Factors influencing the copy number of F-like plasmids in Escherichia coli and Salmonella typhimurium.

The copy numbers of Flac, four F-like plasmids and pLT2 were estimated in two strains of Salmonella typhimurium and (for all except pLT2) one strain of Escherichia coli. For organisms grown in casamino acids minimal medium, the plasmids spanned a 7--8 fold range of copy number with ColB-K98 having the highest copy number in each strain and R124 the lowest. The copy number of ColB-K98 was substantially greater than 1 in each of the strains tested. There was no clear relation between the plasmid size and copy number, although the plasmids studied spanned only a narrow size range. The copy number of individual plasmids was slightly reduced or not affected at all by the presence of a second plasmid in the same strain. Derivatives harbouring each of the plasmids were grown in three different media to ascertain how plasmid copy number responds to changes in growth rate. For each plasmid, the copy number increased with decreasing growth rate. Extracts from each of the three strains harbouring ColB-K98 contained two distinct plasmid species. One appeared to be about twice as large as the other and both were absent from Col- segregants.     

Toxic effects of excess cloned centromeres.

Plasmids carrying a Saccharomyces cerevisiae centromere have a copy number of one or two, whereas other yeast plasmids have high copy numbers. The number of CEN plasmids per yeast cell was made artificially high by transforming cells simultaneously with several different CEN plasmids carrying different, independently selectable markers. Some host cells carried five different CEN plasmids and an average total of 13 extra copies of CEN3. Several effects were noted. The copy number of each plasmid was unexpectedly high. The plasmids were mutually unstable. Cultures contained many dead cells. The viable host cells grew more slowly than control cells, even in nonselective medium. There was a pause in the cell cycle at or just before mitosis. We conclude that an excess of centromeres is toxic and that the copy number of centromere plasmids is low partly because of selection against cells carrying multiple centromere plasmids. The toxicity may be caused by competition between the centromeres for some factor present in limiting quantities, e.g., centromere-binding proteins, microtubules, or space on the spindle pole body.     

Distribution of minichromosomes in individual Escherichia coli cells: implications for replication control

A novel method was devised to measure the number of plasmids in individual Escherichia coli cells. With this method, involving measurement of plasmid-driven expression of the green fluorescent protein gene by flow cytometry, the copy number distribution of a number of different plasmids was measured. Whereas natural plasmids had fairly narrow distributions, minichromosomes, which are plasmids replicating only from a cloned oriC copy, have a wide distribution, suggesting that there is no copy number control for minichromosomes. When the selection pressure (kanamycin concentration) for minichromosomes was increased, the copy number of minichromosomes was also increased. At up to 30 minichromosomes per host chromosome, replication and growth of the host cell was unaffected. This is evidence that there is no negative element for initiation control in oriC and that there is no incompatibility between oriC located on the chromosome and minichromosome. However, higher copy numbers led to integration of the minichromosomes at the chromosomal oriC and to initiation asynchrony of the host chromosome. At a minichromosome copy number of approximately 30, the cell's capacity for synchronous initiation is exceeded and free minichromosomes will compete out the chromosome to yield inviable cells, unless the minichromosomes are incorporated into the chromosome.     

Propagation of an amplifiable recombinant plasmid in Saccharomyces cerevisiae: flow cytometry studies and segregated modeling

Efficient expression of a foreign protein product by the yeastSaccharomyces cerevisiaerequires a stable recombinant vector present at a high number of copies per cell. A conditional centromere yeast plasmid was constructed which can be amplified to high copy number by a process of unequal partitioning at cell division, followed by selection for increased copy number. However, in the absence of selection pressure for plasmid amplification, copy number rapidly drops from 25 plasmids/cell to 6 plasmids/cell in less than 10 generations of growth. Copy number subsequently decreases from 6 plasmids/cell to 2 plasmids/cell over a span of 50 generations. A combination of flow cytometric measurement of copy number distributions and segregated mathematical modeling were applied to test the predictions of a conceptual model of conditional centromereplasmid propagation. Measured distributions of plasmid content displayed a significant subpopulation of cells with a copy number of 4-6, evenin a population whose mean copy number was 13.5. This type of copy number distribution was reproduced by a mathematical model which assumes that amaximum of 4-6 centromere plasmids per cell can be stably partitionedat cell division. The model also reproduces the observed biphasic kinetics of plasmid number instability. The agreement between simulation and experimental results provides support for the proposed model and demonstrates the utility of the flow cytometry/segregated modeling approach for the study of multicopy recombinant vector propagation.     

