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.     

Replication properties of pIM13, a naturally occurring plasmid found in Bacillus subtilis, and of its close relative pE5, a plasmid native to Staphylococcus aureus.

A naturally occurring plasmid from Bacillus subtilis, pIM13, codes for constitutively expressed macrolide-lincosamide-streptogramin B (MLS) resistance, is stably maintained at a high copy number, and exists as a series of covalent multimers. The complete sequence of pIM13 has been reported (M. Monod, C. Denoya, and D. Dubnau, J. Bacteriol. 167:138-147, 1986) and two long open reading frames have been identified, one of which (ermC') is greater than 90% homologous to the ermC MLS resistance determinant of the Staphylococcus aureus plasmid pE194. The second reading frame (repL) shares homology with the only long open reading frame of the cryptic S. aureus plasmid pSN2 and is probably involved in plasmid replication. The map of pIM13 is almost a precise match with that of pE5, a naturally occurring, stable, low-copy-number, inducible MLS resistance plasmid found in S. aureus. pIM13 is unstable in S. aureus but still multimerizes in that host, while pE5 is unstable in B. subtilis and does not form multimers in either host. The complete sequence of pE5 is presented, and comparison between pIM13 and pE5 revealed two stretches of sequence present in pE5 that were missing from pIM13. It is likely that a 107-base-pair segment in the ermC' leader region missing from pIM13 accounts for the constitutive nature of the pIM13 MLS resistance and that the lack of an additional 120-base-pair segment in pIM13 that is present on pE5 gives rise to the high copy number, stability, and multimerization in B. subtilis. The missing 120 base pairs occur at the carboxyl-terminal end of the putative replication protein coding sequence and results in truncation of that protein. It is suggested either that the missing segment contains a site involved in resolution of multimers into monomers or that the smaller replication protein causes defective termination of replication. It is concluded that pIM13 and pE5 are coancestral plasmids and it is probable that pIM13 arose from pE5.     

Stability of ColE1-like and pBR322-like plasmids in Escherichia coli

The average copy number, the level of ampicillin resistance conferred by one plasmid, and the degree of plasmid multimerization were determined for several ColE1-like and pBR322-like plasmids. From the results obtained, the variance of the units of partition corresponding to each plasmid studied was calculated. Experimentally determined plasmid stability was compared with that calculated using the variance of the units of partition and the ratio between the generation times of plasmid-free and of plasmid-carrying cells, assuming that the units of partition are distributed randomly between daughter cells. Stability of the pBR322-like plasmids present mainly as monomers in the bacterial host was consistent with random partitioning, whereas pBR322-like plasmids, present mainly as dimers, and the ColE1-like plasmid showed greater stability than that predicted with random partitioning at cell division.     

An inverted repeat sequence of the IncFI plasmid ColV2-K94 increases multimerization-mediated plasmid instability

A detailed physical map of the region of the IncFI plasmid ColV2-K94 containing the Rep1 replicon, a Tn903 transposon, and an inverted repeat structure (X1) with unknown properties was prepared by cloning restriction fragments into pBR325. Inserts carrying the 1.2 kb repeated sequence of X1, but not the IS903 sequence of Tn903, had a destabilizing effect on pBR325 and pBR322 plasmid maintenance. One of these derivatives, pWS139, was studied further and was shown to have elevated levels of multimeric DNA forms; this resulted in decreased copy number and plasmid instability, as multimerization reduces the effective number of randomly segregating plasmids per cell. A ColV2-K94 miniplasmid, which has a copy number much lower than that of ColE1-derived vectors, was also less stably inherited if it contained the X1 structure. This destabilizing effect of the X1 repeat sequence was dependent on the RecA function, but not the RecB or the RecC functions of the host. These results suggest that the inverted repeat sequence of the X1 structure serves as a 'hot-spot' for generalized recombination. Thus, when present in cis, this sequence can generate plasmid instability because plasmid molecules are readily converted into multimeric forms through enhanced recombination at this site.     

