Location of a second partitioning region (ParL) on the F plasmid

Abstract The leading region of the F plasmid has been found to extend the maintenance of the normally unstable plasmid vector pACYC184. This ability is due to effective partitioning of plasmid molecules at cell division. Cloning, deletion analysis and transposon mutagenesis have located the partitioning region (ParL) to be encoded within 63.65–64.11F. Complementation studies indicated that parL is a cis -acting locus.     

Genetic and physical map of a P1 miniplasmid

The prophage form of bacteriophage P1 is a unit-copy plasmid which is maintained with great fidelity in its Escherichia coli host. The plasmid maintenance functions of P1 are clustered in one region of the genome. An 11.5-kilobase fragment from this region has been cloned into a lambda delta att vector and promotes stable unit-copy plasmid maintenance. The properties of the lambda vector facilitated the isolation of deletion mutants affecting the P1 DNA. Twenty-eight deletion mutants were isolated, and their lesions were mapped by physical techniques. The genetic properties of the mutants with respect to plasmid replication, stability of plasmid maintenance, and ability to exert incompatibility effects against P1 and P7 plasmids were determined. These properties, along with those of several subfragments of the P1 insert cloned into high-copy-number plasmid vectors, allow the construction of an unambiguous genetic and physical map of the maintenance functions. A region of less than 3 kilobases, the rep region, is essential for plasmid replication and contains the incA incompatibility determinant within an 800-base-pair segment. Immediately adjacent to rep is a second region of approximately 3 kilobases which is required for stable plasmid maintenance, but not replication. This region, par, contains a second incompatibility element incB which is approximately 1 kilobase in size. The par region appears to specify equipartition of plasmid copies to daughter cells during cell division.     

The plasmid-maintenance functions of the P7 prophage

The region responsible for the maintenance of the prophage of bacteriophage P7 as a stable, unit-copy plasmid was isolated in a lambda att vector which lysogenizes Escherichia coli as a stable unit-copy plasmid under the control of the P7 replication origin. The P7 plasmid-maintenance region was shown to consist of adjacent replication and partition regions capable of functioning independently. The isolated replication region could support plasmid maintenance but the resulting plasmids were highly unstable unless the partition region was also included. Stable composite plasmids were isolated containing the putative P7 partition region and the origin of replication of the unrelated plasmid F, indicating that P7 encoded an active partition mechanism. The replication regions of P7 and P1 were shown to be highly homologous but the partition regions of the two plasmids appear to be unrelated in sequence. The incompatibility determinants associated with the two replication regions showed the same specificity, whereas the partition-region incompatibility determinants were different, showing no cross-specificity.     

Construction of a newEscherichia coli — Saccharomyces cerevisiae shuttle plasmid cloning vector allowing positive selection for cloned fragments

A new E. coli-S. cerevisiae shuttle plasmid cloning vector (pPW263) with a positive type of selection, was constructed. The selection system, based on the regulatory region of lambda phage controlling the expression of tetracycline resistance, was derived from the cloning vector pUN121 (Nilsson et al. 1983). There are three cloning sites in the cI gene, EcoRI, HindIII and BglII, and, in addition, two unique sites in the neighborhood, BamHI and SalI. The size of the vector is 7.8 kb. The maintenance of the vector and the selection in yeast was ensured by the replication region of the 2 mu plasmid and by the URA3 marker gene, respectively.     

The mcm2 mutation of yeast affects replication, rather than segregation or amplification of the two micron plasmid.

We have studied the maintenance of the endogenous two micron (2 mu) plasmid in a strain of yeast carrying the nuclear mutation mcm2. This mutation, earlier shown to affect the maintenance of yeast minichromosomes in an ARS-dependent manner, also affected the copy number of the 2 mu plasmid. The effect was more pronounced at 35 degrees C leading to the elimination of the plasmid from the cells cultured at this temperature. The mutant cells could be efficiently cured of the circle by transformation with 2 mu ORI-carrying hybrid vectors, an observation consistent with the low copy number of the endogenous plasmid. A chromosomal revertant of this mutant for another ARS(ARS1) was found also to confer stability on the 2 mu ORI-carrying minichromosomes and had elevated levels of the endogenous plasmid. The mutation neither affected the segregation nor the amplification process mediated by site-specific recombination at FRT sites requiring the FLP gene-encoded protein action. ARS131C, an ARS that was unaffected in the mutant at 25 degrees C, could elevate the copy number of a 2 mu hybrid vector in the mutant cells. In view of these results, some aspects of segregation and copy number control of the endogeneous plasmid have been discussed. We propose that the mutation impairs the 2 mu ORI function, leading to its loss.     

