P1 plasmid replication. Role of initiator titration in copy number control

The copy number control locus incA of unit copy plasmid P1 maps in a region containing nine 19 base-pair repeats. Previous results from studies in vivo and in vitro indicated that incA interacts with the plasmid-encoded RepA protein, which is essential for replication. It has been proposed that the repeat sequences negatively control copy number by sequestering the RepA protein, which is rate-limiting for replication. Our results lend further support to this hypothesis. Here we show that the repeats can be deleted completely from P1 miniplasmids and the deletion results in an approximately eightfold increase in plasmid copy number. So, incA sequences are totally dispensable for replication and have only a regulatory role. The copy number of incA-deleted plasmids can be reduced if incA sequences are present in trans or are reincorporated at two different positions in the plasmid. This reduction in copy number is not due to lowered expression of the repA gene in the presence of incA. We show that one repeat sequence is sufficient to bind RepA and can reduce the copy number of incA-deleted plasmids. When part of the repeat was deleted, it lost its ability to bind as well as influence copy number. These results show a strong correlation between the capacity of incA repeats to bind RepA protein both in vivo and in vitro, and the function of incA in the control of copy number.     

Construction of a high-copy "ATG vector" for expression in Escherichia coli.

We report the construction of an inducible, high-copy plasmid for the expression of foreign proteins in Escherichia coli. This plasmid, pPB1, combines the trc promoter, beta-galactosidase translation start site, and polylinker of pKK233-2 with the origin of replication region of pUC19. Replacement of the origin of replication of pKK233-2 results in a threefold increase in plasmid copy number of pPB1 compared with pKK233-2. Subclones of the cDNA for rabbit muscle fructose-1,6-bisphosphate aldolase (E.C. 4.1.2.13) in the two expression plasmids exhibit a comparable difference in copy number. An increase in protein expression measured by SDS-PAGE and aldolase specific activities reflects the increased copy number. Specific activities of aldolases in bacterial extracts differ approximately sixfold between the two expression plasmids in E. coli JM83. Aldolase A can compose up to 40% of the total protein in E. coli JM83 when expressed in pPB1, from which more than 100 mg of purified enzyme can be obtained per liter culture.     

Molecular clocks reduce plasmid loss rates: the R1 case

Plasmids control their replication so that the replication frequency per plasmid copy responds to the number of plasmid copies per cell. High sensitivity amplification in replication response to copy number deviations generally reduces variation in copy numbers between different single cells, thereby reducing the plasmid loss rate in a cell population. However, experiments show that plasmid R1 has a gradual, insensitive replication control predicting considerable copy number variation between single cells. The critical step in R1 copy number control is regulation of synthesis of a rate-limiting cis-acting replication protein, RepA. De novo synthesis of a large number of RepA molecules is required for replication, suggesting that copy number control is exercised at multiple steps. In this theoretical kinetic study we analyse R1 multistep copy number control and show that it results in the insensitive replication response found experimentally but that it at the same time effectively prohibits the existence of only one plasmid copy in a dividing cell. In combination with the partition system of R1, this can lead to very high segregational stability. The R1 control mechanism is compared to the different multistep copy number control of plasmid ColE1 that is based on conventional sensitivity amplification. This implies that while copy number control for ColE1 efficiently corrects for fluctuations that have already occurred, R1 copy number control prevents their emergence in cells that by chance start their cycle with only one plasmid copy. We also discuss how regular, clock-like, behaviour of single plasmid copies becomes hidden in experiments probing collective properties of a population of plasmid copies because the individual copies are out of phase. The model is formulated using master equations, taking a stochastic approach to regulation, but the mathematical formalism is kept to a minimum and the model is simplified to its bare essence. This simplicity makes it possible to extend the analysis to other replicons with similar design principles.     

Analysis of a recessive plasmid copy number mutant: evidence for negative control of Col E1 replication.

The Col E1-derivative copy number mutant plasmid pOP1Δ6 has been used to investigate the control of plasmid replication. pOP1Δ6 normally exists at about 200 copies per chromosome, while the wild-type plasmid from which it was derived (pBGP120) exists at about 15 copies per chromosome. We have observed that in E. coli containing both pOP1Δ6 and pBGP120, the copy number of pOP1Δ6 is lowered to 4–6 copies per chromosome. Thus the mutation in pOP1Δ6 is recessive. The association between the two plasmids is stable in E. coli, indicating that incompatibility properties as well as replication control characteristics have been altered in pOP1Δ6. Co-residence of the unrelated plasmid pSC101 with pOP1Δ6 has no detectable effect on pOP1Δ6 copy number. These results suggest that a plasmid-specific, diffusible repressor may act negatively to control plasmid copy number, and that pOP1Δ6 produces a defective repressor or is altered in repressor synthesis. We have constructed in vitro a plasmid which is identical in size to pQP1Δ6 but contains a replication origin region derived from pBGP120. Since this plasmid, pNOP1, exists stably (like pBGP120) at 10–15 copies per chromosome, the high copy number of pOP1Δ6 is not related to its reduced size relative to pBGP120. To localize the mutation in pOP1Δ6 responsible for DNA overproduction, we have cloned fragments of pBGP120 into pOP1Δ6 and selected for plasmids with wild-type copy number. We find that a 2.0 kb region of pBGP120 DNA surrounding the origin of plasmid DNA replication is capable of suppressing the DNA overproducer phenotype of pOP1Δ6. The 2.0 kb fragment is capable of independent self-replication or can integrate into pOP1Δ6 in vivo to form a composite plasmid with two origins of replication. The overproducer phenotype of pOP1Δ6 is suppressed in either configuration.     

