Impact of plasmid architecture on stability and yEGFP3 reporter gene expression in a set of isomeric multicopy vectors in yeast

Multicopy episomal plasmids in yeast, used whenever elevated levels of foreign or homologous gene expression are necessary, are known to be less stable compared to the endogenous 2-μm plasmid they are based on, at least without selective pressure. Considering that rich medium favors growth rate and, simultaneously, is less expensive than selective medium, enhancing stability in non-selective medium is extremely desirable. In this study, we changed the architecture of a multicopy model expression plasmid, creating six isoforms (same size, same DNA content but different positions and orientations of the expression block) and studied mitotic stability, copy number, as well as reporter yEGFP3 expression between isoforms. With one isoform being significantly more stable than the others and another one exhibiting elevated plasmid copy numbers in rich medium, we show that consideration of the arrangement of the plasmid elements might be crucial for productivity employing Saccharomyces cerevisiae as a host. We strongly believe that the ideal architecture has to be assessed for each case and assembly strategy has to begin by evaluating the stability of the vector backbone before insertion of the desired gene. For the plasmid set studied, yEGFP3 reporter production depends more on mitotic stability than on elevated plasmid copy numbers in a small number of cells retaining the plasmid under non-selective conditions.

     

Replicating plasmids in Schizosaccharomyces pombe: improvement of symmetric segregation by a new genetic element.

We characterized a number of widely used yeast-Escherichia coli shuttle vectors in the fission yeast Schizosaccharomyces pombe. The 2 micron vectors pDB248 and YEp13 showed high frequency of transformation, intermediate mitotic and low meiotic stability, and a low copy number in S. pombe, analogous to their behavior in [cir0] strains of Saccharomyces cerevisiae. The S. cerevisiae integration vectors pLEU2 and pURA3 transformed S. pombe at very low frequencies but, surprisingly, in a nonintegrative fashion. Instead, they replicated autonomously, and they showed very high copy numbers (up to 150 copies per plasmid-containing cell). This could reflect a lack of sequence specificity for replication of plasmid DNA in S. pombe. pFL20, an S. pombe ars vector, and a series of plasmids derived from it were studied to analyze the unusually high stability of this plasmid. Mitotic stability and partitioning of the plasmids was measured by pedigree analysis of transformed S. pombe cells. An S. pombe DNA fragment (stb) was identified that stabilizes pFL20 by improvement of plasmid partitioning in mitosis and meiosis.     

Stability of recombinant plasmids containing the ars sequence of yeast extrachromosomal rDNA in several strains of Saccharomyces cerevisiae

The mitotic stabilities of hybrid plasmid Rcp21/11, which contains the replicator of yeast rDNA, have been compared for four yeast host strains of different origins. In two related strains, Saccharomyces cerevisiae A62-1G-P188 and 1A-P3812 from the Peterhof genetic stocks, the plasmid was much more stable than in strains DC5 and GRF18 from the USA stocks.The enhanced mitotic stability of Rcp21/11 in these two yeast strains is obviously attributable to a higher rate of integration of the plasmid into the chromosomal rDNA repeats of the hosts.The centromeric locus CEN3 was inserted into Rcp21/11 because it provides high mitotic and meiotic stability of plasmids with yeast replicators, due to an ordered distribution of plasmids throughout cell division. Using the new centromeric plasmid RcpCEN3, transformation of the four above-described yeast strains was carried out. It was found that, similarly to centromeric plasmids with other chromosomal replicators, RcpCEN3 remains in the cell as a single copy. In strains DC5, GRF18 and A62-1G-P188 the mitotic stability of RcpCEN3 was 20–50%, i.e., less than half that of plasmids containing locus CEN3 and other yeast repliiators, ars1, ars2 and the 2μ DNA replicator. The mitotic stability of RcpCEN3 in strains 1A-P3812 (from the Peterhof genetic stocks) for individual clones reached 85%, i.e. close to that of the other plasmids. Genetic analysis showed that the capacity of strain 1A-P3812 to stably retain RcpCEN3 has a recessive polygenic character. We suggest that the observed differences in mitotic stability of centromeric plasmid RcpCEN3 between various yeast strains reflects the differences in activity of rDNA replicator in these strains. The nature of extrachromosomal rDNA circles, found in some strains of S. cerevisiae, is discussed from the point of view of the data.     