Functional analysis of ori1 and repA of the R-plasmid pSJ5.6 from Neisseria gonorrhoeae

The functional ori1 of the 5.6kb gonococcal R-plasmid pSJ5.6 contains an A-T rich region followed by four 22bp direct repeats and one 19bp inverted repeat. The replication region of the plasmid also contains a gene encoding for a 39kD RepA protein. We have further assessed the functionality of the replication region in pSJ5.6, an-iteron type plasmid, using in vivo complementation assays in Escherichia coli. A 2.1kb PstI-RsaI fragment containing the ori1 and repA gene of pSJ5.6 was cloned into vector pZErO -2 to obtain pZA-MRR. The pUC origin in pZA-MRR was deleted to render the plasmid dependable on the cis-acting ori1 for replication. The resulting plasmid, pMRR, was capable of replication and maintenance in E. coli. We also cloned the ori1 and repA gene separately to obtain pA-Ori and pZG-Rep, respectively. Using in vivo complementation assays, we demonstrated that the ori1(+) plasmid (pA-Ori) was maintained only when the RepA protein was supplied in trans by the high copy number plasmid pZG-Rep.     

Studies on maintenance of vector pBK2 in Corynebacterium acetoacidophilum by co-culture experiments in a continuous bioreactor

A plasmid vector pBK2 was tested for maintenance in Corynebacterium acetoacidophilum and found to be 100% stable for 90 generations. In co-culture experiments in absence of any selection pressure it was able to cause washout of plasmid free cells. Deletion mutagenesis of the plasmid was done to identify the regions of DNA responsible for its stable maintenance. This led to the identification of a region of DNA in pBB1, an endogenous plasmid used in the construction of pBK2, which is responsible for providing a growth rate advantage to the plasmid containing cell in co-culture with plasmid-free cells. To explain this observed stability we postulate the production of a “growth-inhibiting factor” by the plasmid-containing cells. Inclusion of a death rate term for the plasmid free cells in the computer simulation of the growth kinetics in a chemostat successfully predicts, depending upon the magnitude of the kinetic parameters, a stable coexistence of the two or washout of the plasmid-free cells, an observation that matches the experimental data. Because of its stable maintenance and its ability to select against plasmid-free cells this host-vector system is a natural candidate for cloning and use in large-scale fermentations.     

Cloning the Tra1 region of RP1

The Tra1 region of RP1 from a derivative with Tn7 inserted into the kanamycin resistance determinant was cloned, using EcoRI, into the multicopy vector plasmid pBR325. For one orientation of the cloned fragment the resultant chimeric plasmid was very frequently lost from the cell, but in the other orientation it was much more stable and also compatible with RP1. Complementation by the stable chimeric plasmid, pED800, of a series of RP1 tra mutants showed that the mutations of all those retaining sensitivity to the P-specific phages PRR1, Pf3, and PR4, or only to PR4, mapped in the Tra1 region, while only 2 out of 20 amber mutations leading to full P-specific phage-resistance did so.     

Stable maintenance in chemostat-grownEscherichia coli of pBR322 and pACYC184 by disruption of the tetracycline resistance gene

Plasmid stability was studied in antibiotic-free chemo-stat cultures . Disruption, either by deletion or insertion, of the tetracycline resistance gene in the EcoRl/EcoRV region of the cloning vector pBR322 or in the HindIII]BamHl region of pACYCI84 yields plasmids markedly more stable than the parent plasmids. Thus, at least for these two instances, cloning of a partitioning (par) locus is not prerequisite for plasmid maintenance.Issued as NRCC publication No. 23992.     

Characterization of the minimal replicon of a cryptic Deinococcus radiodurans SARK plasmid and development of versatile Escherichia coli-D. radiodurans shuttle vectors

The nucleotide sequence of a 12-kb fragment of the cryptic Deinococcus radiodurans SARK plasmid pUE10 was determined, in order to direct the development of small, versatile cloning systems for Deinococcus. Annotation of the sequence revealed 12 possible open reading frames. Among these are the repU and resU genes, the predicted products of which share similarity with replication proteins and site-specific resolvases, respectively. The products of both genes were demonstrated using an overexpression system in Escherichia coli. RepU was found to be required for replication, and ResU was found to be required for stable maintenance of pUE10 derivatives. Gel shift analysis using purified His-tagged RepU identified putative binding sites and suggested that RepU may be involved in both replication initiation and autoregulation of repU expression. In addition, a gene encoding a possible antirestriction protein was found, which was shown to be required for high transformation frequencies. The arrangement of the replication region and putative replication genes for this plasmid from D. radiodurans strain SARK is similar to that for plasmids found in Thermus but not to that for the 45.7-kb plasmid found in D. radiodurans strain R1. The minimal region required for autonomous replication in D. radiodurans was determined by sequential deletion of segments from the 12-kb fragment. The resulting minimal replicon, which consists of approximately 2.6 kb, was used for the construction of a shuttle vector for E. coli and D. radiodurans. This vector, pRAD1, is a convenient general-purpose cloning vector. In addition, pRAD1 was used to generate a promoter probe vector, and a plasmid containing lacZ and a Deinococcus promoter was shown to efficiently express LacZ.     

