Partition kinetics and thermosensitive replication of pT169, a naturally occurring multicopy tetracycline resistance plasmid of Staphylococcus aureus

The temperature-sensitivity of a naturally occurring plasmid encoding inducible tetracycline resistance in Staphylococcus aureus has been examined in terms of incorporation of thymidine into plasmid DNA. The plasmid, pT169, has been found to have a multiplicity of about 15 copies per cell and to reduce sharply its replication rate immediately upon a shift to 42.5 °C. Plasmid replication at 42.5 °C continues at a rate equivalent to about 5% of the rate at 32 °C—sufficient to maintain a multiplicity of two to three copies per cell. Since under these conditions the plasmid is hereditarily stable, we conclude that there is a specific segregation mechanism, independent of replication, that ensures equal distribution of plasmid molecules to daughter cells during cell division.     

Random replication and random assortment model for plasmid incompatibility in bacteria

To understand the incompatibility between two related plasmids, both of which replicate in an autonomous state under a common control mechanism, we have developed a model that assumes a random choice mechanism for replication of plasmid copies and their random assortment into daughter cells upon cell division. Segregation kinetics by this model is analyzed mathematically and the number of generations required for segregation is calculated as a function of plasmid copy number per cell. The results obtained offer enough quantitative data to make our model reasonably realistic.     

Quantitative PCR Method for Enumeration of Cells of Cryptic Species of the Toxic Marine Dinoflagellate Ostreopsis spp. in Coastal Waters of Japan

Monitoring of harmful algal bloom (HAB) species in coastal waters is important for assessment of environmental impacts associated with HABs. Co-occurrence of multiple cryptic species such as toxic dinoflagellate Ostreopsis species make reliable microscopic identification difficult, so the employment of molecular tools is often necessary. Here we developed new qPCR method by which cells of cryptic species can be enumerated based on actual gene number of target species. The qPCR assay targets the LSU rDNA of Ostreopsis spp. from Japan. First, we constructed standard curves with a linearized plasmid containing the target rDNA. We then determined the number of rDNA copies per cell of target species from a single cell isolated from environmental samples using the qPCR assay. Differences in the DNA recovery efficiency was calculated by adding exogenous plasmid to a portion of the sample lysate before and after DNA extraction followed by qPCR. Then, the number of cells of each species was calculated by division of the total number of rDNA copies of each species in the samples by the number of rDNA copies per cell. To test our procedure, we determined the total number of rDNA copies using environmental samples containing no target cells but spiked with cultured cells of several species of Ostreopsis. The numbers estimated by the qPCR method closely approximated total numbers of cells added. Finally, the numbers of cells of target species in environmental samples containing cryptic species were enumerated by the qPCR method and the total numbers also closely approximated the microscopy cell counts. We developed a qPCR method that provides accurate enumeration of each cryptic species in environments. This method is expected to be a powerful tool for monitoring the various HAB species that occur as cryptic species in coastal waters.     

Synethesis and integration of viral DNA in chicken cells at different time after infection with various multiplicities of avian oncornavirus.

To see if integration of the provirus resulting from RNA tumor virus infection is limited to specific sites in the cell DNA, the variation in the number of copies of virus-specific DNA produced and integrated in chicken embryo fibroblasts after RAV-2 infection with different multiplicities has been determined at short times, long times, and several transfers after infection. The number of copies of viral DNA in cells was determined by initial hybridization kinetics of single-stranded viral complementary DNA with a moderate excess of cell DNA. The approach took into account the different sizes of cell DNA and complementary DNA in the hybridization mixture. It was found that uninfected chicken embryo fibroblasts have approximately seven copies, part haploid genome of DNA sequences homologous to part of the Rous-association virus 2 (RAV-2) genome. Infection with RAV-2 adds additional copies, and different sequences, of RAV -2- specific DNA. By 13 h postinfection, there are 3 to 10 additional copies per haploid genome. This number can not be increased by increasing the multiplicity of infection, and stays relatively constant up to 20 h postinfection, when some of the additional viral DNA is integrated. Between 20 and 40 h postinfection, the cells accumulated up to 100 copies per haploid genome of viral DNA. Most of these are unintegrated. This number decreases with cell transfer, until cells are left with one to three copies of additional viral DNA sequences per haploid genome, of which most are integrated. The finding that viral infection causes the permanent addition of one to three copies of integrated viral DNA, despite the cells being confronted with up to 100 copies per haploid genome after infection, is consistent with a hypothesis that chicken cells contain a limited number of specific integration sites for the oncornavirus genome.     

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.     