Transformation of 12 different plasmids into soybean via particle bombardment

Particle bombardment offers a simple method for the introduction of DNA into plant cells. Multiple DNA fragments may be introduced on a single plasmid or on separate plasmids (co-transformation). To investigate some of the properties and limits of co-transformation, 12 different plasmids were introduced into embryogenic suspension culture tissue of soybean [Glycine max (L.) Merrill] via particle bombardment. The DNAs used for co-transformation included 10 plasmids containing KFLP markers for maize and 2 plasmids separately encoding hygromycin-resistance and ß-glucuronidase. Two weeks following bombardment with the 12 different plasmids, suspension culture tissue was placed under hygromycin selection. Hygromycin-resistant clones were isolated after an additional 5 to 6 weeks. Southern hybridization analysis of 26 hygromycin-resistant embryogenic clones verified the presence of introduced plasmid DNAs. All of the co-transforming plasmids were present in most of the transgenic soybean clones and there was no preferential uptake and integration of any of the plasmids. The copy number of individual plasmids was approximately equal within clones but highly variable between clones. While some clones contained as few as zero to three copies of each plasmid, others clones contained as many as 10 to 15 copies of each of the 12 different plasmids.     

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.     

The 2-micron plasmid as a nonselectable, stable, high copy number yeast vector

The endogenous 2-microns plasmid of Saccharomyces cerevisiae has been used extensively for the construction of yeast cloning and expression plasmids because it is a native yeast plasmid that is able to be maintained stably in cells at high copy number. Almost invariably, these plasmid constructs, containing some or all 2-microns sequences, exhibit copy number levels lower than 2-microns and are maintained stably only under selective conditions. We were interested in determining if there was a means by which 2-microns could be utilized for vector construction, without forfeiting either copy number or nonselective stability. We identified sites in the 2-microns plasmid that could be used for the insertion of genetic sequences without disrupting 2-microns coding elements and then assessed subsequent plasmid constructs for stability and copy number in vivo. We demonstrate the utility of a previously described 2-microns recombination chimera, pBH-2L, for the manipulation and transformation of 2-microns as a pure yeast plasmid vector. We show that the HpaI site near the STB element in the 2-microns plasmid can be utilized to clone yeast DNA of at least 3.9 kb with no loss of plasmid stability. Additionally, the copy number of these constructs is as high as levels reported for the endogenous 2-microns.     

Stable multicopy integration of vector sequences inHansenula polymorpha

Plasmids without an origin of replication, but bearing theURA3 gene ofSaccharomyces cerevisiae as a selective marker for transformation, are shown to replicate autonomously inHansenula polymorpha, indicating that parts of theS. cerevisiae URA3 gene can fulfil an autonomous replication and stabilization function inH. polymorpha. Such plasmids, replicated in low copy number in monomeric conformation, could be rescued inE. coli, and showed a low mitotic stability under selective and non-selective conditions. Selective propagation of such transformants, however, led to the integration of plasmid sequences into theH. polymorpha genome. The integration event usually occurred in high copy number (approx. 30–50) at a single non-homologous site of the genome. The plasmid sequences were found to be present in tandem array and stable under non-selective conditions. It contrast, the use of homologousURA3 gene under similar conditions led to low-copy-number transformants.     

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.     

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.     

Isolation and characterization of plasmid mutations that enable partitioning of pSC101 replicons lacking the partition (par) locus.

