Maintenance of plasmid pSC101 in Escherichia coli requires the host primase.

The abilities of three Escherichia coli strains with thermosensitive dnaG alleles to maintain plasmids pSC101 or pBR322 or an RP4 derivative were studied at elevated growth temperatures. Under these conditions, pSC101 segregated from cells to a greater extent than did pBR322. No segregation of the primase-encoding RP4 derivative was observed.     

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.     

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.     

Novel mechanism of cell division inhibition associated with the SOS response in Escherichia coli.

Certain Escherichia coli strains were shown to possess a novel system of cell division inhibition, called the SfiC+ phenotype. SfiC+ filamentation had a number of properties similar to those of sfiA-dependent division inhibition previously described: (i) both are associated with the SOS response induced by expression of the recA(Tif) mutation, (ii) both are associated with cell death, (iii) both are amplified in mutants lacking the Lon protease, and (iv) both are suppressed by sfiB mutations. SfiC+ filamentation and sfiA-dependent division inhibition differed in (i) the physiological conditions under which loss of viability is observed, (ii) the extent of amplification in lon mutants, (iii) their genetic regulation (SfiC+ filamentation is not under direct negative control of the LexA repressor), and (iv) their genetic determinants (SfiC+ filamentation depends on a locus, sfiC+, near 28 min on the E. coli map and distinct from sfiA).     

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.     

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.     

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.     

Replication of plasmid pSC101 in Escherichia coli K12: requirement for dnaA function.

Summary Protoplasts of soybean and N. glauca were induced to fuse with polyethylene glycol (PEG 1540). Up to 39% of the protoplasts in the treated population were heterokaryocytes. When the heterokaryocytes were isolated and individually cultivated they divided indefinitely and each produced many millions of cells within 2–3 months.The chromosomal behaviour of soybean and N. glauca in the hybrids were not synchronous in the first few cell generations and the chromosomes of N. glauca had a tendency to stick together and break into pieces. However, some of the N. glauca chromosomes were still retained in the somatic hybrids after 6 months of culturing. The chromosomes of the N. glauca were reconstructed in such a way that in the later cell generations, the movement of the N. glauca chromosomes were in synchrony with the soybean chromosomes.NRCC NO. 15668     

The integration host factor of Escherichia coli binds to bent DNA at the origin of replication of the plasmid pSC101

The integration host factor (IHF) of Escherichia coli is necessary for maintenance of pSC101. The protein binds specifically to the replication origin of the plasmid, in the AT-rich region located immediately adjacent to the left, weak binding site for the plasmid-encoded initiator protein. DNAase I and OH- radical footprinting experiments showed that IHF protects 49 bp of the DNA at the origin region. Methylation protection analyses revealed that IHF contacts purine residues in both the major and minor grooves of the DNA. Electrophoretic analyses showed that IHF binds to bent DNA, and the protein binding further enhances the degree of DNA bending. Site-directed mutagenesis of three of the contact points not only abolished binding of the protein to the DNA but also inactivated the replication origin. Therefore, binding of IHF to the ori sequence most probably is necessary for initiation of plasmid replication.     

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     

A copy-number mutant of plasmid pSC101

Copy-number mutants of plasmid pSC101 were isolated by u.v. mutagenesis and selection for elevated expression of ampicillin resistance. Three independent mutations were identical and mapped in codon 93 of the initiation protein RepA. The mutated plasmids were maintained at a level four to five times higher than that of the wild type. For one of them, it was determined that: (i) the mRNA of the autoregulated repA gene, cloned onto a pUC19 plasmid under the control of its own promoter, was expressed at a level 1.7 times higher than that of the wild type; (ii) the RepA protein, under the same conditions, was expressed at a similarly higher level; (iii) the affinity of the mutated protein for three repeated sequences in the origin region of the plasmid was, on average, 3.4 times higher than that of the wild-type protein. We postulate that the copy-number effect is due to a combination of these two effects, i.e. higher protein concentration and increased affinity of the protein for the repeated sequences.     

