Replication of the mini-R1 plasmid Rsc11 and Rsc11 hybrid plasmids.

Replication of the multicopy mini-R1 plasmid, Rsc11, is dependent on host replication functions dna A, B, C, E and G but independent of polA1. Chloramphenicol immediately stops its replication. A stable relaxation complex is not formed. Composite plasmids were constructed with Rsc11 and other small replicons like pSC101, ColE1 and mini-ColE1. In all combinations the amount of hybrid plasmid DNA in the cell never exceeds the amount of Rsc11 DNA itself. This leads to varying copy numbers of the hybrid plasmids depending on the size of the second plasmid. Replication of the composite plasmids proceeds probably always under the control of the Rsc11 part although the second replicon is still functional. The composite plasmids are incompatible with both the parent replicons.     

Site specific recombination involved in the generation of small plasmids

Summary The physical structures of seven small plasmids, Rsc10, Rsc11, Rsc12, Rsc13, Rsc15, Rsc10-1 and pEM1 were analyzed. Molecular lengths of these plasmids were determined to range from 7.65 to 19.8 kilobases or kb. Electron microscope heteroduplex analysis of these plasmids show that the plasmids were all derived from pKN102 (86.3kb) in a complicated process that takes place by a series of deletion and, in some cases, transposition events. Rsc10 and Rsc11 were each formed by a simple deletion event from the parental plasmid. The physical structures of Rsc13 and pEM1 suggest that these plasmids must have been derived by a single and two successive deletion events from Rsc11. In the formation of these plasmids, all the deletions occured at the ends of the transposon, Tn3, which confers ampicillin resistance (amp) to the plasmid, or at the ends of the insertion sequence, IS1. Rsc15 was assumed to be formed in a two step process. The first step was a deletion event to form Rsc10-1 which occurs at one end of the IS1 present in pKN102. At first, the deletion event leaves out the ampicillin gene but in the second step Tn3 is transposed to the newly formed plasmid, Rsc10-1. Rsc12 is believed to have been formed in a similar fashion; first, a series of deletions and second, the transposition of Tn3.Studies on these small plasmids enabled us to also map the regions of the replication genes and ampicillin resistance on pKN102.     

In vitro system for the replication of the mini R1 factor Rsc11

Summary The in vitro synthesis of the mini R1-factor, Rsc11, was achieved using a soluble Escherichia coli cell-extract system. Triton X-100 lysates of the K 12 strain 1101 (Rsc11) fractionated by Sephadex G25 chromatography supported the incorporation of labeled deoxyribonucleotides into covalently-closed circular (30S) and open-circular (25S) plasmid DNA as well as other molecules of various sizes. DNA synthesis required the presence of all four ribonucleotides and was rifampicin sensitive. Pulse-chase experiments indicated that this reaction is discontinuous. A dependence on ATP and sensitivity to nalidixic acid suggested this system capable of the replicative synthesis of Rsc11 DNA. Density-shift analysis confirmed this. In addition to hybrid, fully-heavy plasmid supercoils were synthesized indicating that more than one round of replication was completed. Approximately one-third of the molecules available to the system participate in this reaction.     

Analysis of plasmid genome evolution based on nucleotide-sequence comparison of two related plasmids of Escherichia coli

Plasmid Rsc13, a small derivative of the plasmid R1, contains a region necessary for replication as well as a complete copy (4957 bp) of the ampicillin resistance transposon, Tn3. We determined the nucleotide sequence of the replication region of Rsc13 to be 2937 bp and then compared this region (designated the 2.9-kb region) to the analogous region of pSM1, a small derivative of the plasmid R100 which has common ancestry with R1. Rsc13 and pSM1 were 96% homologous in this 2.9-kb region except for a discrete region of about 250 bp which showed only 44% homology. The sequence and distribution of nucleotide substitutions between Rsc13 and pSM1 supported a map of possible genes and sites which have previously been seen in the replication region of Rsc13 and pSM1 which showed only 44% homology. Analysis of the amino acid sequence and predicted conformation of the two RepA2 polypeptides, however, suggested that they were very similar. We proposed that the repA2 region of R1 and R100 was replaced by a substitution of a short DNA segment from another plasmid which was evolutionarily related to R1 and R100 but had more divergence. This event may have been mediated by a mechanism similar to that of gene conversion as described in eukaryotic systems.     

