Segregational instability of pUB110-derived recombinant plasmids in Bacillus subtilis

To study plasmid instability in Bacillus subtilis the pUB110-derived hybrid plasmid pLB2 (3.6 kb) and the bifunctional replicon pLB5 (5.9 kb), able to replicate in B. subtilis and Escherichia coli, were constructed. In both vectors homologous B. subtilis, or heterologous E. coli DNA fragments of various lengths were inserted. Irrespective of the source of the cloned DNA, the segregational stability of the recombinant plasmids in B. subtilis was severely affected by the DNA inserts. In contrast, no instability was observed in E. coli. In B. subtilis a steep inverse relationship existed between the size of the inserts and the level of stability. Increased size of the pLB plasmids resulted in strongly reduced copy numbers. This seems to be the primary cause of the size-dependent segregational instability.     

Cloning and stable maintenance of DNA fragments over 300 kb in Escherichia coli with conventional plasmid-based vectors.

Bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) systems were previously developed for cloning of very large eukaryotic DNA fragments in bacteria. We report the feasibility of cloning very large fragments of eukaryotic DNA in bacteria using conventional plasmid-based vectors. One conventional plasmid vector (pGEM11), one conventional binary plasmid vector (pSLJ1711) and one conventional binary cosmid vector (pCLD04541) were investigated using the widely used BAC (pBeloBAC11 and pECBAC1) and BIBAC (BIBAC2) vectors as controls. The plasmid vector pGEM11 yielded clones ranging in insert sizes from 40 to 100 kb, whereas the two binary vectors pCLD04541 and pSLJ1711 yielded clones ranging in insert sizes from 40 to 310 kb. Analysis of the pCLD04541 and pSLJ1711 clones indicated that they had insert sizes and stabilities similar to the BACs and BIBACs. Our findings indicate that conventional plasmid-based vectors are capable of cloning and stably maintaining DNA fragments as large as BACs and PACs in bacteria. These results suggest that many existing plasmid-based vectors, including plant and animal transformation and expression binary vectors, could be directly used for cloning of very large eukaryotic DNA fragments. The pCLD04541 and pSLJ1711 clones were shown to be present at at least 4-5 copies/cell. The high stability of these clones indicates that stability of clones does not seem contingent on single-copy status. The insert sizes and the copy numbers of the pCLD04541 and pSLJ1711 clones indicate that Escherichia coli can stably maintain at least 1200 kb of foreign DNA per cell. These results provide a new conceptual and theoretical basis for development of improved and new vectors for large DNA fragment cloning and transformation. According to this discovery, we have established a system for large DNA fragment cloning in bacteria using the two binary vectors, with which several very large-insert DNA libraries have been developed.     

Characterization of a region of the Enterococcus faecalis plasmid pAM beta 1 which enhances the segregational stability of pAM beta 1-derived cloning vectors in Bacillus subtilis.

The nucleotide sequence of a 2.13-kb EcoRI-HindIII, pAM beta 1-derived fragment, isolated from the gram-positive cloning vector pHV1431, has been determined and shown to encode two ORFs. ORF H encodes for a protein of 23,930 Da which exhibits substantial homology to bacterial site-specific recombinases, particularly the resolvases of the gram-positive transposons Tn917 (30.3% identity) and Tn552 (31.6% identity) and the clostridial plasmid pIP404 (27.1% identity). The second ORF (I) is incomplete and encodes a polypeptide which has significant homology with Escherichia coli topoisomerase I (26.0% identity). Insertion of either the entire 2.13-kb EcoRI-HindIII fragment or a 0.73-kb EcoRI-DraI subfragment encoding only the resolvase into the pAM beta 1-based cloning vector pMTL500E causes a significant enhancement of segregational stability (from 6.5 X 10(-2) to 3.0-4.0 X 10(-3) plasmid loss per cell per generation). Improved segregational stability is mirrored by a reduction in plasmid polymerization. The introduction of a stop codon into the resolvase coding region negates its ability to promote segregational stability. It is proposed that the identified determinant stabilizes pAM beta 1-based vectors in Bacillus subtilis by maintaining the plasmid population in the monomeric state, thereby reducing the chances of producing plasmid-free segregants.     

Replication in Saccharomyces cerevisiae of plasmid pBR313 carrying DNA from the yeast trpl region.

