Role of DNA superhelicity in partitioning of the pSC101 plasmid

Previous work has shown that a cis-acting locus (termed par for partitioning) on the pSC101 plasmid accomplishes its stable inheritance in dividing cell populations. We report here that the DNA of pSC101 derivatives lacking the par region shows a decrease in overall superhelical density as compared with DNA of wild-type pSC101. Chemicals and bacterial mutations that reduce negative DNA supercoiling increase the rate of loss of par plasmids and convert normally stable plasmids that have minimal par region deletions into unstable replicons. topA gene mutations, which increase negative DNA supercoiling, reverse the instability of partition-defective plasmids that utilize the pSC101, p15A, F, or oriC replication systems. Our observations show that the extent of negative supercoiling of plasmid DNA has major effects on the plasmid's inheritance and suggest a mechanism by which the pSC101 par region may exert its stabilizing effects.     

The replication of plasmid pSC101

The origin of replication of plasmid pSC101 presents features reminiscent of those found in a number of plasmids. As for those plasmids, many details about the way it initiates its replication are beginning to be known, but the regulation of this process will not be easily understood.     

Separate roles of Escherichia coli replication proteins in synthesis and partitioning of pSC101 plasmid DNA

We report here that the Escherichia coli replication proteins DnaA, which is required to initiate replication of both the chromosome and plasmid pSC101, and DnaB, the helicase that unwinds strands during DNA replication, have effects on plasmid partitioning that are distinct from their functions in promoting plasmid DNA replication. Temperature-sensitive dnaB mutants cultured under conditions permissive for DNA replication failed to partition plasmids normally, and when cultured under conditions that prevent replication, they showed loss of the entire multicopy pool of plasmid replicons from half of the bacterial population during a single cell division. As was observed previously for DnaA, overexpression of the wild-type DnaB protein conversely stabilized the inheritance of partition-defective plasmids while not increasing plasmid copy number. The identification of dnaA mutations that selectively affected either replication or partitioning further demonstrated the separate roles of DnaA in these functions. The partition-related actions of DnaA were localized to a domain (the cell membrane binding domain) that is physically separate from the DnaA domain that interacts with other host replication proteins. Our results identify bacterial replication proteins that participate in partitioning of the pSC101 plasmid and provide evidence that these proteins mediate plasmid partitioning independently of their role in DNA synthesis.     

Negative control of plasmid pSC101 replication by increased concentrations of both initiator protein and iterons

Increased intracellular concentrations of the initiator protein Rep (or RepA) interfere with pSC101 DNA replication, and mutated Rep proteins that result in an increase in plasmid copy numbers do not inhibit the replication. A rep mutant (rep(inh)) defective in the inhibitory activity was isolated and found to be a new high copy number mutant. The inhibitory function of Rep was enhanced by the coexistence of directly repeated sequences (DR; iterons) in the replication origin region (ori), but not by the inverted repeat sequences (IR) in ori and the rep promoter. This synergistic effect of Rep and DR sequences for the replication inhibition was dependent on their intracellular concentrations. Considering that DR sequences are the specific binding sites of the Rep monomer form, the Rep monomer-DR complex might be responsible for the inhibition of the plasmid replication. Furthermore, the Rep monomer in the crude cell extracts facilitated dimerization of DR DNA fragments by DNA ligase. Neither synergistic inhibitory function with DR nor Rep mediated dimerization of DR DNA was observed in high copy number mutant Rep proteins. The role of the Rep-iteron complex in the copy number control of pSC101 is discussed.     

Autogenous regulation of synthesis of the replication protein in plasmid pSC101

Summary A 1.3-kb segment of plasmid pSC101 includes the replication origin (ori) and the gene (rep) encoding the 37 kilodalton (K) protein required for autonomous replication of the plasmid. The present work describes the regulation of the rep gene expression. The promoters P _R and P _L fail to promote rep gene expression when located upstream of a sequence with dyad symmetry overlapping the rep promoter, whereas elimination of this sequence allows expression and results in over-production of the rep protein. When expression of trpA-lacZ is controlled under the rep promoter, -galactosidase is produced without the lac inducer. However, this enzyme synthesis is effectively reduced when the complete rep sequence is provided in trans. A partial disruption of the sequence with dyad symmetry relieves the repression. These results suggest that expression of the rep gene is negatively regulated by its own product and that the sequence with dyad symmetry plays the role of a receptor site for the rep protein.Abbreviations bp base pairs - kb kilobase pairs - ORF open reading frame - SDS sodium dodecyl sulphate - ts temperature sensitive     

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.     