Replication of the Streptomyces plasmid pSN22 through single-stranded intermediates

The replication of the 11 kb conjugative multicopy Streptomyces plasmid pSN22 was analyzed. Mutation and complementation analyses indicated that the minimal region essential for plasmid replication was located on a 1.9 kb fragment of pSN22, containing a trans-acting element encoding a replication protein and a cis-acting sequence acting as a replication origin. Southern hybridization showed that minimal replicon plasmids accumulated much more single-stranded plasmid molecules than did wild-type pSN22. Only one strand was accumulated. A 500 by fragment from the pSN22 transfer region was identified which reduced the relative amount of single-stranded DNA, when added in the native orientation to minimal replicon plasmids. This 500 by DNA sequence may be an origin for second-strand synthesis. It had no effect on the efficiency of co-transformation, plasmid incompatibility, or stability. The results indicate that pSN22 replicates via single-stranded intermediates by a rolling circle mechanism.     

Direction of transcription affects the replication mode of lambda in an in vitro system

Summary pMV158 is a 5.4 kb broad host range multicopy plasmid specifying tetracycline resistance. This plasmid and two of its derivatives, pLS1 and pLS5, are stably mantained and express their genetic information in gram-positive and gram-negative hosts. The in vitro replication of plasmid pMV158 and its derivatives was studied in extracts prepared from plasmid-free Escherichia coli cells and the replicative characteristics of the streptococcal plasmids were compared to those of the E. coli replicons, ColE1 and the mini-R1 derivative pKN182. The optimal replicative activity of the E. coli extracts was found at a cellular phase of growth that corresponded to 2 g wet weight of cells per litre. Maximal synthesis of streptococcal plasmid DNA occurred after 90 min of incubation and at a temperature of 30° C. The optimal concentration of template DNA was 40 g/ml. Higher plasmid DNA concentrations resulted in a decrease in the incorporation of dTMP, indicating that competition of specific replication factor(s) for functional plasmid origins may occur. In vitro replication of plasmid pMV158 and its serivatives required the host RNA polymerase and de novo protein synthesis. The final products of the streptococcal plasmid DNAs replicated in the E. coli in vitro system were monomeric supercoiled DNA forms that had completed at least one round of replication, although a set of putative replicative intermediates could also be found. The results suggest that a specific plasmid-encoded factor is needed for the replication of the streptococcal plasmids.     

Properties of hybrid plasmids, consisting of parts of the mini-R1 factor Rsc11 and ColE1.

Summary In vitro joining of the two small multicopy plasmidsRsc11 andColE1 by a poly dAdT linker resulted in hybrid plasmids, which determine resistance to ampicillin and immunity to colicin E1. Isolation of the plasmid DNA from single colonies revealed that a variety of hybrid plasmids was formed. Cleavage of these plasmids with restriction endonucleasesHincII,HindIII,EcoR1,SmaI andBamI and hybridization withColE1 demonstrated that they contain different parts of the parent plasmids,Rsc11 andColE1. Their copy number in the cell is between 6 and 15 per chromosome depending on the plasmid. None of these plasmids can replicate inpolA mutants. Replication continues in the presence of chloramphenicol. This suggests that replication can only occur from theColE1 origin and that the replication function of theRsc11 part is lost. The hybrid plasmids are compatible withRsc11 but not withColE1. The comparison of the physical maps of theseRsc11-ColE1 hybrids with their functions allows a partial determination of the location of ampicillin resistance, replication and incompatibility on theRsc11 genome.Schering AG, Müllerstr. 170–178, D-1000 Berlin 65Gesellschaft für Biotechnologische Forschung mbH, Abt. Genetik, Mascheroder Weg 1, D-3300 Braunschweig-Stöckheim     

Genetic interaction between the Ras-CAMP pathway and the Dis2s1/Glc7 protein phosphatase in Saccharomyces cerevisiae

The Saccharomyces cerevisiae DIS2S1/GLC7 gene encodes a type 1 protein phosphatase indispensable for cell proliferation. We found that introduction of a multicopy DIS2S1 plasmid impaired growth of cells with reduced activity of the cAMP-dependent protein kinase. In order to understand further the interaction between the two enzymes, a temperature-sensitive mutation in the DIS2S1 gene was isolated. The mutant accumulated less glycogen than wild type at the permissive temperature, indicating that activity of the Dis2s1 protein phosphatase is attenuated by the mutation. Furthermore, the dis2s1 ^ts mutation was shown to be suppressed by a multicopy plasmid harboring PDE2, a gene for cAMP phosphodiesterase. These results indicate that the Ras-cAMP pathway interacts genetically with the DIS2S1/GLC7 gene.     