Control of plasmid DNA replication by iterons: no longer paradoxical

Replication origins of a family of bacterial plasmids have multiple sites, called iterons, for binding a plasmid-specific replication initiator protein. The iteron-initiator interactions are essential for plasmid replication as well as for inhibition of plasmid over-replication. The inhibition increases with plasmid copy number and eventually shuts plasmid replication off completely. The mechanism of inhibition appears to be handcuffing, the coupling of origins via iteron-bound initiators that block origin function. The probability of a trans-reaction such as handcuffing is expected to increase with plasmid copy number and diminish with increases in cell volume, explaining how the copy number can be maintained in a growing cell. Control is also exerted at the level of initiator synthesis and activation by chaperones. We propose that increases in active initiators promote initiation by overcoming handcuffing, but handcuffing dominates when the copy number reaches a threshold. Handcuffing should be ultrasensitive to copy number, as the negative control by iterons can be stringent (switch-like).     

Control of replication of FII plasmids: comparison of the basic replicons and of the copB systems of plasmids R100 and R1

The copy numbers of the FII plasmids R1 and R100 were determined in four different ways and found to be identical. Deletion of one of the copy number control genes, copB, together with its promoter gives rise to plasmid copy mutants with an increased copy number. The increase was found to be 8- and 3.5-fold for plasmids R1 and R100, respectively. These deletion derivatives were found to be extremely sensitive to the presence of CopB activity from their own parent plasmid but not to that of the other plasmid. Hence, the CopB protein and its target are plasmid-specific and not FII-group-specific. These results are consistent with the high degree of nonhomology between plasmids R1 and R100 in a 250-bp region covering the distal part of the copB gene and the repA promoter region, which contains the target for the CopB protein.     

Increased stability of pBR322-related plasmids in Escherichia coli W3101 grown in carrageenan gel beads

Abstract The stability and the copy number of pBR322, pBR325 and pBR328 were studied during continous cultures of free and immobilized E. coli W3101 without selective pressure. In the free-cell system, it was found that pBR328 and pBR325-free E. coli cells appeared after a lag period. They rapidly overgrew the cultures and the plasmid copy number subsequently declined. On the other hand, an increase in the proportion of pBR322- carrying cells during a free continuous culture was observed. This increase correlated with that of plasmid copy number. By contrast, in the immobilized- cell system, plasmid free segregants were not detected in all the cases even after 250 generations. We have also shown that plasmid copy number remained constant and phenomena such as fluctuations or genetic modifications which occured after long term growth of bacteria in a free continuous culture could be avoided throughout cell immobilization.     

Regulation of plasmid R1 replication: PcnB and RNase E expedite the decay of the antisense RNA, CopA

The replication frequency of plasmid R1 is controlled by an unstable antisense RNA, CopA, which, by binding to its complementary target, blocks translation of the replication rate-limiting protein RepA. Since the degree of inhibition is directly correlated with the intracellular concentration of CopA, factors affecting CopA turnover can also alter plasmid copy number. We show here that PcnB (PAP I — a poly(A)polymerase of Escherichia coli  ) is such a factor. Previous studies have shown that the copy number of ColE1 is decreased in pcnB mutant strains because the stability of the RNase E processed form of RNAI, the antisense RNA regulator of ColE1 replication, is increased. We find that, analogously, the twofold reduction in R1 copy number caused by a pcnB lesion is associated with a corresponding increase in the stability of the RNase E-generated 3' cleavage product of CopA. These results suggest that CopA decay is initiated by RNase E cleavage and that PcnB is involved in the subsequent rapid decay of the 3' CopA stem-loop segment. We also find that, as predicted, under conditions in which CopA synthesis is unaffected, pcnB mutation reduces RepA translation and increases CopA stability to the same extent.     