Plasmid pBR313 carrying a 1.4 kb EcoRI fragment from the yeast TRP1 region (designated pLC544) is capable of transforming yeast trp1 mutants to Trp+ at high frequency (10(3)--10(4) transformants/micrograms DNA). Transformation can be achieved either by using purified plasmid DNA or by fusion of yeast spheroplasts with partially lysed Escherichia coli [pLC544] protoplast preparations. The Trp+ yeast transformants are highly unstable, segregating Trp- cells at frequencies of 0.18 per cell per generation (haploids) and 0.056 per cell per generation (diploids) in media containing tryptophan. Plasmid pLC544 replicates autonomously in the nucleus of yeast cells and segregation of Trp-cells is associated with the complete loss of plasmid sequences. In genetic crosses, pLC544 is randomly assorted during meiosis and is carried unchanged through the mating process into haploid recombinants.     

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.     

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.     

Characterization of a region of the Enterococcus faecalis plasmid pAMβ1 which enhances the segregational stability of pAMβ1-derived cloning vectors in Bacillus subtilis

The nucleotide sequence of a 2.13-kb EcoRI-HindIII, pAMβ1-derived fragment, isolated from the gram-positive cloning vector pHV1431, has been determined and shown to encode two ORFs. ORF H encodes for a protein of 23,930 Da which exhibits substantial homology to bacterial site-specific recombinases, particularly the resolvases of the gram-positive transposons Tn917 (30.3% identity) and Tn552 (31.6% identity) and the clostridial plasmid pIP404 (27.1% identity). The second ORF (I) is incomplete and encodes a polypeptide which has significant homology with Escherichia coli topoisomerase I (26.0% identity). Insertion of either the entire 2.13-kb EcoRI-HindIII fragment or a 0.73-kb EcoRI-DraI subfragment encoding only the resolvase into the pAMβ1-based cloning vector pMTL500E causes a significant enhancement of segregational stability (from 6.5 × 10⁻² to 3.0–4.0 × 10⁻³ plasmid loss per cell per generation). Improved segregational stability is mirrored by a reduction in plasmid polymerization. The introduction of a stop codon into the resolvase coding region negates its ability to promote segregational stability. It is proposed that the identified determinant stabilizes pAMβ1-based vectors in Bacillus subtilis by maintaining the plasmid population in the monomeric state, thereby reducing the chances of producing plasmid-free segregants.     

Canine Adenovirus Type 2 Vector Generation via I-Sce1-Mediated Intracellular Genome Release

When canine adenovirus type 2 (CAdV-2, or also commonly referred to as CAV-2) vectors are injected into the brain parenchyma they preferentially transduce neurons, are capable of efficient axonal transport to afferent regions, and allow transgene expression for at last >1 yr. Yet, translating these data into a user-friendly vector platform has been limited because CAV-2 vector generation is challenging. Generation of E1-deleted adenovirus vectors often requires transfection of linear DNA fragments of >30 kb containing the vector genome into an E1-transcomplementing cell line. In contrast to human adenovirus type 5 vector generation, CAV-2 vector generation is less efficient due, in part, to a reduced ability to initiate replication and poor transfectibility of canine cells with large, linear DNA fragments. To improve CAV-2 vector generation, we generated an E1-transcomplementing cell line expressing the estrogen receptor (ER) fused to I-SceI, a yeast meganuclease, and plasmids containing the I-SceI recognition sites flanking the CAV-2 vector genome. Using transfection of supercoiled plasmid and intracellular genome release via 4-OH-tamoxifen-induced nuclear translocation of I-SceI, we improved CAV-2 vector titers 1,000 fold, and in turn increased the efficacy of CAV-2 vector generation.     

Repair of UV-irradiated plasmid DNA in mutants of Saccharomyces cerevisiae and Escherichia coli deficient in repair of pyrimidine dimers

The repair of in vitro UV-irradiated DNA of plasmid pBB29 was studied in excision defective yeast mutants rad1, rad2, rad3, rad4, rad10 and in Escherichia coli mutants uvr- and recA-, by measuring the cell transformation frequency. Rad2, rad3, rad4, and rad10 mutants could repair plasmid DNA despite their inability to repair nuclear DNA, whereas the reduced ability of rad1 mutant for plasmid DNA repair demonstrated alone the same dependence on the host functions that are needed for nuclear DNA repair. In E. coli the repair of UV-irradiated plasmid DNA is carried out only by the excision-repair system dependent on uvr genes. Treatment of UV-irradiated plasmid DNA with UV endonuclease from Micrococcus luteus greatly enhances the efficiency of transformation of E. coli uvr- mutants. Similar treatment with cell-free extracts of yeast rad1 mutant or wild-type strains as well as with nuclease BaL31, despite their ability for preferential cutting of UV damaged DNA, showed no influence on cell transformation.     

