Support Plasmid Replication Research Articles

Characterization of an accessory plasmid of Sinorhizobium meliloti and its two replication-modules.

Rhizobia are Gram-negative bacteria known for their ability to fix atmospheric N2 in symbiosis with leguminous plants. Current evidence shows that rhizobia carry in most cases a variable number of plasmids, containing genes necessary for symbiosis or free-living, a common feature being the presence of several plasmid replicons within the same strain. For many years, we have been studying the mobilization properties of pSmeLPU88b from the strain Sinorhizobium meliloti LPU88, an isolate from Argentina. To advance in the characterization of pSmeLPU88b plasmid, the full sequence was obtained. pSmeLPU88b is 35.9 kb in size, had an average GC % of 58.6 and 31 CDS. Two replication modules were identified in silico: one belonging to the repABC type, and the other to the repC. The replication modules presented high DNA identity to the replication modules from plasmid pMBA9a present in an S. meliloti isolate from Canada. In addition, three CDS presenting identity with recombinases and with toxin-antitoxin systems were found downstream of the repABC system. It is noteworthy that these CDS present the same genetic structure in pSmeLPU88b and in other rhizobial plasmids. Moreover, in all cases they are found downstream of the repABC operon. By cloning each replication system in suicide plasmids, we demonstrated that each of them can support plasmid replication in the S. meliloti genetic background, but with different stability behavior. Interestingly, while incompatibility analysis of the cloned rep systems results in the loss of the parental module, both obtained plasmids can coexist together.

Lactococcus Ceduovirus Phages Isolated from Industrial Dairy Plants—From Physiological to Genomic Analyses

LactococcusCeduovirus (formerly c2virus) bacteriophages are among the three most prevalent phage types reported in dairy environments. Phages from this group conduct a strictly lytic lifestyle and cause substantial losses during milk fermentation processes, by infecting lactococcal host starter strains. Despite their deleterious activity, there are limited research data concerning Ceduovirus phages. To advance our knowledge on this specific phage group, we sequenced and performed a comparative analysis of 10 new Lactococcus lactis Ceduovirus phages isolated from distinct dairy environments. Host range studies allowed us to distinguish the differential patterns of infection of L. lactis cells for each phage, and revealed a broad host spectrum for most of them. We showed that 40% of the studied Ceduovirus phages can infect both cremoris and lactis strains. A preference to lyse strains with the C-type cell wall polysaccharide genotype was observed. Phage whole-genome sequencing revealed an average nucleotide identity above 80%, with distinct regions of divergence mapped to several locations. The comparative approach for analyzing genomic data and the phage lytic spectrum suggested that the amino acid sequence of the orf8-encoded putative tape measure protein correlates with host range. Phylogenetic studies revealed separation of the sequenced phages into two subgroups. Finally, we identified three types of phage origin of replication regions, and showed they are able to support plasmid replication without additional phage proteins.

The Origin of Mutants Under Selection: How Natural Selection Mimics Mutagenesis (Adaptive Mutation).

Selection detects mutants but does not cause mutations. Contrary to this dictum, Cairns and Foster plated a leaky lac mutant of Escherichia coli on lactose medium and saw revertant (Lac(+)) colonies accumulate with time above a nongrowing lawn. This result suggested that bacteria might mutagenize their own genome when growth is blocked. However, this conclusion is suspect in the light of recent evidence that revertant colonies are initiated by preexisting cells with multiple copies the conjugative F'lac plasmid, which carries the lac mutation. Some plated cells have multiple copies of the simple F'lac plasmid. This provides sufficient LacZ activity to support plasmid replication but not cell division. In nongrowing cells, repeated plasmid replication increases the likelihood of a reversion event. Reversion to lac(+) triggers exponential cell growth leading to a stable Lac(+) revertant colony. In 10% of these plated cells, the high-copy plasmid includes an internal tandem lac duplication, which provides even more LacZ activity—sufficient to support slow growth and formation of an unstable Lac(+) colony. Cells with multiple copies of the F'lac plasmid have an increased mutation rate, because the plasmid encodes the error-prone (mutagenic) DNA polymerase, DinB. Without DinB, unstable and stable Lac(+) revertant types form in equal numbers and both types arise with no mutagenesis. Amplification and selection are central to behavior of the Cairns-Foster system, whereas mutagenesis is a system-specific side effect or artifact caused by coamplification of dinB with lac. Study of this system has revealed several broadly applicable principles. In all populations, gene duplications are frequent stable genetic polymorphisms, common near-neutral mutant alleles can gain a positive phenotype when amplified under selection, and natural selection can operate without cell division when variability is generated by overreplication of local genome subregions.

