RNAI with its target, so that at high copy number the frequency of pseudoknot formation is lowered, reducing the proportion of repA mRNA that are translated. At low copy number, when concentration of RNAI is low, repA is translated with increased frequency, leading to increased frequency of plasmid replication.     

Characterization of the replicon of a 51-kb native plasmid from the gram-positive bacterium Leifsonia xyli subsp. cynodontis

The 4992-bp replicon of a large cryptic plasmid in the gram-positive bacterium Leifsonia xyli subsp. cynodontis was identified and sequenced. The replicon encoded two proteins essential for plasmid replication and stability. The putative replication protein (RepA) is homologous to that of the plasmids in mycobacterial pLR7 family, while the putative ParA protein immediately downstream of RepA is significantly homologous to the Walker-type ATPase required for partition of plasmid and chromosome of the gram-positive bacteria. These two proteins and other ORFs are clustered with the putative promoters and other regulatory sequences, illustrating an efficient organization of the replicon for this novel plasmid.     

Hexamerization of RepA from the Escherichia coli plasmid pKL1

The Escherichia coli plasmid pKL1 is one of the smallest bacterial plasmids. It encodes a single, autoregulating structural gene, repA, responsible for replication and copy number control. The oligomerization of RepA was previously proposed as the basis of a strategy for pKL1 copy number control. To elucidate the oligomerization properties of RepA in solution, RepA was expressed in E. coli; purified by ion exchange and hydrophobic chromatography; and examined in solution by spectrapolarimetry, light scattering, sedimentation velocity, and equilibrium ultracentrifugation. RepA behaved as a concentration-dependent equilibrium of dimers and hexamers. Conformational parameters of the RepA hexameric complex were determined. These results support the proposed autogenous regulatory model whereby RepA hexamers negatively regulate repA expression thereby affecting the copy number control of pKL1. RepA of pKL1 is the first plasmid replication initiation protein documented to be in dimeric-hexameric forms.     

Sequence analysis and characterization of plasmids from Streptococcus thermophilus

The nucleotide sequences of eight plasmids isolated from seven Streptococcus thermophilus strains have been determined. Plasmids pSt04, pER1-1, and pJ34 are related and replicate via a rolling circle mechanism. Plasmid pJ34 encodes for a replication initiation protein (RepA) and a small polypeptide with unknown function. Plasmids pSt04 and pER1-1 carry in addition to repA genes coding for small heat shock proteins (sHsp). Expression of these proteins is induced at elevated temperatures or low pH and increases the thermo- and acid resistance. Plasmids pER1-2 and pSt22-2 show identical sequences with five putative open reading frames (ORFs). The gene products of ORF1 and ORF4 reveal some similarities to transposon encoded proteins of Bacillus subtilis and Tn916. ORF1 of plasmid pSt106 encodes a protein similar to resolvases of different Gram-positive bacteria. Integrity of ORF2 and 3, encoding a putative DNA primase and a replication protein, is essential for replication. ORF1 to 3 of plasmid pSt08, which are organized in a tricistronic operon, encode a RepA protein, an adenosine-specific methyltransferase, and a type II restriction endonuclease. Another type II restriction-modification (R/M) system is encoded on plasmid pSt0 which is highly similar to those encoded on lactococcal plasmid pHW393 and B. subtilis plasmid pXH13. Plasmid-free derivatives of strains St0 and St08 show increased phage sensitivity, indicating that in the wild-type strains the R/M systems are functionally expressed. Recombinant plasmids based on the replicons of plasmids pSt04, pJ34, pSt106, pSt08, and pSt0, are able to replicate in Lactococcus lactis and B. subtilis, respectively, whereas constructs carrying pER1-2 only replicate in S. thermophilus.