The effect of procaine on the partitioning of plasmid pSC101

Abstract An examination of samples obtained from a commercial fish smoker, using seawater agar with incubation at 4°, 15° and 37°C for up to 28 days, revealed the presence of large bacterial populations in smoked fish. However, initially only low bacterial numbers, i.e., 2 × 10³/g, were present in the muscle of fresh, whole haddock ( Melanogrammus aeglefinus ). With filleting, there was a sudden increase in numbers to 9.2 × 10⁵/g. Yet immediately after smoking, the bacterial populations decreased (5 × 10⁵/g), followed by a gradual increase with storage (e.g., 2 × 10⁶/g after 24 h). Representative colonies were presumptively identified as Acinetobacter, Alcaligenes , coryneforms, Pseudomonas and Vibrio spp.     

P1 plasmid replication. Purification and DNA-binding activity of the replication protein RepA

The minimal P1 replicon encompasses an open reading frame for the essential replication protein, RepA, bracketed by two sets of multiple 19-base pair repeated sequences, incA and incC. This study focused on the interaction of RepA with the incC and incA repeated sequences because earlier studies suggested that incA might control P1 copy number by titrating limiting amounts of RepA and because the incC repeats, which are part of the origin of replication, contain the promoter for repA. RepA is essential for origin function, autoregulates its own synthesis from the promoter, and, when overproduced, blocks origin function. In this study, RepA was overproduced from an expression vector and purified to 90% homogeneity. The binding of RepA to the DNA encompassing repeat sequences was assayed by monitoring the mobility of protein-DNA complexes on polyacrylamide gels. Distinct species of retarded bands were seen with the maximum number of bands corresponding to the number of repeats present in the target fragment. No evidence was found for RepA binding to fragments not containing the repeats. This suggests that the specific binding of RepA to the repeats may be involved in each of the diverse activities of RepA.     

A louse involved in the regulation of replication in plasmid pSC 101

The origin of replication of plasmid pSC101 contains three directly repeated sequences RS1, RS2, and RS3 separated by 22 bp from two palindromic sequences, IR1 and IR2, which are partially homologous to the direct repeats. These inverted repeat (IR) sequences overlap the promoter of the repA gene which encodes a protein essential for plasmid replication. We have shown that RepA binds to the RS sites as a monomer and to the IR sites as a dimer. The influence of the IR1 site, and of the DNA segment that separates it from RS3, on plasmid copy number control has been studied in detail. We show that the integrity of IR1 is essential for efficient replication and plasmid stability, the critical site extending to the left of IR1 proper. We also show that the presence of IR1 modifies profoundly the binding properties of purified RepA protein to a segment of DNA containing the RS sequences. IR1 is separated from its homologous site on RS3 by approximately four turns of the DNA helix. Replication is abolished if this distance is increased by half a turn of the helix but it is restored if the distance is increased by a whole turn. These results suggest a DNA looping interaction, in the initiation of replication, between the RepA dimer that binds iR1 and the RepA monomers that bind the RS sequences.     

Effects of the pSC101 partition (par) locus on in vivo DNA supercoiling near the plasmid replication origin.

Previous work has shown that deletion of the partition (par) locus of plasmid pSC101 results in decreased overall superhelical density of plasmid DNA and concommitant inability of the plasmid to be stably inherited in populations of dividing cells. We report here that the biological effects of par correlate specifically with its ability to generate supercoils in vivo near the origin of pSC101 DNA replication. Using OsO4 reactivity of nucleotides adjoining 20 bp (G-C) tracts introduced into pSC101 DNA to measure local DNA supercoiling, we found that the wild type par locus generates supercoiling near the plasmid's replication origin adequate to convert a (G-C) tract in the region to Z form DNA. A 4 bp deletion that decreases par function, but produces no change in the overall superhelicity of pSC101 DNA as determined by chloroquine/agarose gel analysis, nevertheless reduced (G-C) tract supercoiling sufficiently to eliminate OsO4 reactivity. Mutation of the bacterial topA gene, which results in stabilized inheritance of par-deleted plasmids, restored supercoiling of (G-C) tracts in these plasmids and increased OsO4 reactivity in par+ replicons. Removal of par to a site more distant from the origin decreased supercoiling in a (G-C) tract adjacent to the origin and diminished par function. Collectively, these findings indicate that par activity is dependent on its ability to produce supercoiling at the replication origin rather than on the overall superhelical density of the plasmid DNA.     