Second-site mutations that allow stable inheritance of partition-defective pSC101 plasmids mapped to seven distinct sites in the 5' half of the plasmid repA gene. While the mutations also elevated pSC101 copy number, there was no correlation between copy number increase and plasmid stability. Combinations of mutations enabled pSC101 DNA replication in the absence of integration host factor and also stabilized par-deleted plasmids in cells deficient in DNA gyrase or defective in DnaA binding. Our findings suggest that repA mutations compensate for par deletion by enabling the origin region RepA-DNA-DnaA complex to form under suboptimal conditions. They also provide evidence that this complex has a role in partitioning that is separate from its known ability to promote plasmid DNA replication.     

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.     

Yeast telomere repeat sequence (TRS) improves circular plasmid segregation, and TRS plasmid segregation involves the RAP1 gene product.

Telomere repeat sequences (TRSs) can dramatically improve the segregation of unstable circular autonomously replicating sequence (ARS) plasmids in Saccharomyces cerevisiae. Deletion analysis demonstrated that yeast TRSs, which conform to the general sequence (C(1-3)A)n, are able to stabilize circular ARS plasmids. A number of TRS clones of different primary sequence and C(1-3)A tract length confer the plasmid stabilization phenotype. TRS sequences do not appear to improve plasmid replication efficiency, as determined by plasmid copy number analysis and functional assays for ARS activity. Pedigree analysis confirms that TRS-containing plasmids are missegregated at low frequency and that missegregated TRS-containing plasmids, like ARS plasmids, are preferentially retained by the mother cell. Plasmids stabilized by TRSs have properties that distinguish them from centromere-containing plasmids and 2 microns-based recombinant plasmids. Linear ARS plasmids, which include two TRS tracts at their termini, segregate inefficiently, while circular plasmids with one or two TRS tracts segregate efficiently, suggesting that plasmid topology or TRS accessibility interferes with TRS segregation function on linear plasmids. In strains carrying the temperature-sensitive mutant alleles rap1grc4 and rap1-5, TRS plasmids are not stable at the semipermissive temperature, suggesting that RAP1 protein is involved in TRS plasmid stability. In Schizosaccharomyces pombe, an ARS plasmid was stabilized by the addition of S. pombe telomere sequence, suggesting that the ability to improve the segregation of ARS plasmids is a general property of telomere repeats.     

Identification and characterization of novel ColE1-type, high-copy number plasmid mutants in Legionella pneumophila

ColE1-type plasmids are commonly used in bacterial genetics research, and replication of these plasmids is regulated by interaction of RNA I and RNA II. Although these plasmids are narrow-host-range, they can be maintained in Legionella pneumophila under antibiotic selection, with low-copy number and instability. Here, we have described the isolation of two novel spontaneous mutants of pBC(gfp)Pmip, pBG307 and pBG309, which are able to mark the L. pneumophila with strong green fluorescence when exposed to visible light. One of the mutants, pBG307, has a single CG-->TA mutation in RNA II promoter located 2-bases upstream the - 10 region. Another one, pBG309, has the same mutation, as well as an additional CG-->AT mutation in the 76th nucleotide of RNA I, or in the 6th nucleotide of RNA II. A plasmid with the single mutation in RNA I, pBG308, was also constructed. Characterization of these plasmids carrying the enhanced green fluorescent protein (gfpmut2) gene revealed that the green fluorescence intensities of these plasmids were 2- to 30-fold higher than that of the wild type and both of the mutations contribute to increase the plasmid copy number and/or plasmid stability. The mutation located in RNA II promoter played a more dominant role in elevating the copy number, compared to the mutation in RNA I. We also tested the mutant plasmids for replication in Escherichia coli, and found that their copy number and stability were dramatically decreased, except pBG307. Our data suggest that these plasmids might be useful and convenient in genetic studies in L. pneumophila.     

