Plasmid stability in budding yeast populations: Steady-state growth with selection pressure

Plasmid gene product accumulation in a cell population depends on the fraction of plasmid-containing cells and the distribution of single-cell plasmid content. These important population properties have been related to plasmid replication regulation and kinetics and to plasmid segregation rules at the single-cell level using population balance mathematical models. Budding yeast populations are considered in detail because of the practical potential of yeast host-vector systems and because of the model complications introduced by the asymmetric division pattern observed for Saccharomyces cerevisiae at all but the largest growth rates. Solutions are presented for several different reasonable models of plasmid replication and segregation. The results offer potential for identification of important qualitative features of yeast plasmid replication and of model parameter values from average and segregated experimental data on yeast populations.     

Analysis of unstable recombinant Saccharomyces cerevisiae population growth in selective medium

Widely applied selection strategies for plasmid-containing cells in unstable recombinant populations are based upon synthesis in those cells of an essential, selection gene product. Regular partitioning of this gene product combined with asymmetric plasmid segregation produces plasmid-free cells which retain for some time the ability to grow in selective medium. This theory is elaborated here in terms of a segregated model for an unstable recombinant population which predicts population growth characteristics and composition based upon experimental data for stable strain growth kinetics, plasmid content, and selection gene product stability. Analytical solutions from this model are compared with an unsegregated phenomenological model to evaluate the effective specific growth rate of plasmid-free cells in selective medium. Model predictions have been validated using experimental growth kinetics and flow cytometry data for Saccharomyces cerevisiae D603 populations containing one of the plasmids YCpG1ARS1, YCpG1DeltaR8, YCpG1DeltaR88, YCpG1DeltaH103, YCpG1DeltaH200, pLGARS1, and pLGSD5. The recombinant strains investigated encompass a broad range of plasmid content (from one to 18 plasmids per cell) and probability alpha of plasmid loss at division (0.05 相似文献   

A structured, segregated model for genetically modified Escherichia coli cells and its use for prediction of plasmid stability

A structured, segregated model is presented for an asynchronously growing population of genetically modified Escherichia coli cells. A finite representation method was modified so that 272 cells could be used to represent a microbial population. The concept of a "limbo" compartment was introduced to allow random plasmid distribution to daughter cells upon cell division while restricting the number of computer cells included in the calculation. This scheme enabled us to predict plasmid instability and distribution of plasmid-originated properties in a population without a priori determination of growth rates or probability of forming plasmid-free cells from plasmid-containing cells. Predictions of population behavior using a single-cell model requires no adjustable parameters. The results comparing different induction strategies suggest that in continuous culture, there exists an optimum efficiency of partial induction that maximizes the long-term productivity of the gene product due to plasmid stability. With the optimum efficiency of partial induction, constant induction appears to prove more stable than cycling induction.     

Cell cycle analysis of plasmid-containing Escherichia coli HB101 populations with flow cytometry

The relationship between cell mass and cell number dynamics for bacteria such as Escherichia coli depends on the cell cycle parameters C and D. Effects of plasmid copy number on these cell cycle parameters have been studied for Escherichia coli HB101 containing pMB1 plasmids propagated at different copy numbers ranging from 12 to 122. Determination of cell cycle and cell size parameters was accomplished using flow cytometry data on single-cell light scattering and DNA content frequency functions in conjunction with a mathematical model of cell population statistics. Two independent methods for estimating C and D intervals based on flow cytometry were developed and applied with essentially identical results. The presence of plasmids decreases the C and D periods, mean cell sizes, and initiation masses for chromosome replication by 14, 24, 38, and 18%, respectively, relative to corresponding values for plasmid-free host cells. Plasmid copy number has a negligible influence on these parameters, suggesting that host-plasmid inter actions which determine these properties are centered on the single plasmid selected for replication according to the random selection model established for ColE1-type plasmids.     