The pSC101 par locus alters protein-DNA interactions in vivo at the plasmid replication origin.

We report here direct evidence that mutations in the par locus affect protein-DNA interactions in vivo at the replication origin of plasmid pSC101. Concomitant with par-mediated plasmid stabilization, two sites in the origin region show an altered methylation pattern as detected by in vivo footprinting with dimethyl sulfate. One site is located near an integration host factor-binding sequence adjacent to the first of three direct repeats known to be involved in the initiation of pSC101 replication; the second site is within the third direct repeat.     

DNA bending of pSC101 ori induced by Rep,a replication initiator protein and IHF

Purified Rep (or RepA) protein, a replication initiator of plasmid pSC101, is present almost solely in the dimer form, and its binding activity for the directly repeated sequences (iterons) in the replication origin (ori) is very low. When Rep protein was treated with guanidine hydrochloride followed by renaturation, it was shown to bind to the iterons with very high efficiency. A gel shift experiment suggested that guanidine-treated Rep bound to iterons as a monomer form. The Rep monomer bound noncooperatively to the three iterons and induced bending of the DNA helix axis in the same direction (about 100 degrees ). The configuration of the IHF box that is a binding site of another DNA bending protein IHF, the three iterons and an AT-rich region between these sequences was important for efficient bending of the ori region. Furthermore, a mutant Rep protein (Rep(IHF)) which can support the plasmid replication in IHF-deficient host cells was purified, and it was found that affinity of the Rep(IHF) monomer for iterons was similar to that of wild-type Rep and bent DNA only 14 degrees more strongly than did the wild-type Rep. Rep(IHF)-dependent plasmid replication, however, required both enhancer regions, par and IR-1, in addition to "core ori" as a minimal essential ori, whereas only one of these two enhancers was necessary for wild-type Rep-dependent replication. How Rep(IHF) can support plasmid replication in the absence of IHF is discussed.     

Effect of dna mutations on the replication of plasmid pSC101 in Escherichia coli K-12.

Escherichia coli strains with mutations in genes dnaB, dnaC, and dnaG were tested for their capacity to replicate pSC101 deoxyribonucleic acid (DNA) at a nonpermissive temperature. Only a small amount of radioactive thymine was incorporated into pSC101 DNA in the dna mutants at 42 degrees C, whereas active incorporation into plasmid DNA took place in wild-type strains under the same conditions. The effects of the dnaB and dnaC mutations were greater on plasmid DNA synthesis than on host chromosomal DNA synthesis, suggesting that these gene products are directly involved in the process of pSC101 DNA replication. In dnaG mutants, both plasmid and chromosomal DNA synthesis were blocked soon after the shift to high temperature; although the extent of inhibition of the plasmid DNA synthesis was greater during the early period of temperature shift to 42 degrees C as compared with that of the host DNA synthesis, during the later period it was less. It was found that the number of copies of pSC101 per chromosome in dnaA and dnaC strains, grown at 30 degrees C, was considerably lower than that in wildtype strains, suggesting that the replication of pSC101 in these mutant strains was partially suppressed even under the permissive conditions. No correlation was found between the number of plasmid copies and the tetracycline resistance level of the host bacterium.     

Importance of the C terminus of plasmid Rts1 RepA protein for replication and incompatibility of the plasmid.

RepA protein, essential for replication of plasmid Rts1, was found to bind in vivo immediately upstream of the repA promoter in studies with mini-Rts1 derivatives with deletions in the upstream region of repA. We constructed another series of repA mutants that would encode RepA derivatives containing oligopeptide substitutions in place of the carboxyl-terminal six amino acids. These modified RepA proteins could not activate ori (Rts1) at all and showed various degrees of incompatibility, or no incompatibility, toward a mini-Rts1 plasmid. These results suggest that the carboxyl-terminal six (or fewer) amino acids of RepA are important for exerting replication and incompatibility functions. One of the RepA derivatives, which showed an evident incompatibility without initiating replication, was examined for its ability to repress the repA gene.