Copy number control by a yeast centromere

Plasmids containing a cloned yeast (Saccharomyces cerevisiae) centromere (CEN3) in combination with a suitable DNA replication system are maintained in yeast at the low copy number typical of a chromosome. In composite plasmids containing CEN3 plus the yeast 2 mu plasmid, the CEN3 copy number control is dominant over the amplification system that normally drives the 2 mu plasmids to high copy number. The CEN3-2 mu composite plasmids are relatively stably maintained in yeast at a copy number of about one per haploid genome, and segregate through meiosis in a typical Mendelian pattern. Some of the CEN3-2 mu composite plasmids isolated from yeast contain deletions of variable size that remove the functional centromere, resulting in loss of the CEN3 control and reversion to high copy number. Formation of the CEN3 deletions requires the specialized recombination system (inverted repeat sequences and FLP gene) of the yeast 2 mu plasmid.     

Copy number and the stability of 2-micron circle-based artificial plasmids of Saccharomyces cerevisiae

The copy number and stability of artificial 2-micron circle-based plasmids have been accurately measured in [Cir+] and [Cir0] strains of Saccharomyces cerevisiae. We conclude that (i) instability and copy number vary greatly from plasmid to plasmid; (ii) instability and copy number are negatively correlated--that is, high copy number is associated with low instability; (iii) it is difficult to reconcile this variability with a strict and direct system of copy number control; (iv) instabilities are much higher than expected from random partition and the observed copy numbers: this may imply partition which is less efficient than random. Even so, (v) the partitioning of 2-micron circle-like plasmids is more efficient than that of ARS-based plasmids, which hints at the existence of a system for the (inefficient) distribution of 2-micron circles.     

The presence of a defective LEU2 gene on 2 mu DNA recombinant plasmids of Saccharomyces cerevisiae is responsible for curing and high copy number.

The copy number of 2 mu DNA-derived plasmids in CIR+ Saccharomyces cerevisiae transformants is determined by its selective marker and is usually much lower than that of the endogenous plasmid. Only plasmids containing the leu2 allele of pJDB219, designated as leu2-d, under selective conditions displayed a higher copy number than did endogenous 2 mu DNA and by displacement generated cured cells. Spontaneous loss of 2 mu DNA occurred with a frequency of about 0.02% per generation. Curing plasmids, like pMP78, have copy numbers of 35; noncuring plasmids, like pDB248 or YEp6, have copy numbers of 4 to 8. The 2 mu DNA copy number in strains AH22 and YNN27 were determined to be 40 and 100, respectively. The high copy number of leu2-d-containing plasmids can be explained by its weak expression of less than 5% that of the wild-type LEU2 gene. The leu2-d allele has a deletion of the 5'-end sequence starting from 29 base pairs before the ATG initiation codon, but surprisingly, its expression is still regulated. On YRp7, which contains the chromosomal autonomic replication sequence ARS1, the defective leu2-d allele could not complement a leu2 host strain. This suggests a more stringent control of replication of ARS1-containing plasmids than of 2 mu-containing plasmids.     