Cloning and mapping of fourteen different DNA fragments containing Aspergillus nidulans 5S rRNA genes

Fourteen different plasmids hybridizing to Aspergillus nidulans 5S rRNA were isolated from a gene bank obtained after cloning Sau3A partial digests of A. nidulans DNA in a yeast--Escherichia coli vector, pBB29. The restriction maps of these plasmids were determined. The size of the cloned fragments was 2.7-9.5 kb, 12 of the plasmids were found to code for single 5S rRNA genes and 2 coded for 2 genes. No similarity of the sequences surrounding the 5S rRNA genes was found by restriction mapping.     

Stability and transmissibility of the cryptic plasmids of Rhizobium meliloti GR4

Rhizobium meliloti strain GR4 harbours two cryptic plasmids sharing extensive regions of homology between them and with other non-symbiotic plasmids of different strains of R. meliloti. They both are very stable showing a segregation rate of less than 0.1% loss per generation. pRmeGR4a (115 MDa) is a self-transmissible plasmid at a variable frequency to other species, and it is also responsible for promoting, at low frequency, the contransfer of pRmeGR4b (140 MDa), the other cryptic plasmid of the strain. A 4.8 kb PstI fragment of pRmeGR4a, responsible for the high stability in cis of this plasmid, has been isolated and several recombinant plasmids have been constructed showing different segregation rates in the strains used in this study. Their stabilities can be considerably improved by insertion of the stabilization mrs/par region of RK2.     

Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously

A procedure for precise assembly of linear DNA constructs as long as 20 kb is proposed. The method, which we call long multiple fusion, has been used to assemble up to four fragments simultaneously (for a 10.8 kb final product), offering an additional improvement on the combination of long PCR and overlap extension PCR. The method is based on Pfu polymerase mix, which has a proofreading activity. We successfully assembled (and confirmed by sequencing) seven different linear constructs ranging from 3 to 20 kb, including two 20 kb products (from fragments of 11, 1.7 and 7.5 kb), two 10.8 kb constructs, and two constructs of 6.1 and 6.2 kb, respectively. Accuracy of the PCR fusion is greater than or equal to one error per 6.6 kb, which is consistent with the expected error rate of the PCR mix. The method is expected to facilitate various kinds of complex genetic engineering projects that require precise in-frame assembly of multiple fragments, such as somatic cell knockout in human cells or creation of whole genomes of viruses for vaccine research.     

Increased missegregation and chromosome loss with decreasing chromosome size in vertebrate cells

Chromosome engineering has allowed the generation of an extensive and well-defined series of linear human X centromere-based minichromosomes, which has been used to investigate the influence of size and structure on chromosome segregation in vertebrate cells. A clear relationship between overall chromosome size and mitotic stability was detected, with decreasing size associated with increasing loss rates. In chicken DT40, the lower size limit for prolonged mitotic stability is approximately 550 kb: at 450 kb, there was a dramatic increase in chromosome loss, while structures of approximately 200 kb could not be recovered. In human HT1080 cells, the size threshold for mitotic stability is approximately 1.6 Mb. Minichromosomes of 0.55–1.0 Mb can be recovered, but display high loss rates. However, all minichromosomes examined exhibited more segregation errors than normal chromosomes in HT1080 cells. This error rate increases with decreased size and correlates with reduced levels of CENP-A and Aurora B. In mouse LA-9 and Indian muntjac FM7 cells, the size requirements for mitotic stability are much greater. In mouse, a human 2.7-Mb minichromosome is rarely able to propagate a kinetochore and behaves acentrically. In Indian muntjac, CENP-C associates with the human minichromosome, but the mitotic apparatus appears unable to handle its segregation.     

Determination and in vivo characterization of the basic replicon of natural plasmids of Methylomonas clara

The basic replicon of the endogenous Methylomonas clara plasmid pBE-2 and its derivatives was defined to a region of 2.7 kb by in vivo deletions and conjugative transfer experiments using Escherichia coli-M. clara hybrid plasmids. Origin activity was found to be confined to a maximal length of 1.3 kb. The origin consists of two fragments which can be separated more than 4 kb by the integration of foreign DNA fragments without loss of function. A fragment having a maximum size of 2.1 kb supports in trans replication initiation at the origin. In addition, two incompatibility determinants were revealed, one localized in the origin fragment and the other outside the origin. Incompatibility between two basic replicons of the natural M. clara plasmids can be overcome by the integration of one of them in the compatible IncP plasmid R68-Kms. No homology was found between the plasmid basic replicon and the chromosomal DNA of M. clara.     