Mutations affecting replication and copy number control in plasmid mini-F both reside in the gene for the 29-kDa protein

We have isolated and characterized cop, copts, and repam mutants of plasmid mini-F after in vitro mutagenesis with hydroxylamine. cop mutants exhibit a copy number of about 10 per cell. The copts mutants are cold-sensitive and have, at 25 degrees C, a copy number of about 30-40 copies per cell, which drops to 4 copies at 42 degrees C. The cop and repam mutations affect the 29-kDa E protein. The Copts phenotype results from the simultaneous occurrence of two mutations, a cop mutation in the E protein and a temperature-dependent mutation (termed ecp) enhancing the Cop phenotype at low temperature. The latter new type of mutation is located within the DNA region 44.1-44.85F. Complementation experiments with plasmid cointegrates show that the wild-type gene is dominant over the cop allele. The nucleotide sequences of the cop and the repam mutations have been determined.     

Effect ofEscherichia coliIHF Mutations on Plasmid p15A Copy Number

Four isogenic strains (himAhimDdouble mutant,himAandhimDsingle mutants, and their wild type counterpart) harboringorip15A plasmid (pACYC184 or pACYC184Amp or pACYC177) show different copy numbers of that plasmid in the early stationary phase of cultivation. The copy number oforip15A plasmid increases about four times in thehimAhimDdouble (65–70 copies per cell) andhimDsingle mutant cells (50–56 copies per cell) and was almost the same inhimAmutant (17–18 copies per cell) and wild type cells (14–16 copies per cell). The results suggest that HimD can form homodimers, which are functionally competent for the regulation oforip15A plasmid copy number. Complementation experiments ofhimAhimDdouble mutant cells using plasmid carryinghimAandhimDgenes (pP_LhiphimA-5) confirm the effect of integration host factor (IHF) absence on increasing the copy number oforip15A plasmid (plasmid producing IHF complemented the defect of IHF mutant). The absence of IHF (usinghimAhimDdouble mutant as host) had no effect on the copy number of the pBR322 (oripMB1) plasmid.     

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.     

Time-course determination of plasmid content in eukaryotic and prokaryotic cells using Real-Time PCR

A Real-Time PCR method was developed to monitor the plasmid copy number (PCN) in Escherichia coli and Chinese hamster ovary (CHO) cells. E. coli was transformed with plasmids containing a ColE1 or p15A origin of replication and CHO cells were transfected with a ColE1 derived plasmid used in DNA vaccination and carrying the green fluorescent protein (GFP) reporter gene. The procedure requires neither specific cell lysis nor DNA purification and can be performed in <30 min with dynamic ranges covering 0.9 pg–55 ng, and 5.0 pg–2.5 ng of plasmid DNA (pDNA) for E. coli and CHO cells, respectively. Analysis of PCN in E. coli batch cultures revealed that the maximum copy number per cell is attained in mid-exponential phase and that this number decreases on average 80% towards the end of cultivation for both types of plasmids. The plasmid content of CHO cells determined 24 h post-transfection was around 3 × 10⁴ copies per cell although only 37% of the cells expressed GFP one day after transfection. The half-life of pDNA was 20 h and around 100 copies/cell were still detected 6 days after transfection.     

Isolation and Characterization of a Copy Mutant of the Bacteriocinogenic Plasmid Clo DF13

After nitrosoguanidine mutagenesis, strain Escherichia coli P678-54, bacteriocinogenic for Clo DF13, yielded a mutant strain that showed an enhanced bacteriocin production. The results from conjugation experiments indicated that the mutation, responsible for the enhanced bacteriocin production, is located on the Clo DF13 plasmid. The following properties of strains harboring the mutant Clo DF13 plasmid could be observed. (i) The bacteriocin production in these strains can be further enhanced at least fourfold by mitomycin C. (ii) The fraction of spontaneously induced cells, as revealed by lacunae experiments, in cultures of these strains is about nine times higher than in cultures of wild-type Clo DF13-harboring strains. (iii) Chromosomeless minicells from strain P678-54 harboring the mutant Clo DF13 plasmid synthesize about six times more deoxyribonucleic acid, ribonucleic acid, and protein as compared to wild-type Clo DF13-harboring minicells. (iv) Analysis of this mutant Clo DF13-specific ribonucleic acid and protein on polyacrylamide gels revealed mainly the same ribonucleic acid and polypeptide species as synthesized by the wild-type Clo DF13 minicells, but in larger amounts (Kool et al., 1974). (v) Segregation experiments, using a strain with temperature-sensitive polymerase I, show that mutant Clo DF13-harboring cells contain an average of 70 Clo DF13 copies per cell, whereas wild-type Clo DF13-harboring cells contain only about 10 Clo DF13 copies per cell. The data presented in this paper indicate that the mutation on the Clo DF13 plasmid leads to an altered control of Clo DF13 replication and results in an enhanced number of Clo DF13 copies per cell. As a secondary effect, this enhanced number of Clo DF13 copies enhances the probability of "spontaneous" induction per cell. Since the mutation is plasmid specific and affects the number of plasmid copies produced, one can conclude that the Clo DF13 plasmid is not dependent solely on chromosomal information, but that at least plasmid base sequences are involved in Clo DF13 plasmid replication.     