Adenosine monophosphate-induced amplification of ColE1 plasmid DNA in Escherichia coli

ColE1 plasmid copy number was analyzed in relaxed (relA) and stringent (relA(+)) Escherichia coli cells after supplementation of culture media with adenosine monophosphate (AMP). When a relaxed E. coli strain bearing ColE1 plasmid was cultured in LB medium for 18 h and induced with AMP for 4h, the plasmid DNA yield was significantly increased, from 2.6 to 16.4 mgl(-1). However no AMP-induced amplification of ColE1 plasmid DNA was observed in the stringent host. Some plasmid amplification was observed in relA mutant cultures in the presence of adenosine, while adenine, ADP, ATP, ribose, potassium pyrophosphate and sodium phosphate caused a minor, if any, increase in ColE1 copy number. A mechanism for amplification of ColE1 plasmid DNA with AMP in relA mutant bacteria is suggested, in which AMP interferes with the aminoacylation of tRNAs, increases the abundance of uncharged tRNAs, and uncharged tRNAs promote plasmid DNA replication. According to this proposal, in relA(+) cells, the AMP induction could not increase ColE1 plasmid copy number because of lower abundance of uncharged tRNAs. Our results suggest that the induction with AMP can be used as an effective method of amplification of ColE1 plasmid DNA in relaxed strains of E. coli.     

Plasmid R1 in Salmonella typhimurium: Molecular instability and gene dosage effects

Plasmid R1 drd-19 and two of its copy mutants (pKN102 and pKN103) were transferred from Escherichia coli to Salmonella typhimurium, where the expression of the copy mutations was studied further. The copy number (ratio of plasmid DNA to chromosomal DNA) was the same in S. typhimurium and in E. coli. The activities of the plasmid-coded antibiotic-metabolizing enzymes β-lactamase, chloramphenicol acetyltransferase, and streptomycin adenylyltransferase as well as the resistances to ampicillin and streptomycin were proportional to the gene dosage up to at least a threefold increase in the steady state plasmid copy number, whereas resistance to chloramphenicol showed no increase with increased number of plasmid copies per chromosome equivalent. Also the resistance to rifampicin was affected since S. typhimurium cells became more sensitive the higher the copy number of the resident plasmid. Furthermore, plasmid R1 showed molecular instability in S. typhimurium cells since there was a tendency to dissociate into resistance transfer factors and resistance determinants and also to form miniplasmids. This tendency to instability was more pronounced the higher the plasmid copy number.     

Chromatin organization of the Saccharomyces cerevisiae 2 microns plasmid depends on plasmid-encoded products.

We have used gene disruptions and nuclease probes to assess the roles of yeast 2 micron plasmid genes in plasmid chromatin organization. The chromatin structure at the replication origin is not dependent on any of the four major open reading frames, A, B, C, or D. While stable plasmid maintenance is known to depend on a cis-acting locus STB and genes B and C, we find that only gene B influences STB chromatin. Other interactions between plasmid gene products and sequences may reflect gene regulation: the chromatin organization at the 5' end of gene A, which codes for a site-specific recombinase, depends on both gene B and gene C. Since disruption of gene C results in an increase in plasmid copy number that is dependent on gene A, we propose that gene C (and probably gene B) control copy number by regulating the level of the gene A recombinase.     

The mcm2 mutation of yeast affects replication, rather than segregation or amplification of the two micron plasmid.

We have studied the maintenance of the endogenous two micron (2 mu) plasmid in a strain of yeast carrying the nuclear mutation mcm2. This mutation, earlier shown to affect the maintenance of yeast minichromosomes in an ARS-dependent manner, also affected the copy number of the 2 mu plasmid. The effect was more pronounced at 35 degrees C leading to the elimination of the plasmid from the cells cultured at this temperature. The mutant cells could be efficiently cured of the circle by transformation with 2 mu ORI-carrying hybrid vectors, an observation consistent with the low copy number of the endogenous plasmid. A chromosomal revertant of this mutant for another ARS(ARS1) was found also to confer stability on the 2 mu ORI-carrying minichromosomes and had elevated levels of the endogenous plasmid. The mutation neither affected the segregation nor the amplification process mediated by site-specific recombination at FRT sites requiring the FLP gene-encoded protein action. ARS131C, an ARS that was unaffected in the mutant at 25 degrees C, could elevate the copy number of a 2 mu hybrid vector in the mutant cells. In view of these results, some aspects of segregation and copy number control of the endogeneous plasmid have been discussed. We propose that the mutation impairs the 2 mu ORI function, leading to its loss.     