Analysis of the initiating events in HIV-1 particle assembly and genome packaging

HIV-1 Gag drives a number of events during the genesis of virions and is the only viral protein required for the assembly of virus-like particles in vitro and in cells. Although a reasonable understanding of the processes that accompany the later stages of HIV-1 assembly has accrued, events that occur at the initiation of assembly are less well defined. In this regard, important uncertainties include where in the cell Gag first multimerizes and interacts with the viral RNA, and whether Gag-RNA interaction requires or induces Gag multimerization in a living cell. To address these questions, we developed assays in which protein crosslinking and RNA/protein co-immunoprecipitation were coupled with membrane flotation analyses in transfected or infected cells. We found that interaction between Gag and viral RNA occurred in the cytoplasm and was independent of the ability of Gag to localize to the plasma membrane. However, Gag:RNA binding was stabilized by the C-terminal domain (CTD) of capsid (CA), which participates in Gag-Gag interactions. We also found that Gag was present as monomers and low-order multimers (e.g. dimers) but did not form higher-order multimers in the cytoplasm. Rather, high-order multimers formed only at the plasma membrane and required the presence of a membrane-binding signal, but not a Gag domain (the CA-CTD) that is essential for complete particle assembly. Finally, sequential RNA-immunoprecipitation assays indicated that at least a fraction of Gag molecules can form multimers on viral genomes in the cytoplasm. Taken together, our results suggest that HIV-1 particle assembly is initiated by the interaction between Gag and viral RNA in the cytoplasm and that this initial Gag-RNA encounter involves Gag monomers or low order multimers. These interactions per se do not induce or require high-order Gag multimerization in the cytoplasm. Instead, membrane interactions are necessary for higher order Gag multimerization and subsequent particle assembly in cells.     

Multicopy plasmid instability: the dimer catastrophe hypothesis

Multimer formation reduces plasmid copy number and is an established cause of segregational instability. Nevertheless, it is difficult to rationalize observations that low levels of dimers can cause severe instability, if we assume they are distributed evenly in cell populations. We report here that dimer distribution is in fact heterogeneous in recombination-proficient strains. Most cells in the population contain only monomers; dimers are confined to a small sub-population from which plasmid-free daughters arise at high frequency. In a rec⁺ culture where 4% of pBR322 molecules are dimers, more than half are in dimer-only cells. We show that this situation is inevitable because dimers replicate at twice the rate of monomers. Runaway multimerization is avoided because dimer-containing cells grow more slowly than their monomer-containing counterparts. A computer simulation is used to show how dimers proliferate after formation by homologous recombination. The equilibrium concentration of dimers is proportional to the inter-plasmid recombination rate and is essentially independent of the rate at which homologous recombination converts dimers to monomers.     

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.     

Concerted action of plasmid maintenance functions: partition complexes create a requirement for dimer resolution

Partition of prokaryotic DNA requires formation of specific protein-centromere complexes, but an excess of the protein can disrupt segregation. The mechanisms underlying this destabilization are unknown. We have found that destabilization by the F plasmid partition protein, SopB, of plasmids carrying the F centromere, sopC, results from the capacity of the SopB-sopC partition complex to stimulate plasmid multimerization. Mutant SopBs unable to destabilize failed to increase multimerization. Stability of wild-type mini-F, whose ResD/rfsF site-specific recombination system enables it to resolve multimers to monomers, was barely affected by excess SopB. Destabilization of plasmids lacking the rfsF site was suppressed by recF, recO and recR, but not by recB, mutant alleles, indicating that multimerization is initiated from single-strand gaps. SopB did not alter the amounts or distribution of replication intermediates, implying that SopB-DNA complexes do not create single-strand gaps by blocking replication forks. Rather, the results are consistent with SopB-DNA complexes channelling gapped molecules into the RecFOR recombination pathway. We suggest that extended SopB-DNA complexes increase the likelihood of recombination between sibling plasmids by keeping them in close contact prior to SopA-mediated segregation. These results cast plasmid site-specific resolution in a new role - compensation for untoward consequences of partition complex formation.     

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.     

Maintenance of the 2 microns circle plasmid in populations of Saccharomyces cerevisiae.