Roles of Escherichia coli heat shock proteins DnaK, DnaJ and GrpE in mini-F plasmid replication

Summary A subset of Escherichia coli heat shock proteins, DnaK, DnaJ and GrpE were shown to be required for replication of mini-F plasmid. Strains of E. coli K12 carrying a missense mutation or deletion in the dnaK, dnaJ, or grpE gene were virtually unable to be transformed by mini-F DNA at the temperature (30° C) that permits cell growth. When excess amounts of the replication initiator protein (repE gene product) of mini-F were provided by means of a multicopy plasmid carrying repE, these mutant bacteria became capable of supporting mini-F replication under the same conditions. However, the copy number of a high copy number mini-F plasmid was reduced in these mutant bacteria as compared with the wild type in the presence of excess RepE protein. Furthermore, mini-F plasmid mutants that produce altered initiator protein and exhibit a very high copy number were able to replicate in strains deficient in any of the above heat shock proteins. These results indicate that the subset of heat shock proteins (DnaK, DnaJ and GrpE) play essential roles that help the functioning of the RepE initiator protein in mini-F DNA replication.     

Replication of the Streptomyces plasmid pSN22 through single-stranded intermediates

The replication of the 11 kb conjugative multicopy Streptomyces plasmid pSN22 was analyzed. Mutation and complementation analyses indicated that the minimal region essential for plasmid replication was located on a 1.9 kb fragment of pSN22, containing a trans-acting element encoding a replication protein and a cis-acting sequence acting as a replication origin. Southern hybridization showed that minimal replicon plasmids accumulated much more single-stranded plasmid molecules than did wild-type pSN22. Only one strand was accumulated. A 500 by fragment from the pSN22 transfer region was identified which reduced the relative amount of single-stranded DNA, when added in the native orientation to minimal replicon plasmids. This 500 by DNA sequence may be an origin for second-strand synthesis. It had no effect on the efficiency of co-transformation, plasmid incompatibility, or stability. The results indicate that pSN22 replicates via single-stranded intermediates by a rolling circle mechanism.     

Chromosome replication in Escherichia coli induced by oversupply of DnaA

Summary Overexpression of DnaA protein from a multicopy plasmid accompanied by a shift to 42°C causes initiation of one extra round of replication in a dnaA ⁺ strain grown in glycerol minimal medium. This extra round of replication does not lead to an extra cell division, such that cells contain twice the normal number of chromosomes.     

Par site of the ColN plasmid: Structural and functional organization

Summary A par site involved in the resolution of multimeric plasmid DNA forms was localized in a 679 by SalI-KpnI fragment of the small colicinogenic plasmid CoIN. It was shown that replication of the monomeric pUC19 recombinant plasmid carrying the par region of ColN does not result in the formation of significant numbers of multimers. In order to function properly, the Co1N multimer resolution mechanism requires the product of the xerA gene, just as in the case of ColEI. Nucleotide sequence analysis of the par region of CoIN revealed substantial homology with the par locus of the ColE1 plasmid. The results of this study and data from the literature indicate that the par sites of ColEl-type plasmids have substantial homology and the same mechanism of action, and in fact represent a universal stability module for small multicopy colicinogenic plasmids.     

Mitotic gene conversion of large DNA heterologies in Saccharomyces cerevisiae

Summary Gene conversion of large DNA heterologous fragments has been shown to take place efficiently in Saccharomyces cerevisiae. It has been found that a 2.6 kb LEU2 DNA fragment in a multicopy plasmid was replaced by a 3.1 kb PG11 chromosomal DNA fragment, when both fragments were flanked by homologous DNA regions. Gene conversion was asymmetric in a total of 481 recombinants analyzed. In contrast, truncated PG11 or LEU2 genes in multicopy plasmids, gave no recombinants that restored a complete plasmid copy of these genes in a total of 242 recombinants studied, confirming that a conversion tract is disrupted by a heterologous region. The asymmetry of the events detected suggest that gene conversion of large DNA heterologies involves a process whereby a gap first covers one heterologous fragment and then this is followed by new DNA synthesis using the other heterologous fragment as a template. Therefore, it is likely that large DNA heterologies are converted by a double-strand gap repair mechanism.     

Construction and characterization of multicopy plasmids containing the entire F transfer region

The restriction endonucleases HindIII and/or BamI have been used to clone the entire F transfer region into pBR322 to create a series of transfer-proficient multicopy plasmids. Despite the insertion of 40.7- to 55.9-kilobase F fragments, the plasmid copy numbers remained high, at 25–40 copies per cell. One of the chimeric plasmids contained the F replication and incompatibility region, and its high copy number confirmed that replication of the cointegrate was governed by pBR322. Despite the 30- to 40-fold increase in tra gene copy number compared to Flac, the transfer frequencies, number of pili per cell, and syntheses of the individual traT and traI proteins were increased only by about 5-fold. The level of tra mRNA in cells carrying the multicopy transfer-proficient plasmids was also increased only by about 5-fold, suggesting that its relative synthesis or stability was reduced in this situation. Nonetheless, the increased production of tra DNA, mRNA, and protein makes cells carrying the multicopy conjugative plasmids excellent sources of these products.     