Genetic transformation of mouse cultured cells with the help of high-velocity mechanical DNA injection

NIH 3T3 mouse cells were transfected by the plasmid pSV3neo (G418-resistant) with the help of high-velocity mechanical DNA injection based on the principle of bombarding cells with tungsten particles covered with the DNA. Stable transformants were obtained. Dot-hybridization and Southern analysis revealed the integration into the genome of 5-20 copies per cell of original plasmid DNA. The plasmid DNA was shown to have tandem organization.     

Copy number amplification of the 2 micron circle plasmid of Saccharomyces cerevisiae

The 2 micron circle is a small double stranded DNA plasmid that occurs at about 60 copies per cell in the nuclei of virtually all strains of Saccharomyces cerevisiae. The plasmid has no apparent phenotypic effect on host cells, and is the basis of many useful vectors for the transformation of yeast. Under certain circumstances, the plasmid is apparently able to replicate more than once per cell cycle; this over-replication allows the maintenance of the plasmid at high copy number. The plasmid has two inverted repeat sequences, and encodes a product that catalyses intra-molecular recombination between these two repeats. Models are proposed whereby recombination leads to copy number amplification. In particular, it is proposed that intra-molecular recombination during replication flips the orientation of one replication fork with respect to the other, so that both forks travel in the same direction around a circular monomer template, generating a large multimer from a monomer and a single initiation of replication.     

Kinetics of plasmid segregation in Escherichia coli

Low copy-number bacterial replicons occupy specific locations in their host cells. Production of a GFP-Lac repressor hybrid protein in cells carrying F or P1 plasmids tagged with a lac operator array reveals that in smaller (younger) cells these plasmids are seen mainly as a single fluorescent focus at mid-cell, whereas larger cells tend to have two foci, one at each quarter-cell position. Duplication of the central focus is presumed to represent active partition of plasmid copies. We report here our investigation by time-lapse microscopy of the subsequent movement of these copies to the quarter positions. Following duplication of the central focus, the new foci migrated rapidly and directly to their quarter-cell destinations, where they remained until the next cell cycle. The speed of movement was about five times faster than poleward migration of oriC and 50 times faster than cell elongation. Aberrant positioning of mini-F lacking its sopC centromere demonstrated the requirement for the partition system in this localization process. From the measured number of F plasmid copies per cell it appears that each migrating focus contains two or more plasmid molecules. The molecular basis of this clustering, and evidence for phasing of the partition event in the cell cycle, are discussed.     

Nonintegrated plasmid-chromosome complexes in Escherichia coli.

A number of plasmid systems have been examined for the ability of their covalently closed circular deoxyribonucleic acid (CCC DNA) forms to cosediment in neutral sucrose gradients with the folded chromosomes of their respective hosts. Given that cosedimentation of CCC plasmid and chromosomal DNA represents a bound or complexed state between these replicons, our results can be expressed as follows. (i) All plasmid systems complex, on the average, at least one plasmid per chromosomal equivalent. (ii) Stringently controlled plasmids exist predominantly in the bound state, whereas the opposite is true for plasmids that exist in multiple copies or are under relaxed control of replication. (iii) The degree to which a plasmid population binds to host chromosomes appears to be a function of plasmid genotype and not of plasmid size. (iv) For the colicin E1 plasmid the absolute number of plasmids bound per folded chromosome equivalent does increase as the intracellular plasmid/chromosome ratio increases in cells starved for required amino acids or in cells treated with chloramphenicol; however, the ratio of bound to free plasmids remains constant during plasmid copy number amplification.     

Epstein-Barr virus DNA is amplified in transformed lymphocytes.

Leukocytes isolated from two adult donors who lacked detectable antibodies to antigens associated with Epstein-Barr virus were exposed to an average of 0.02 to 0.1 DNA-containing particles of Epstein-Barr virus per cell and immediately clones in agarose. Within about 30 generations all transformed cell clones contained between 5 and 800 copies of viral DNA per cell. Only 1 in 10(4) to less than 1 in 10(5) of the cells of each clone release virus, and the frequency of release did not correlate with the average number of copies of viral DNA in the cells of each clone. One clone that had an average of five copies of viral DNA per cell was recloned, and the average number of copies in four of six subclones increased 15-to 50-fold while the subclones were being propagated sufficiently to study them. These results indicate that Epstein-Barr virus DNA can undergo amplification relative to cell DNA at different times after it transforms cells.     