Involvement of integration host factor (IHF) in maintenance of plasmid pSC101 in Escherichia coli: characterization of pSC101 mutants that replicate in the absence of IHF

Escherichia coli mutants defective in the stable maintenance of plasmid pSC101 have been isolated following Tn10 insertion mutagenesis. One class of mutations affecting pSC101 replication was located in the genes himA and himD (hip), which encode the two subunits of integration host factor (IHF), a small histonelike DNA-binding protein that has multiple cellular functions. Mutants of pSC101 that could replicate in the absence of IHF were isolated and characterized; four independent mutational alterations were found to affect the third codon of the pSC101 rep gene, resulting in the replacement of glutamic acid by lysine. The compensating alteration appears to function by altering the activity of the pSC101 rep protein in him mutants.     

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.     

Inhibition of the SOS response of Escherichia coli by the Ada protein.

Induction of the adaptive response by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) caused a decrease in the UV-mediated expression of both recA and sfiA genes but not of the umuDC gene. On the other hand, the adaptive response did not affect the temperature-promoted induction of SOS response in a RecA441 mutant. The inhibitory effect on the UV-triggered expression of the recA and sfiA genes was not dependent on either the alkA gene or the basal level of RecA protein, but rather required the ada gene. Furthermore, an increase in the level of the Ada protein, caused by the runaway plasmid pYN3059 in which the ada gene is regulated by the lac promoter, inhibited UV-mediated recA gene expression even in cells to which the MNNG-adaptive treatment had not been applied. This inhibitory effect of the adaptive pretreatment was not observed either in RecBC- strains or in RecBC mutants lacking exonuclease V-related nuclease activity. However, RecF- mutants showed an adaptive response-mediated decrease in UV-promoted induction of the recA gene.     

Involvement of integration host factor (IHF) in maintenance of plasmid pSC101 in Escherichia coli: mutations in the topA gene allow pSC101 replication in the absence of IHF.

Integration host factor (IHF), encoded by the himA and himD genes, is a histonelike DNA-binding protein that participates in many cellular functions in Escherichia coli, including the maintenance of plasmid pSC101. We have isolated and characterized a chromosomal mutation that compensates for the absence of IHF and allows the maintenance of wild-type pSC101 in him mutants, but does not restore IHF production. The mutation is recessive and was found to affect the gene topA, which encodes topoisomerase I, a protein that relaxes negatively supercoiled DNA and acts in concert with DNA gyrase to regulate levels of DNA supercoiling. A previously characterized topA mutation, topA10, could also compensate for the absence of IHF to allow pSC101 replication. IHF-compensating mutations affecting topA resulted in a large reduction in topoisomerase I activity, and plasmid DNA isolated from such strains was more negatively supercoiled than DNA from wild-type strains. In addition, our experiments show that both pSC101 and pBR322 plasmid DNAs isolated from him mutants were of lower superhelical density than DNA isolated from Him+ strains. A concurrent gyrB gene mutation, which reduces supercoiling, reversed the ability of topA mutations to compensate for a lack of him gene function. Together, these findings indicate that the topological state of the pSC101 plasmid profoundly influences its ability to be maintained in populations of dividing cells and suggest a model to account for the functional interactions of the him, rep, topA, and gyr gene products in pSC101 maintenance.     

Characterization of copy number mutants of plasmid pSC101

We have characterized three copy number mutants of the plasmid pSC101. These mutations caused single amino acid substitutions at the 46th, 83rd and 115th codons in the rep gene and an increase in the copy number by 4- to 8-fold. Although the in vivo and in vitro repressor activities of these mutated Rep proteins were quite different from each other, the intracellular concentrations of the proteins were maintained at higher levels than the wild-type protein. It has been reported that excess amounts of Rep inhibit pSC101 replication (Ingmer and Cohen, 1993). This inhibitory activity of Rep was markedly decreased in all three mutants. When both the wild-type and one of the mutated rep genes were retained in the same plasmids, the copy number of these plasmids was decreased compared with plasmids retaining a single mutated rep gene. These results support the theory that the inhibitory activity of Rep for its own replication plays an important role in copy number regulation.