Tagging of Bacillus subtilis soil isolates through illegitimate recombination for PCR detection

Although there are numerous bacteria of the genus Bacillus of great importance for biological control, little is known about their ecology in the soil. We wanted to test illegitimate recombination as a tagging system that would allow us to study selected or genetically engineered Bacillus soil isolates. Strains carrying the plasmid integrated into the chromosome were obtained by growing at a non-permissive temperature after transformation with a plasmid carrying a thermo-sensitive replication origin with selection for erythromycin. A laboratory strain, a commercial strain (Kodiak), and four other soil isolates were generated through this procedure and analysed. In all of these strains the integrated plasmid was maintained in multicopy. The erythromycin resistance gene (ermB) placed on the plasmid was used as a target for polymerase chain reaction (PCR). The tagged strains could be then detected when inoculated into microcosms prepared with non-sterile soil.     

Isolation of the RAD18 gene of Saccharomyces cerevisiae and construction of rad18 deletion mutants

Summary The RAD18 gene of Saccharomyces cerevisiae is involved in mutagenic DNA repair. We describe its isolation from a yeast library introduced into the centromeric YCp50 vector, a low copy number plasmid. The insert was sublconed into YCp50 and into the multicopy YRp7 plasmid. RAD18 is not toxic when present in multiple copies but the UV survival response indicates an heterogeneity in the cell population, a fraction of it being more sensitive. A DNA segment, close to RAD18, is toxic on the multicopy plasmid and may correspond to the tRAN sup61 known to be tightly linked to RAD18. Chromosomal deletions of RAD18 were constructed. The gene is not essential and the deleted strains have the properties of single site mutants. Thus, RAD18 appears to be essentially involved in DNA repair metabolism.     

Origin plasticity during budding yeast DNA replication in vitro

The separation of DNA replication origin licensing and activation in the cell cycle is essential for genome stability across generations in eukaryotic cells. Pre‐replicative complexes (pre‐RCs) license origins by loading Mcm2‐7 complexes in inactive form around DNA. During origin firing in S phase, replisomes assemble around the activated Mcm2‐7 DNA helicase. Budding yeast pre‐RCs have previously been reconstituted in vitro with purified proteins. Here, we show that reconstituted pre‐RCs support replication of plasmid DNA in yeast cell extracts in a reaction that exhibits hallmarks of cellular replication initiation. Plasmid replication in vitro results in the generation of covalently closed circular daughter molecules, indicating that the system recapitulates the initiation, elongation, and termination stages of DNA replication. Unexpectedly, yeast origin DNA is not strictly required for DNA replication in vitro, as heterologous DNA sequences could support replication of plasmid molecules. Our findings support the notion that epigenetic mechanisms are important for determining replication origin sites in budding yeast, highlighting mechanistic principles of replication origin specification that are common among eukaryotes.     

The cost of copy number in a selfish genetic element: the 2‐μm plasmid of Saccharomyces cerevisiae

Many autonomously replicating genetic elements exist as multiple copies within the cell. The copy number of these elements is often assumed to have important fitness consequences for both element and host, yet the forces shaping its evolution are not well understood. The 2 μm is a multicopy plasmid of Saccharomyces yeasts, encoding just four genes that are solely involved in plasmid replication. One simple model for the fitness relationship between yeasts and 2 μm is that plasmid copy number evolves as a trade‐off between selection for increased vertical transmission, favouring high copy number, and selection for decreased virulence, favouring low copy number. To test this model, we experimentally manipulated the copy number of the plasmid and directly measured the fitness cost, in terms of growth rate reduction, associated with high plasmid copy number. We find that the fitness burden imposed by the 2 μm increases with plasmid copy number, such that each copy imposes a fitness burden of 0.17% (± 0.008%), greatly exceeding the cost expected for it to be stably maintained in yeast populations. Our results demonstrate the crucial importance of copy number in the evolution of yeast per 2 μm associations and pave the way for future studies examining how selection can shape the cost of multicopy elements.     

Enhanced plasmid stability through post-segregational killing of plasmid-free cells

Summary In order to develop an extremely stable, inducible host/vector system, the following genetic manipulations were made: a recA mutation was introduced into the chromosome of the host strain, the plasmid selectable marker was changed from ampicillin to kanamycin, and the parB stability locus of plasmid R1 was added to the plasmid. The stability of the new vector, pTKW106, was increased such that the fraction of plasmid-bearing cells present during chemostat fermentations under selective pressure increased from 1.75% to 100% when plasmid protein production was fully induced. At this level of induction, -galactosidase represents 10% of the total cell protein. In addition, the frequency of generation of plasmid-free cells was shown to decrease from 1.0 per generation to less than 10^–11 with full promoter induction under non-selective conditions.