Study of recombinant micro-organism populations characterized by their plasmid content per cell using a segregated model

Numerous observations from recombinant systems have shown that properties such as the specific cell growth rate and the plasmid-free cell formation rate are related, not only to the average plasmid content per cell, but also to the plasmid distribution within a population. The plasmid distribution in recombinant cultures can have an effect on the culture productivity that cannot be modelled using average values of the overall culture. The prediction of the behaviour of a plasmid content distribution and its causes and effects can only be studied using segregated models. A segregated model that describes populations of recombinant cells characterized by their plasmid content distribution has been developed. This model includes critical causes of recombinant culture instability such as the plasmid partition mechanism at cell division, plasmid replication kinetics and the effect of the plasmid content on the specific growth rate. The segregated model allows investigation of the effect of each of these causes and that of the plasmid content distribution on the observable behaviour of a recombinant culture.The effect of two partitioning mechanisms (Gaussian distribution and binomial distribution) on culture stability was investigated. The Gaussian distribution is slightly more stable. A small plasmid replication rate constant results in a very unstable culture even after short periods of time. This instability is dramatically improved for a larger value of this constant, hence improving protein synthesis. For a very narrow initial plasmid distribution, a given plasmid replication rate and partitioning mechanism can become broad even after a relatively short period of time. In contrast, a very "broad" initial distribution gave rise to a "Gamma-like" distribution profile. If we compare the results obtained in the simulations of the segregated model with those of the non-segregated one (average model), the latter model predicts much more stable behaviour, thus these average models cannot predict culture instability with the same precision.When compared with the experimental results, the segregated model was able to predict the practical behaviour with accuracy even in a system with a high plasmid content per cell and a high rate of plasmid-free cell formation which could not be achieved with a non-segregated model.     

A mathematical model for prediction of plasmid copy number and genetic stability in Escherichia coli

The design of bioreactors for genetically modified bacterial cultures would benefit from predictive models. Of particular importance is the interaction of the external environment, cell physiology, and control of plasmid copy number. We have recently developed a model based on the molecular mechanisms for control of replication of Co1E1 type plasmids. The inclusion of the plasmid model into a single-cell E. coli model allows the explicit prediction of the interaction of cell physiology and plasmid-encoded functions. The model predictions of the copy number of plasmids with the Co1E1 origin of replication carrying a variety of regulatory mutations is very close to that observed experimentally.All of the model parameters for plasmid replication control can be obtained independently and no adjustable parameters are needed for the plasmid model. In this article we discuss the model's use in predicting the effect of operating conditions on production of a protein from a plasmid encoded gene and the stability of the recombinant cells in a continuous culture.     

Effects of plasmid amplification and recombinant gene expression on the growth kinetics of recombinant E. coli

An experimental study was undertaken to identify and quantitate the effects of plasmid amplification and recombinant gene expression on Escherichia coli growth kinetics. Identification of these effects was possible because recombinant gene expression and plasmid copy number were controlled by different mechanisms on plasmid pVH106/172. Recombinant gene expression of the lactose operon structural genes was under the control of the lac promoter and was activated by the addition of the chemicals, IPTG and cyclic AMP, to the fermentation medium. Plasmid content was amplified in a separate fermentation by increasing culture temperature since the plasmid replicon was temperature-sensitive. A final fermentation was performed in which both plasmid content and recombinant gene expression were induced simultaneously by adding chemicals and raising the culture temperature. Recombinant growth rates were found to be reduced by the expression of high levels of recombinant lac proteins in the chemical induction experiments and by the amplification of plasmid levels in the temperature induction experiment. High expression of recombinant lac proteins following chemical induction was accompanied by a loss in recombinant cell viability. In the plasmid amplification experiment, the recombinant cells did not lose viability but the recombinant product yields were much lower than those achieved in the chemical induction experiments. Combining temperature and chemical induction increased the recombinant product yield by a factor of 4400 but also lowered cellular growth rates by 70%.     