Multimerization of high copy number plasmids causes instability: CoIE1 encodes a determinant essential for plasmid monomerization and stability

Although the natural multicopy plasmid CoIE1 is maintained stably under most growth conditions, plasmid cloning vectors related to it are relatively unstable, being lost at frequencies of 10(-2)-10(-5) per cell per generation. Evidence suggests that CoIE1 and related plasmids are partitioned randomly at cell division and that plasmid stability is correlated inversely with plasmid multimerization; factors or conditions that reduce multimerization increase stability. Cells containing plasmid multimers segregate plasmid-free cells because the multimers are maintained at lower copy numbers than monomers, as predicted by origin-counting models for copy number control. CoIE1 is stable because it encodes a determinant, cer, that is necessary for recA-, recF-, and recE-independent recombination events that efficiently convert any multimers to monomers. We have localized monomerizing and stability determinants of CoIE1 to within a 0.38 kb region that, when cloned into plasmid vectors, greatly increases their stability.     

Improving synthetic lethal screens by regulating the yeast centromere sequence.

The synthetic lethal screen is a useful method in identifying novel genes functioning in an alternative pathway to the gene of interest. The current synthetic lethal screen protocol in yeast is based on a colony-sectoring assay that allows direct visualization of mutant colonies among a large population by their inability to afford plasmid loss. This method demands an appropriate level of stability of the plasmid carrying the gene of interest. YRp-based plasmids are extremely unstable and complete plasmid loss occurs within a few generations. Consequently, YCp plasmids are the vector of choice for synthetic lethal screens. However, we found that the high-level stability of YCp plasmids resulted in a large number of false positives that must be further characterized. In this study, we attempt to improve the existing synthetic lethal screen protocol by regulating the plasmid stability and copy number. It was found that by placing a yeast centromere sequence under the control of either inducible or constitutive promoters, plasmid stability can be significantly decreased. Hence, altering the conditions under which yeast cells carrying the plasmid PGAL1-CEN4 were cultivated allowed us to develop a method that eliminated virtually 100% of false positives and drastically reduced the time required to carry out a synthetic lethal screen.     

Pedigree analysis of plasmid segregation in yeast

We have used pedigree analysis to investigate the mitotic segregation of circular and linear DNA plasmids in Saccharomyces cerevisae. Circular ARS plasmids, which bear putative chromosomal replication origins, have a high segregation frequency and a strong bias to segregate to the mother cell at mitosis. The segregation bias explains how the fraction of plasmid-bearing cells can be small despite the high average copy number of circular ARS plasmids. Linear ARS plasmids do not show strong segregation bias, nor does the 2 mu ori-containing plasmid YEp 13, when it is present in strains containing intact 2 mu circles. In the absence of endogenous 2 mu circles, YEp 13 behaves like an ARS plasmid, showing a strong maternal segregation bias. The presence of a centromere on circular ARS plasmids eliminates segregation bias. We discuss a model for plasmid segregation, which explains these findings and the possible biological significance of mother-daughter segregation bias.     

Identification and characterization of a novel allele of Escherichia coli dnaB helicase that compromises the stability of plasmid P1

Bacteriophage P1 lysogenizes Escherichia coli cells as a plasmid with approximately the same copy number as the copy number of the host chromosome. Faithful inheritance of the plasmids relies upon proper DNA replication, as well as a partition system that actively segregates plasmids to new daughter cells. We genetically screened for E. coli chromosomal mutations that influenced P1 stability and identified a novel temperature-sensitive allele of the dnaB helicase gene (dnaB277) that replaces serine 277 with a leucine residue (DnaB S277L). This allele conferred a severe temperature-sensitive phenotype to the host; dnaB277 cells were not viable at temperatures above 34 degrees C. Shifting dnaB277 cells to 42 degrees C resulted in an immediate reduction in the rate of DNA synthesis and extensive cell filamentation. The dnaB277 allele destabilized P1 plasmids but had no significant influence on the stability of the F low-copy-number plasmid. This observation suggests that there is a specific requirement for DnaB in P1 plasmid maintenance in addition to the general requirement for DnaB as the replicative helicase during elongation.