Effect of plasmid copy number and lac operator sequence on antibiotic-free plasmid selection by operator-repressor titration in Escherichia coli

The Escherichia coli strain DH1lacdapD enables plasmid selection and maintenance that is free from antibiotics and selectable marker genes. This is achieved by using only the lac operator sequence as a selectable element. This strain is currently used to generate high copy number plasmids with no antibiotic resistance genes for use as DNA vaccines and for expression of recombinant proteins. Until now these have been limited to pUC-based plasmids containing a high copy number pMB1-derived origin of replication, and the principle lacO(1) and auxiliary lacO(3) operators. In this study we have shown that this system can also be used to select and maintain pBR322-based plasmids with the lower copy number pMB1 origin of replication, and that lacO(1) alone or a palindromic version of lacO(1) can provide a sufficient level of repressor titration for plasmid selection. This is advantageous for recombinant protein production, where low copy number plasmids are often used and plasmid maintenance is important. The degree of repressor titration due to these plasmids was measured using the natural lactose operon in E. coli DH1 as a model.     

Salinity-dependent copy number change of endogenous plasmids in Synechococcus sp. strain PCC 7002

Marine cyanobacterium Synechococcus sp. strain. PCC 7002 has at least six endogenous plasmids. When it was cultured in 1 m NaCl medium, the copy numbers of the smallest (4.5 kb) and the second smallest (9.7 kb) plasmids decreased to one-third and one-tenth of those in control culture (0.3 m NaCl) respectively. In medium without NaCl, the copy numbers of those plasmids also decreased but the changes were much smaller. On the other hand, copy numbers of 15.4-kb, 30.0-kb, and 36.9-kb plasmids did not change among the three different NaCl concentrations (0 m, 0.3 m, 1 m). The copy number changes of the two plasmids were reversible. A similar copy number change was also observed in medium with 0.3 m NaCl+0.7 m KCl. These observations suggest that copy number controls are different among endogenous plasmids, and some of them are affected by salinity in the medium.     

Linear- and circular-plasmid copy numbers in Borrelia burgdorferi.

Borrelia burgdorferi, the Lyme disease agent, and other members of the spirochetal genus Borrelia have double-stranded linear plasmids in addition to supercoiled circular plasmids. The copy number relative to the chromosome was determined for 49- and 16-kb linear plasmids and a 27-kb circular plasmid of the type strain, B31, of B. burgdorferi. All three plasmids were present in low copy number, about one per chromosome equivalent, as determined by relative hybridizations of replicon-specific DNA probes. The low copy number of Borrelia plasmids suggests that initiation of DNA replication and partitioning are carefully controlled during the cell division cycle. The copy numbers of these three plasmids of strain B31 were unchanged after approximately 7,000 generations in continuous in vitro culture. A clone of B. burgdorferi B31 that did not contain the 16-kb linear plasmid was obtained after exposure of a culture to novobiocin, a DNA gyrase inhibitor. The plasmid-cured strain contains only one linear plasmid, the 49-kb plasmid, and thus has the smallest genome reported to date for B. burgdorferi.     

Directed evolution of copy number of a broad host range plasmid for metabolic engineering

Random mutagenesis and directed evolution has been successfully used to improve desired properties of enzymes for biocatalysis and metabolic engineering. Here we employ the method to increase copy number of a pBBR-based broad host range plasmid, which can be used to express desired enzymes in a variety of microbial hosts. Localized random mutagenesis was performed in the replication control region of a pBBR-derived plasmid containing a beta-carotene reporter. Mutant plasmids were isolated that showed increased beta-carotene production. Real-time PCR analysis confirmed that the copy number of the mutant plasmids increased 3-7 fold. Sequence of the 10 mutant plasmids indicated that each plasmid contained single or multiple mutations in the rep gene or the flanking regions. Single amino acid change of serine to leucine at codon 100 of the replication protein and single nucleotide change of C to T at 46 bp upstream of the rep gene caused the increase of plasmid copy number. The utility of the mutant plasmids for metabolic engineering were further demonstrated by increased beta-carotene production, when an isoprenoid pathway gene (dxs) was co-expressed on a compatible plasmid. The mutant plasmids were tested in Agrobacterium tumefaciens. Increase of plasmid copy number and beta-carotene production was also observed in the non-Escherichia coli host.     