Partition of unit-copy miniplasmids to daughter cells. I. P1 and F miniplasmids contain discrete, interchangeable sequences sufficient to promote equipartition

Hybrids formed by insertion of the plasmid maintenance regions of P1 or F into a lambda delta att vector form stable unit-copy plasmids in their Escherichia coli host. They must therefore both be substrates for an accurate cellular partition apparatus that ensures that all daughter cells inherit a plasmid copy. Analysis of deletion mutants of both types of hybrid showed that, although the P1 and F plasmid maintenance regions differ in sequence and specificity, they are similar in general organization. Each contains an approximately 3 X 10(3) base-pair region that is essential for replication (rep) and an adjacent but separable 3 X 10(3) base-pair region that is essential for the stability of plasmid maintenance (par). Each par region is thought to specify the recognition of the plasmid as a substrate for equipartition. The deletion mutants provide sources of isolated rep and par sequences from both P1 and F DNA. These elements were then used to construct composite plasmids with novel combinations and arrangements of rep and par sequences. Heterologous constructions containing P1 rep and F par or F rep and P1 par sequences were maintained faithfully. We conclude that par regions are both necessary and sufficient to promote equipartition of replicating plasmid DNA. This activity is exerted only in cis but otherwise seems to be independent of the position or orientation of the par sequences within the DNA. Both P1 and F par regions include DNA sequences (incB of P1, incD of F) that we propose are analogues of the centromeres of eukaryotic chromosomes. The remaining portions of the par regions are known to encode protein products that, we believe, act at the inc sites. Extra copies of these inc sites appear to exert incompatibility by competition for the cellular partition apparatus.     

Strategy to approach stable production of recombinant nattokinase in Bacillus subtilis

Bacillus subtilis (B. subtilis) is widely accepted as an excellent host cell for the secretory production of recombinant proteins. In this study, a shuttle vector was constructed by fusion of Staphylococcus aureus (S. aureus) plasmid pUB110 with Escherichia coli (E. coli) plasmid pUC18 and used for the expression of nattokinase in B. subtilis. The pUB110/pUC-based plasmid was found to exhibit high structural instability with the identification of a DNA deletion between two repeated regions. An initial attempt was made to eliminate the homologous site in the plasmid, whereas the stability of the resulting plasmid was not improved. In an alternative way, the pUC18-derived region in this hybrid vector was replaced by the suicidal R6K plasmid origin of E. coli. As a consequence, the pUB110/R6K-based plasmid displayed full structural stability, leading to a high-level production of recombinant nattokinase in the culture broth. This was mirrored by the detection of a very low level of high molecular weight DNAs generated by the plasmid. Moreover, 2-fold higher nattokinase production was obtained by B. subtilis strain carrying the pUB110/R6K-based plasmid as compared to the cell with the pAMbeta1-derived vector, a plasmid known to have high structural stability. Overall, it indicates the feasibility of the approach by fusing two compatible plasmid origins for stable and efficient production of recombinant nattokinase in B. subtilis.     

The complete sequence and functional analysis of pANL, the large plasmid of the unicellular freshwater cyanobacterium Synechococcus elongatus PCC 7942

Two endogenous plasmids are present in Synechococcus elongatus PCC 7942, a model organism for studying photosynthesis and circadian rhythms in cyanobacteria. The large plasmid, pANL, was shown previously to be involved in adaptation of S. elongatus cells to sulfur starvation, which provided the first evidence of cellular function of a cyanobacterial plasmid. Here, we report the complete sequence of pANL, which is 46,366 bp in length with 53% GC content and encodes 58 putative ORFs. The pANL plasmid can be divided into four structural and functional regions: the replication origin region, a signal transduction region, a plasmid maintenance region, and a sulfur-regulated region. Cosmid-based deletion analysis suggested that the plasmid maintenance and replication origin regions are required for persistence of pANL in the cells. Transposon-mediated mutagenesis and complementation-based pANL segregation assays confirmed that two predicted toxin-antitoxin cassettes encoded in the plasmid maintenance region, belonging to PemK and VapC families, respectively, are necessary for plasmid exclusion. The compact and efficient organization of sulfur-related genes on pANL may provide selective advantages in environments with limited sulfur.     