Effects of recombinant plasmid content on growth properties and cloned gene product formation in Escherichia coli

Plasmid-host cell interactions have been investigated experimentally using Escherichia coli HB101, plasmid RSF1050 which contains the origin of replication of pMB1, and four other closely related copy number mutant plasmids. Growth characteristics of these recombinant strains and beta-lactamase activity expressed from a plasmid gene were investigated in Luria broth (LB) and in minimal medium (M9) containing in some cases casamino acids or different concentrations of alpha-methylglucoside, a competitive inhibitor of glucose transport. Maximum specific growth rates in LB and minimal media were reduced for increasing plasmid content per cell. Plasmid copy number increased when specific growth rate was reduced by changing medium composition. Growth rates of high copy number strains were less sensitive to alpha-methylglucoside than lower copy number strains and the plasmidfree host. The overall efficiency of plasmid gene expression, measured as the ratio of beta-lactamase specific activity to plasmid content, decreased significantly with increasing plasmid content in LB medium.     

Multileveled selection on plasmid replication

The replication control genes of bacterial plasmids face selection at two conflicting levels. Plasmid copies that systematically overreplicate relative to their cell mates have a higher chance of fixing in descendant cells, but these cells typically have a lower chance of fixing in the population. Apart from identifying the conflict, this mathematical discussion characterizes the efficiency of the selection levels and suggests how they drive the evolution of kinetic mechanisms. In particular it is hypothesized that: (1) tighter replication control is more vulnerable to selfishness; (2) cis-acting replication activators are relics of a conflict where a plasmid outreplicated its intracellular competitors by monopolizing activators; (3) high-copy plasmids with sloppy replication control arise because intracellular selection favors overreplication, thereby relieving intercellular selection for lower loss rates; (4) the excessive synthesis of cis-acting replication activators and trans-acting inhibitors is the result of an arms race between cis selfishness and trans retaliations; (5) site-specific recombination of plasmid dimers is equivalent to self-policing; and (6) plasmids modify their horizontal transfer to spread without promoting selfishness. It is also discussed how replication control may be subject to a third level of selection acting on the entire population of plasmid-containing cells.     

Effect of growth rate on the maintenance of a recombinant plasmid inBacillus subtilis

Summary The rate of fall in the proportion of plasmid-containing cells in a population ofBacillus subtilis 1A297[pVC102] grown in continuous culture was independent of growth rate. Plasmid loss could not be ascribed to faulty partitioning during cell division. At a low dilution rate, the specific rate of plasmid loss exceeded the specific growth rate of the plasmid-containing cells.     

Expression of tetracycline resistance in pBR322 derivatives reduces the reproductive fitness of plasmid-containing Escherichia coli

Plasmid pBR322 and its numerous derivatives are used extensively for research and in biotechnology. The tetracycline-resistance (TcR) genes in these plasmids are expressed constitutively and cells carrying these plasmids are resistant to tetracycline. We have shown that expression of the TcR gene has an adverse effect on the reproductive fitness of plasmid-containing bacteria in both glucose-limited batch and chemostat cultures. If the TcR genes are inactivated at any one of three different restriction sites, mixed cultures of plasmid-free and plasmid-containing bacteria grow at the same rate.     

Stability studies of recombinant Saccharomyces cerevisiae in the presence of varying selection pressure

A recombinant yeast plasmid carrying the Ieu2 gene for auxotrophic complementation and a reporter gene for beta-galactosidase under the control of Gal10 promoter was studied in Saccharomyces cerevisiae. Growth, product formation, and plasmid stability were studied in defined, semi-defined, and complex media. The biomass concentration and specific activity were higher in complex medium than in defined medium, which was selective for the growth of plasmid-containing cells, leading to a 10-fold increase in volumetric activity. However, plasmid instability was very high in complex media with 50% plasmid-free cells emerging in the culture within 75 h of cultivation. In order to control instability, the growth rates of the plasmid-containing and plasmid-free cells were determined in semi-defined media, which consisted of defined medium supplemented with different concentrations of yeast extract. Below a critical concentration of yeast extract (0.05 g/L), the plasmid-containing cells had a growth rate advantage over the plasmid-free cells. This was possibly because, at this concentration of yeast extract, the availability of leucine became the rate-determining factor in the specific growth rate of plasmid-free cells. A feeding strategy was designed which maintained a low concentration of the residual yeast extract in the medium and thus continuously provided the plasmid-containing cells with a competitive advantage over the plasmid-free cells. This resulted in high stability as well as high cell density under non-selective conditions, which led to a 10-fold increase in the volumetric activity compared to that achieved in defined selective media. A simple mathematical model was formulated to verify the experimental data. The important state variables and process parameters, i.e., biomass concentration, beta-galactosidase expression, sucrose consumption, yeast extract consumption, and specific growth rates of the two cell populations, were evaluated. These variables and parameters along with the differential equations based on material balances as well as the experimental results obtained were used in a mathematical model for the fed-batch cultivation. These correctly verified the experimental data and clearly illustrated the concept behind the success of the fed-batch strategy under yeast extract starvation.     