Plasmid copy number control: isolation and characterization of high-copy-number mutants of plasmid pE194.

A plasmid, pE194, obtained from Staphylococcus aureus confers resistance to macrolide, lincosamide, and streptogramin type B ("MLS") antibiotics. For full expression, the resistance phenotype requires a period of induction by subinhibitory concentrations of erythromycin. A copy number in the range of 10 to 25 copies per cell is maintained during cultivation at 32 degrees C. It is possible to transfer pE194 to Bacillus subtilis by transformation. In B. subtilis, the plasmid is maintained at a copy number of approximately 10 per cell at 37 degrees C, and resistance is inducible. Tylosin, a macrolide antibiotic which resembles erythromycin structurally and to which erythromycin induces resistance, lacks inducing activity. Two types of plasmid mutants were obtained and characterized after selection on medium containing 10 microgram of tylosin per ml. One mutant class appeared to express resistance constitutively and maintained a copy number indistinguishable from that of the parent plasmid. The other mutant type had a 5- to 10-fold-elevated plasmid copy number (i.e., 50 to 100 copies per cell) and expressed resistance inducibly. Both classes of tylosin-resistant mutants were shown to be due to alterations in the plasmid and not to modifications of the host genome.     

Evaluating quantitative methods for measuring plasmid copy numbers in single cells

The life of plasmids is a constant battle against fluctuations: failing to correct copy number fluctuations can increase the plasmid loss rate by many orders of magnitude, as can a failure to more evenly divide the copies between daughters at cell division. Plasmids are therefore long-standing model systems for stochastic processes in cells, much thanks to the efforts of Kurt Nordstr?m to whose memory this issue is dedicated. Here we analyze a range of experimental methods for measuring plasmid copy numbers in single cells, focusing on challenges, trade-offs, and necessary experimental controls. In particular we analyze published and unpublished strategies to infer copy numbers from expression of plasmid-encoded reporters, direct labeling of plasmids with fluorescent probes or DNA binding proteins fused to fluorescent reporters, PCR based methods applied to single cell lysates, and plasmid-specific replication arrest. We conclude that no method currently exists to measure plasmid copy numbers in single cells, and that most methods are overwhelmed by various types of experimental noise. We also discuss how accurate methods can be developed.     

Clustering versus random segregation of plasmids lacking a partitioning function: a plasmid paradox?

Plasmids lacking a functional partition system are randomly distributed to the daughter cells; plasmid-free daughter cells are formed with a frequency of (1/2)2n per cell and cell generation where 2n is the (average) copy number at cell division. Hence, the unit of segregation is one plasmid copy. However, plasmids form clusters in the cells. A putative solution to this potential paradox is presented: one plasmid copy at a time is recruited from the plasmid clusters to the replication factories that are located in the cell centres. Hence, replication offers the means of declustering that is necessary in a growing host population. The daughter copies diffuse freely and each copy may with equal probability end up in either of the two cell halves. In this way, the random segregation of the plasmids is coupled to replication and occurs continuously during the cell cycle, and is not linked to cell division. The unit of segregation is the plasmid copy and not the plasmid clusters. In contrast, the two daughters of a Par+ plasmid are directed in opposite directions by the plasmid-encoded partition system, thereby assuring that each daughter cell receives the plasmid.     

Quantitative studies on ultrathin sections of bacteria infected with bacterio phage

A quantitative relationship has been established between the number of particles, for example bacteriophages, counted in ultrathin sections of bacteria and the total number present in the whole bacterial cells. The factor F relating particles counted per section with the total number of these particles per entire bacterium could be arrived at by two methods, which proved to give results in close agreement. The first involves knowledge of the average volume of a bacterial section in proportion to the average volume of a whole bacterium; if the mean number of appearances of the same particle on consecutive sections is also known, F may then be calculated. The thickness of sections and, therefore, their volume, as well as the average number of times a single particle is sectioned could be learned by examination of serial sections. By counting the relative number of T2 phage particles which had been intersected once or twice, and relating this proportion to the known phage dimensions, the thickness of the sections was determined to be about 400 A. The second measurement of F could be made in a particular case of late phage development where the number of particles per cell was countable or titratable directly in the bacterial lysate, this number being compared with the number seen in sections of the bacteria just before lysis. The different sources of errors are discussed. The statistical error is under 20 per cent, while the systematic errors are higher and cannot yet be indicated precisely. After a very cautious estimation of the upper limits, we can state, however, that the counts made with this method are certainly reliable to well within a factor of two.