The 2 microns circle plasmid is maintained at high frequencies in populations of yeast cells. To find out how the plasmid is maintained, three forces were measured: the selective advantage or disadvantage conferred by 2 microns circles, the rate of generation of [Cir0] cells, and the rate of illegitimate transfer of 2 microns circles from cell to cell. It was found that under the conditions used, 2 microns circles confer a selective disadvantage of about 1%, that [Cir0] cells are generated at the rate of 7.6 x 10(-5) per [Cir+] cell per generation, and that illegitimate transfer of 2 microns circles occurs at a rate less than 10(-7) per recipient cell per generation. The most likely explanation of 2 microns circle maintenance is that the plasmid is sexually transmitted at such a rate that it spreads through populations despite selection against it.     

Interplay of SOS induction,recombinant gene expression,and multimerization of plasmid vectors in Escherichia coli

Using pBR322- and pUC-derived plasmid vectors, a homologous (Escherichia coli native esterase) and three heterologous proteins (human interleukin-2, human interleukin-6, and Zymomonas levansucrase) were synthesized in E. coli IC2015(recA::lacZ) and GY4786 (sfiA::lacZ) strains. Via time-course measurement of beta-galactosidase activity in each recombinant culture, the SOS induction was estimated in detail and the results were systematically compared. In recombinant E. coli, the SOS response did not happen either with the recombinant insert-negative plasmid backbone alone or the expression vectors containing the homologous gene. Irrespective of gene expression level and toxic activity of synthesized foreign proteins, the SOS response was induced only when the heterologous genes were expressed using a particular plasmid vector, indicating strong dependence on the recombinant gene clone and the selection of a plasmid vector system. It is suggested that in recombinant E. coli the SOS response (i.e., activation of recA expression and initial sfiA expression) may be related neither to metabolic burden nor toxic cellular event(s) by synthesized heterologous protein, but may be provoked by foreign gene-specific interaction between a foreign gene and a plasmid vector. Unlike in E. coli XL1-blue(recA(-)) strains used, all expression vectors encoding each of the three heterologous proteins were multimerized in E. coli IC2015 strains in the course of cultivation, whereas the expression vectors containing the homologous gene never formed the plasmid multimers. The extent of multimerization was also dependent on a foreign gene insert in the expression vector. As a dominant effect of the SOS induction, recombinant plasmid vectors used for heterologous protein expression appear to significantly form various multimers in the recA(+) E. coli host.     

The Mode of Replication Is a Major Factor in Segregational Plasmid Instability in Lactococcus lactis

The effects of the rolling-circle and theta modes of replication on the maintenance of recombinant plasmids in Lactococcus lactis were studied. Heterologous Escherichia coli or bacteriophage λ DNA fragments of various sizes were inserted into vectors based on either the rolling-circle-type plasmid pWV01 or the theta-type plasmid pAMβ1. All pAMβ1 derivatives were stably maintained. pWV01 derivatives, however, showed size-dependent segregational instability, in particular when large DNA fragments were inserted. All recombinant pWV01 derivatives generated high-molecular-weight plasmid multimers (HMW) in amounts that were positively correlated with plasmid size and inversely correlated with the copy numbers of the monomeric plasmid forms. Formation of HMW or reductions in copy numbers were not observed with pAMβ1 derivatives. The results indicate that HMW formation and/or reduction in plasmid copy numbers is an important factor in the maintenance of pWV01 derivatives. It is concluded that theta-type plasmids are superior to rolling-circle-type plasmids for cloning in lactococci.     

Nuclear plasmids in the Dictyostelium slime molds

Cellular slime molds are one of only three types of eukaryotes known to contain circular nuclear plasmids. Unlike the 2-microns circle in Saccharomyces, different strains of Dictyostelium can carry different, nonhomologous plasmids. Covalently closed, circular DNA plasmids have been identified in D. discoideum, D. mucoroides, D. giganteum, and D. purpureum. These plasmids range in size from 1.3-27 kb and in copy number from 50-300 molecules per cell. Plasmids have been identified in approximately one-fifth of all isolates examined. The organization of their DNA in nucleosomes establishes their presence in the nucleus. We have successfully cotransformed endogenous Dictyostelium plasmids into D. discoideum using the G418 resistance shuttle vector B10S. Transformants carrying D. discoideum plasmids are recovered at much higher frequency than those carrying plasmids from the other Dictyostelium species. We have constructed recombinant plasmids based on the D. discoideum plasmid Ddp2 and the G418 resistance gene. With these extrachromosomal vectors, transformed cells are recovered at frequencies of up to 10(-4) per input cell, the vectors are stably maintained at high copy number in the absence of selection, and the vectors can be used to introduce foreign DNA sequences into D. discoideum cells.     