Control of Plasmid R1 Replication: Kinetics of Replication in Shifts Between Different Copy Number Levels

Plasmid R1 replication was studied in shifts between two steady states of copy number. The copy number was varied in two ways. First, we utilized the fact that it decreases with increasing growth rate. To minimize the metabolic effects of changes in the growth rate, the downshifts were obtained by adding α-methylglucoside to cultures growing in glucose-minimal medium, and the upshifts were obtained by adding glucose to cultures growing in the presence of glucose plus α-methylglucoside. Second, we used a temperature-dependent copy mutant of plasmid R1 (pKN301). Plasmid pPK301 shows a threefold higher copy number at 40 than at 30°C. In both types of shift, plasmid replication immediately adjusted to the postshift differential rate. The copy number asymptotically adjusted to the new steady state. Hence, the system that controls plasmid R1 replication sets the frequency of replication without measuring the actual copy number. It has been suggested that plasmid R1 replication is under negative control by an R1-mediated repressor protein. Among the replication control models that involve negative control, the Pritchard inhibitor dilution model, the Sompayrac-Maaløe autorepressor model, and the plasmid λdv system all predict gene dose-independent copy number control.     

The construction and monitoring of genetically marked, plasmid-containing, naphthalene-degrading strains in soil

A genetically marked, plasmid-containing, naphthalene-degrading strain, Pseudomonas putida KT2442(pNF142::TnMod-OTc), has been constructed. The presence of the gfp gene (which codes for green fluorescent protein) and the kanamycin and rifampicin resistance genes in the chromosome of this strain allows the strain's fate in model soil systems to be monitored, whereas a minitransposon, built in naphthalene biodegradation plasmid pNF142, contains the tetracycline resistance gene and makes it possible to follow the horizontal transfer of this plasmid between various bacteria. Plasmid pNF142::TnMod-OTc is stable in strain P. putida KT2442 under nonselective conditions. The maximal specific growth rate of this strain on naphthalene was found to be higher than that of the natural host of plasmid pNF142. When introduced into a model soil system, the genetically marked strain is stable and competitive for 40 days. The transfer of marked plasmid pNF142::TnMod-OTc to natural soil bacteria, predominantly fluorescent pseudomonads, has been detected.     

Maintenance stability of the pBR322 and pBR327 vector plasmids in Escherichia coli cells during multiple passages

Stability of pBR322 and pBR327 plasmids was studied. Plasmid-containing Escherichia coli strains were grown in liquid growth medium without selection pressure. Plasmid pBR327 was shown to be more stable in E. coli CSH54 cells than pBR322. Essential heterogenity of individual plasmid-containing clones was recognized by the maintenance stability of plasmid DNA. The indicated clones with high stability failed to be cured from pBR327 plasmid by means of acridine orange. High stability of plasmid maintenance and the failure to cure cells containing this plasmid are suggested to correlate with and to be essentially determined by the cell functions.     