Drug resistance and R plasmids in Bordetella bronchiseptica isolates from pigs

A total of 207 strains of Bordetella bronchiseptica isolated from pigs in 1978 and 1979 were tested for drug resistance and for the properties of their R plasmids. Apart from intrinsic resistance to spectinomycin, single (sulfadimethoxine), double (sulfadimethoxine and streptomycin), andt riple (sulfadimethoxine, streptomycin, and ampicillin) resistance were found in 54.1%, 1.0%, and 15.9% of the strains, respectively. All of the triple-resistance determinants were associated with mercury resistance and were conjugative. pBB1, one of these R plasmids, was identified as Fi- (F) and Spp- (no suppression of phage multiplication) type, and as a member of incompatability group IncP. The single- and double-resistance determinants were nonconjugative. pBB2, one of the double-resistance determinants, was mobilized by an R plasmid, RP4, with the high efficiency of 80% and at a frequency of 3.3% without cotransfer of RP4. The molecular weight of pBB1 and pBB2 was estimated at 36 X 10(6) and 13 X 10(6) daltons, respectively, by electron microscopy and agarose gel electrophoresis. pBB1 had five cleavage sites for EcoRI endonuclease, and four sites for HindIII. pBB2 had two EcoRI sites, one HindIII, and one BamHI site. Cells carrying pBB1 or pBB2 produced enzymic activity tha inactivated streptomycin in the presence of ATP.     

Effects of genes exerting growth inhibition and plasmid stability on plasmid maintenance.

Plasmid stabilization mediated by the parA+ and parB+ genes of the R1 plasmid and the ccd+ and sop+ genes of the F plasmid was tested on a mini-R1 plasmid and a pBR322 plasmid derivative. The mini-R1 plasmid is thought to be unstably inherited owing to a low copy number and to random segregation of the plasmid at cell division, whereas cells harboring the pBR322 derivative used in this work are lost through competition with plasmid-free cells, mainly as a result of the shorter generation time of cells without plasmids. The pBR322 derivative carries a fusion between part of the atp operon of Escherichia coli and the bacteriophage lambda pR promoter, and the cI857 repressor gene. The insertion of sop+ from the F plasmid or parB+ from the R1 plasmid reduced the loss frequency by a factor of 10(3) for the pBR322 derivative and by at least a factor of 10(2) for the mini-R1 plasmid. Insertion of parA+ from the R1 plasmid decreased the loss frequency of the pBR322 derivative by a factor of 10 and that of the mini-R1 plasmid by a factor of 50. When ccd+ from the F plasmid was inserted, the loss frequency of the pBR322 derivative was decreased by a factor of 10, but it had only a marginal effect on the stability of the mini-R1 plasmid. In no case was any significant structural instability of the plasmids observed.     

The insertion of large pieces of foreign genetic material reduces the stability of bacterial plasmids

The stability of genetically engineered bacterial plasmids under continuous culture fermentation is of crucial importance in the application of microbiology to many processes of potential industrial importance. In order to determine the effect of inserting large pieces of foreign DNA on the stability of bacterial plasmids we have studied the behavior of pAT153 with DNA inserts of various sizes derived from cytomegalovirus. Foreign DNA up to 2 kb in length had no effect on stability, whereas the insertion of an 8-kb fragment resulted in a transient instability. This instability was overcome by the spontaneous appearance of leu+ cells in the culture. Insertion of a 21-kb DNA fragment resulted in a rapid loss of plasmid, which was not prevented by the appearance of leu+ cells. In all cases copy number analyses indicated that plasmid loss was due to segregational instability, probably because the plasmid placed an unacceptable metabolic load on the cell.     

Identification of a maintenance system on rolling circle replicating plasmid pVT736-1

Distribution of plasmid molecules to the two daughter cells at cell division is of major importance for their stable inheritance. Several mechanisms that control equipartitioning of low-copy-number plasmids have been described in molecular terms. However, no homologous or analogous systems have been identified for intermediate or high-copy-number plasmids, including rolling circle replicating (RCR) plasmids. It has been suggested that distribution of such plasmids at cell division relies solely on random segregation. Plasmid pVT736-1 is a 2 kb RCR plasmid that was isolated from the Gram-negative capnophilic coccobacillus Actinobacillus actinomycetemcomitans . The plasmid contains a DNA region of approximately 0.8 kb that is associated with its segregational stability. An operon that consists of two genes ( orf3 and orf2 ) is followed by a putative cis -acting site that contains an integration host factor (IHF) binding site, flanked by several repeats. Mutations in orf 2 resulted in plasmid instability. In addition, this DNA region was able to stabilize partially a heterologous replicon, p15A. Homologues or analogues of the pVT736-1 stabilization system have been detected on numerous plasmid and bacterial genomes.