Kinetic analysis of the effects of plasmid multimerization on segregational instability of CoIE1 type plasmids in Escherichia coli B/r

The effect of plasmid multimerization on segregational instability was investigated using a structured, segregated model of genetically modified Escherichia coli cells. By including the multimerization of plasmids, the model can predict the proportion of each multimer in the total plasmid population. Simulation results suggest that the plasmid copy number is controlled by the total plasmid content (i.e., total number of plasmid origins) in the host cell and that multimerization reduces the total number of independent, monomeric segregation units. However, multimerization is found to have a minor effect on decreasing plasmid segregational stability for multicopy plasmids with average copy number per cell greater than about 25. Also model predictions were used to test whether or not a nonrandom plasmid distribution at cell fission could cause segregational instability. Even in the case of severely biased partitioning, plasmids whose copy number is above 45 per cell do not show significant segregational instability. The results suggest that when the ColE1-type plasmid does not encode and express any large or disruptive foreign proteins, the copy number of 45 per cell may be the threshold at which only growth rate-dependent instability is responsible for overall plasmid instability.     

Participation of the lytic replicon in bacteriophage P1 plasmid maintenance.

P1 bacteriophage carries at least two replicons: a plasmid replicon and a viral lytic replicon. Since the isolated plasmid replicon can maintain itself stably at the low copy number characteristic of intact P1 prophage, it has been assumed that this replicon is responsible for driving prophage replication. We provide evidence that when replication from the plasmid replicon is prevented, prophage replication continues, albeit at a reduced rate. The residual plasmid replication is due to incomplete repression of the lytic replicon by the c1 immunity repressor. Incomplete repression was particularly evident in lysogens of the thermoinducible P1 c1.100 prophage, whose replication at 32 degrees C remained almost unaffected when use of the plasmid replicon was prevented. Moreover, the average plasmid copy number of P1 in a P1 c1.100 lysogen was elevated with respect to the copy number of P1 c1+. The capacity of the lytic replicon to act as an auxiliary in plasmid maintenance may contribute to the extraordinary stability of P1 plasmid prophage.     

The effect of the partition locus on plasmid stability and expression of a prolonged chemostat culture.

The stability, copy number, and gene expression of the pBR322 plasmid containing the par-locus under prolonged cultivation were studied. In the initial stage of the experiment it was observed that the par-locus had a stabilization effect on plasmid maintenance. This observation was consistent with previously reported results. However, after approximately 225 h, a mixed population of plasmid-containing and plasmid-free cells appeared. The mixed culture was stably maintained for approximately 200 h. In addition, the relative plasmid copy number showed an increase as compared to the par- culture. After 100 h the copy number decreased, reached a minimum, then stabilized. The beta-lactamase activity, was not significantly affected by the par-locus.     

Suppression of ColE1 high-copy-number mutants by mutations in the polA gene of Escherichia coli.

We isolated three Escherichia coli suppressor strains that reduce the copy number of a mutant ColE1 high-copy-number plasmid. These mutations lower the copy number of the mutant plasmid in vivo up to 15-fold; the wild-type plasmid copy number is reduced by two- to threefold. The suppressor strains do not affect the copy numbers of non-ColE1-type plasmids tested, suggesting that their effects are specific for ColE1-type plasmids. Two of the suppressor strains show ColE1 allele-specific suppression; i.e., certain plasmid copy number mutations are suppressed more efficiently than others, suggesting specificity in the interaction between the suppressor gene product and plasmid replication component(s). All of the mutations were genetically mapped to the chromosomal polA gene, which encodes DNA polymerase I. The suppressor mutational changes were identified by DNA sequencing and found to alter single nucleotides in the region encoding the Klenow fragment of DNA polymerase I. Two mutations map in the DNA-binding cleft of the polymerase region and are suggested to affect specific interactions of the enzyme with the replication primer RNA encoded by the plasmid. The third suppressor alters a residue in the 3'-5' exonuclease domain of the enzyme. Implications for the interaction of DNA polymerase I with the ColE1 primer RNA are discussed.     

The cost of copy number in a selfish genetic element: the 2‐μm plasmid of Saccharomyces cerevisiae

Many autonomously replicating genetic elements exist as multiple copies within the cell. The copy number of these elements is often assumed to have important fitness consequences for both element and host, yet the forces shaping its evolution are not well understood. The 2 μm is a multicopy plasmid of Saccharomyces yeasts, encoding just four genes that are solely involved in plasmid replication. One simple model for the fitness relationship between yeasts and 2 μm is that plasmid copy number evolves as a trade‐off between selection for increased vertical transmission, favouring high copy number, and selection for decreased virulence, favouring low copy number. To test this model, we experimentally manipulated the copy number of the plasmid and directly measured the fitness cost, in terms of growth rate reduction, associated with high plasmid copy number. We find that the fitness burden imposed by the 2 μm increases with plasmid copy number, such that each copy imposes a fitness burden of 0.17% (± 0.008%), greatly exceeding the cost expected for it to be stably maintained in yeast populations. Our results demonstrate the crucial importance of copy number in the evolution of yeast per 2 μm associations and pave the way for future studies examining how selection can shape the cost of multicopy elements.