Nuclear plasmids in the simple eukaryotes Saccharomyces cerevisiae and Dictyostelium discoideum

High copy number nuclear plasmids are becoming recognized as common genetic components of simple eukaryotes. Like bacterial plasmids, eukaryotic plasmids ensure their persistence in dividing cells by having a partitioning system and a regulated means of amplifying copy number to correct inherent fluctuations in partitioning. By virtue of their small size and autonomy from the chromosomes, eukaryotic plasmids are useful for studying not only features of eukaryotic replicons but many aspects of gene regulation and DNA organization in nucleated cells.     

Nuclear plasmids in the simple eukaryotes Saccharomyces cerevisiae and Dictyostelium discoideum

High copy number nuclear plasmids are becoming recognized as common genetic components of simple eukaryotes. Like bacterial plasmids, eukaryotic plasmids ensure their persistence in dividing cells by having a partitioning system and a regulated means of amplifying copy number to correct inherent fluctuations in partitioning. By virtue of their small size and autonomy from the chromosomes, eukaryotic plasmids are useful for studying not only features of eukaryotic replicons but many aspects of gene regulation and DNA organization in nucleated cells.     

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.     

Regulation of DNA replication by iterons: an interaction between the ori2 and incC regions mediated by RepE-bound iterons inhibits DNA replication of mini-F plasmid in Escherichia coli.

In bacteria, plasmids and some DNA viruses, DNA replication is initiated and regulated by binding of initiator proteins to repetitive sequences. To understand the control mechanism we used the plasmid mini-F, whose copy number is stringently maintained in Escherichia coli, mainly by its initiator protein RepE and the incC region. The monomers of RepE protein bound to incC iterons, which exert incompatibility in trans and control the copy number of mini-F plasmid in cis. Many incompatibility defective mutants carrying mutations in their incC iterons had lost the affinity to bind to RepE, while one mutant retained high level binding affinity. The mutated incC mini-F plasmids lost the function to control the copy number. The copy number of the wild-type mini-F plasmid did not increase in the presence of excess RepE. These results suggested that the control of replication by incC iterons does not rely on their capacity to titrate RepE protein. Using a ligation assay, we found that RepE proteins mediated a cross-link structure between ori2 and incC, for which the dimerization domain of RepE and the structure of incC seem to be important. The structure probably causes inhibition of extra rounds of DNA replication initiation on mini-F plasmids, thereby keeping mini-F plasmid at a low copy number.     

Molecular cloning and functional characterization of a copy number control gene (copB) of plasmid R1.

Deletions or insertions in the copB gene of plasmid R1 result in a copy mutant phenotype. The wild-type copB gene has been cloned on various plasmid vectors. The presence of such chimeric plasmids reduced the copy number of R1 copB mutant plasmids to normal or subnormal levels, indicating the expression of a trans-acting inhibitor activity from the copB chimeras. However, the cloned copB gene did not affect the copy number of wild-type R1, and no incompatibility was exerted by the cloned copB gene against wild-type R1 (or R100). Although the copB gene is not normally required for the incompatibility exerted by copA, it is shown that the CopB function is required for expression of incompatibility by the copA gene from some types of chimeric plasmids. Mutant plasmids that have lost both Cop functions replicate in an uncontrolled fashion.     

Some stochastic models for plasmid copy number

Some stochastic models for the copy number of plasmids in a cell line are studied. When considering the behavior of copy number in the whole cell line, the theory of multitype branching processes is appropriate. Attention is paid to the cure rate in the cell line, and the asymptotic fractions of cells containing a given number of plasmids. These quantities are used to compare the models numerically.     

Genetic control of the number of copies of type K2 killer plasmids in Saccharomyces cerevisiae

13 non-linked chromosomal mutations derepress the negative genetic control of copy number of K2 yeast killer plasmids and lead to 1.5-2-fold elevating of copy number of that type plasmids -L2A and M2 virus-like dsRNA. The content of both plasmids is increased 3-5-fold in cells with chromosomal ski5 mutation, as compared to the strains of wild type. Expression of ski5 allele is recessive. The dose effect of this allele is observed on haploid and diploid levels. Dominant ochre nonsense suppressors suppress the action of ski5 and the ski6 allele epistates that of ski5.