Mammalian and viral DNA sequences which interfere with the maintenance of a centromeric vector in yeast.

We constructed a recombinant plasmid by inserting into the pRS314 yeast centromeric plasmid vector the mouse DNA sequence responsible for the maintenance in transgenic mice of plasmid p12B1 (1). Such constructs could constitute convenient shuttle vectors between yeast and mouse cells. However, the recombinant molecule could not be established as a stable plasmid in Saccharomyces cerevisiae. A region with a limited similarity to the yeast centromere (CEN element) is present in this mouse sequence as well as in two other sequences subsequently identified in a data bank search using the CEN consensus. One of them is localized in Bovine Papillomavirus Type 1 DNA, and the other one in the human beta-globin locus. Once inserted in pRS314, these two sequences showed the same inhibitory effect on plasmid maintenance as the p12B1 mouse DNA fragment. This effect appears to depend on the simultaneous presence in the construct of one of the "CEN-like regions" and of an authentic CEN element. Non-centromeric yeast plasmids carrying one of the three sequences could replicate autonomously, and were even stabilized to a significant extent. These results identify in the genomes of higher eukaryotes and their viruses a family of sequences which cannot be simply cloned in centromeric yeast vectors.     

A novel expression vector for high-level synthesis and secretion of foreign proteins in Escherichia coli: overproduction of bovine pancreatic phospholipase A2

A novel expression plasmid (pTO-N) has been constructed that allows for the production of large quantities of foreign proteins (or fragments thereof) in an unfused state. The vector has a strong and tightly regulated T7 gene 10 promoter and the ompA Shine-Dalgarno (SD) sequence, followed by the ompA sequence and a cloning linker region. The mRNAs produced by the vector are protected by secondary structures at both ends of the mRNAs. The OmpA signal peptide directed the synthesized proteins into the periplasmic space of Escherichia coli. Phospholipase A2 and prophospholipase A2 from bovine pancreas have been produced to a high level by using this expression vector. One additional feature, which is essential for the stable maintenance of the plasmid in the E. coli expression host, BL21 (DE3)[pLysS], is the shortened distance between the 5' secondary structure sequence (immediately following the gene 10 promoter) and the SD sequence. This vector could be particularly useful for synthesis of toxins in E. coli.     

Effective generation of transgenic mice by Bovine papillomavirus type 1 based self-replicating plasmid that is maintained as extrachromosomal genetic element in three generations of animals

The objective of our study was to analyze the efficiency and the properties of the inheritance of the Bovine papillomavirus type 1 (BPV1) replicator-based plasmid used as vector system for generation of transgenic animals. Previously, we have characterized a series of self-replicating plasmid vectors containing all viral factors necessary and sufficient for stable extrachromosomal replication of the BPV1 genome in the tissue culture system. We also demonstrated that the designed replicating vector system has a considerable benefit in the transgene expression, if compared to the regular expression vector. The vector, which showed the highest stability and maintenance function in the tissue culture was chosen for generation of the transgenic mice by pronuclear injections of the circular supercoiled plasmid. This method resulted in successful production of transgenic animals. Transmission efficiency of the vectors into the F(1) generation of animals varied between 0 and 48%, whereas transmission into the F(2) generation was uniformly near 50%. The maintenance of the vector-plasmids in the F(2) generation of transgenic animals as extrachromosomal genetic element was demonstrated by rescue of the plasmid into the Escherichia coli.     

Origin and direction of in vitro replication of Haemophilus ducreyi and Neisseria gonorrhoeae ampicillin resistance plasmids.

The origin of replication of Haemophilus ducreyi and Neisseria gonorrhoeae ampicillin resistance plasmids was located by cloning BamHI restriction fragments into vector plasmid pAT153 and a derivative plasmid, pAT2. Selection was made for plasmid maintenance in a polA mutant. Direction of replication was determined by in vitro replication of plasmid DNA in the presence of radiolabeled deoxynucleotide.     

Replication and partitioning of the broad-host-range plasmid RK2

The broad-host-range plasmid RK2 has been a model for studying DNA metabolism in bacteria for many years. It is used as a vector allowing genetic manipulations in numerous bacterial species. The RK2 genome encodes several genes providing the plasmid with diverse functions allowing for its stable maintenance in a variety of bacterial hosts. This review will focus on two processes indispensable for plasmid DNA maintenance. We will summarize recent understanding of the molecular mechanisms contributing to the RK2 DNA replication and partitioning.