Chromosomal targeting of replicating plasmids in the yeast Hansenula polymorpha.

Using an optimized transformation protocol we have studied the possible interactions between transforming plasmid DNA and the Hansenula polymorpha genome. Plasmids consisting only of a pBR322 replicon, an antibiotic resistance marker for Escherichia coli and the Saccharomyces cerevisiae LEU2 gene were shown to replicate autonomously in the yeast at an approximate copy number of 6 (copies per genome equivalent). This autonomous behaviour is probably due to an H. polymorpha replicon-like sequence present on the S. cerevisiae LEU2 gene fragment. Plasmids replicated as multimers consisting of monomers connected in a head-to-tail configuration. Two out of nine transformants analysed appeared to contain plasmid multimers in which one of the monomers contained a deletion. Plasmids containing internal or flanking regions of the genomic alcohol oxidase gene were shown to integrate by homologous single or double cross-over recombination. Both single- and multi-copy (two or three) tandem integrations were observed. Targeted integration occurred in 1-22% of the cases and was only observed with plasmids linearized within the genomic sequences, indicating that homologous linear ends are recombinogenic in H. polymorpha. In the cases in which no targeted integration occurred, double-strand breaks were efficiently repaired in a homology-independent way. Repair of double-strand breaks was precise in 50-68% of the cases. Linearization within homologous as well as nonhomologous plasmid regions stimulated transformation frequencies up to 15-fold.     

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.     

A host-vector system for an Arthrobacter species

An efficient host-vector system has been developed for an industrial strain of Arthrobacter sp. (NRRL B3728)used for glucose isomerase production. Protoplasts of Arthrobacter were generated by treating the cells with 0.5 mg lysozyme ml(-1) for 60 min in a solution containing 0.5 M-sucrose. Around 30% of the protoplasts regenerated on agar containing 0.5 M-sodium succinate as osmotic stabilizer. Three hybrid vectors, PBL2100, pCG1100 and pCG2100, were constructed by combining the Escherichia coli plasmid pBR322, a kanamycin- resistance gene from pNCAT4 and a cryptic plasmid from either Brevibacterium lactofermentum NCIB 9567 or Corynebacterium glutamicum NCIB 10026. These vectors transformed the protoplasts and expressed the kanamycin-resistance gene for screening. They contain a number of unique restrictions sites for cloning of foreign DNA. The transformation frequency of this system was 10(5)-10(6) transformants per micrograms of input plasmid and ws constant up to 5 micrograms of DNA. the probability of a plasmid transforming a plasmid transforming a protoplast was in the range 10(-5)-10(-6). The copy number of pBL2100 was around 5 per cell and those of pCG1100 and pCG2100 were around 33 per cell. Deletion mutants were generated from pCG2100. One of them, pCG2120, was able to transform protoplasts of strain NRRL B3728. Plasmids pBL2100 and pCG2100 were structurally stable in cells of NRRL B3728 but could not be maintained in non-selective medium. They segregated at a rate of 12.2 and 2.2% per generation respectively.     

Effect of high density cultivation on plasmid copy number in recombinantEscherichia coli cells

Summary The copy number of plasmid harboringE. coli K 12 strains was examined in fed batch cultivations in semisynthetic and synthetic media. Under conditions of high cell density (45–50 g dry weight/I) the plasmid copy number reached a maximum level between 200 and 400 copies per cell. A decrease of phosphate concentration in the medium was obtained similar to the increase of copy number. A high segregational and structural stability of vectors used in this work was observed.