Plasmid instability kinetics of the yeast S288C pUCKm8 [cir+] in non-selective and selective media

Plasmid loss kinetics for Saccharomyces cerevisiae transformed with the 2-mum DNA-based-plasmid pUCKm8 were measured in nonselective and selective media. The plasmid pUCKm8 gives the organism two new phenotypes: resistance to the wide spectrum antibiotic G418 sulfate, and the ability to produce the enzyme, beta-lactamase. Plasmid stability was determined using the production of beta-lactamase as a marker. The effect of G418 on the growth rates of all organisms present in the culture and on plasmid stability was also determined. Mathematical models describing plasmid loss kinetics during exponential growth for both nonselective and selective conditions are used to simulate the experimental data. In nonselective medium, over 80% of the cells still exhibited the desired phenotype after 50 doublings. In medium containing G418, improvements in plasmid stability were only marginal due to the appearance of antibiotic-resistant cells.     

Kinetics of benzoate-induced loss of the TOL plasmid from Pseudomonas putida MT15 during growth in chemostat culture

Potassium-limited chemostat cultures of Pseudomonss putida MT15, grown on excess glucose, displayed approximately 100% plasmid loss after 60 generations of growth in the presence of 5 mM benzoate. The kinetics of plasmid loss indicated that plasmid-free cells displayed a growth rate advantage, which we attribute to selective inhibition of the growth of plasmid-containing cells by benzoate. However, stable, mixed populations of plasmid-free cells, deletants and plasmid-containing cells were selected during growth under glucose limitation in the presence of benzoate. This behaviour indicated that the plasmid-free cells in these cultures displayed a growth rate disadvantage and that their appearance was due entirely to benzoate-induced segregational instability of the plasmid.     

Fate of cloned bacteriophage T4 DNA after phage T4 infection of clone-bearing cells

Plasmid pBR322 replication is inhibited after bacteriophage T4 infection. If no T4 DNA had been cloned into this plasmid vector, the kinetics of inhibition are similar to those observed for the inhibition of Escherichia coli chromosomal DNA. However, if T4 DNA has been cloned into pBR322, plasmid DNA synthesis is initially inhibited but then resumes approximately at the time that phage DNA replication begins. The T4 insert-dependent synthesis of pBR322 DNA is not observed if the infecting phage are deleted for the T4 DNA cloned in the plasmid. Thus, this T4 homology-dependent synthesis of plasmid DNA probably reflects recombination between plasmids and infecting phage genomes. However, this recombination-dependent synthesis of pBR322 DNA does not require the T4 gene 46 product, which is essential for T4 generalized recombination. The effect of T4 infection on the degradation of plasmid DNA is also examined. Plasmid DNA degradation, like E. coli chromosomal DNA degradation, occurs in wild-type and denB mutant infections. However, neither plasmid or chromosomal degradation can be detected in denA mutant infections by the method of DNA--DNA hybridization on nitrocellulose filters.     

An adjustable expression system for controlling growth rate, plasmid maintenance, and culture dynamics

Recombinant bacterial cells express various levels of model product proteins if the genes of interest are regulated by controllable promoters. The level of gene expression influences the growth-rate differential between plasmid-bearing and plasmid-free cells, and thereby affects the culture dynamics of a plasmid-containing cell population. An expression system has been designed in which host Escherichia coli cells contain the pil operon controlled by a tac promoter; these cells are transformed with plasmids that contain the repressor gene, lacl, for the tac promoter, in combination with an expression system for a model protein, chloramphenicol acetyl tranferase (CAT). Experimental and theoretical results show that plasmid-bearing cells can be maintained as dominant in continuous cultures without selective pressure when 12% or less of the cells' total protein is the model product protein, CAT. This is because the segment cells produce pili greatly in excess of normal wild-type levels, and thus have more of a metabolic burden than do the plasmid-bearng cells that overproduce CAT. However, when the level of the plasmid-directed CAT expression is increased above 12% of the cells' total protein, the growth rate of the plasmid-bearing cells decreases to a value lower than that of the segregant cells. Therefore, plasmid-containing cells lose their selective advantage at this expression level, and cannot be maintained as the dominant cell type in a continuous culture unless antibiotic or other positive selection methods are used. By controlling the growth rate differential of this bacterial host/plasmid system, a variety of interesting competitive culture dynamics is investigated. All experimental measurements for continuous cultures are in very good agreement with theory using kinetic parameters determined from independent batch experiments. (c) 1992 John Wiley & Sons, Inc.     

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.