Murine Gammaherpesvirus 68 Contains Two Functional Lytic Origins of Replication

A 1.25-kbp DNA fragment from the right side of the genome containing the lytic origin of replication (oriLyt) of murine gammaherpesvirus 68 (MHV-68) has been identified by a plasmid replication assay. Here we show that a mutant MHV-68 with a deletion of an essential part of this oriLyt, generated by using an MHV-68 bacterial artificial chromosome, was only slightly attenuated and still able to replicate but that a mutant containing an additional deletion on the left side of the genome was replication deficient. The newly identified region was sufficient to support plasmid replication, thus providing evidence for a second oriLyt.

Chromosomal deletion formation system based on Tn5 double transposition: use for making minimal genomes and essential gene analysis.

In this communication, we describe the use of specialized transposons (Tn5 derivatives) to create deletions in the Escherichia coli K-12 chromosome. These transposons are essentially rearranged composite transposons that have been assembled to promote the use of the internal transposon ends, resulting in intramolecular transposition events. Two similar transposons were developed. The first deletion transposon was utilized to create a consecutive set of deletions in the E. coli chromosome. The deletion procedure has been repeated 20 serial times to reduce the genome an average of 200 kb (averaging 10 kb per deletion). The second deletion transposon contains a conditional origin of replication that allows deleted chromosomal DNA to be captured as a complementary plasmid. By plating cells on media that do not support plasmid replication, the deleted chromosomal material is lost and if it is essential, the cells do not survive. This methodology was used to analyze 15 chromosomal regions and more than 100 open reading frames (ORFs). This provides a robust technology for identifying essential and dispensable genes.

Components of human papillomavirus that activate transcription and support plasmid replication in human airway cells.

Human papillomaviruses (HPVs) such as types 6 and 11 can establish lifelong infections in airway epithelial cells in patients, and long-term infection can lead to pulmonary involvement and death. The mechanisms underlying this persistence depend on both the transcriptional activity of the viral enhancers and promoters and the ability of this virus to maintain its double-stranded circular DNA genome in infected tissues. We investigated the transcription and replication properties of HPV sequence elements and protein products in a human airway cell line. We showed that incorporation of the upstream regulatory region and cotransfection with expression vectors of two virus-encoded proteins, E1 and E2, conferred approximately 5,000-fold stimulation of reporter gene expression. Transient plasmid replication in transfected human airway cells and lungs of FVB/N-C57BL/6 mice was demonstrated by a modified transient replication assay. These results have important implications for viral pathogenesis in airway cells and the potential of HPV-based replicons for gene transfer into airway epithelium.

An embryonic demethylation mechanism involving binding of transcription factors to replicating DNA

In vertebrates, transcriptionally active promoters are undermethylated. Since the transcription factor Sp1, and more recently NF-kappaB, have been implicated in the demethylation process, we examined the effect of transcription factors on demethylation by injecting in vitro methylated plasmid DNA into Xenopus fertilized eggs. We found that various transactivation domains, including a strong acidic activation domain from the viral protein VP16, can enhance demethylation of a promoter region when fused to a DNA binding domain which recognizes the promoter. Furthermore, demethylation occurs only after the midblastula transition, when the general transcription machinery of the host embryo becomes available. Nevertheless, transcription factor binding need not be followed by actual transcription, since demethylation is not blocked by alpha-amanitin treatment. Finally, replication of the target DNA is a prerequisite for efficient demethylation since only plasmids that carry the bovine papilloma virus sequences which support plasmid replication after the midblastula transition are demethylated. No demethylation is detectable in the oocyte system where DNA is not replicated. These results suggest that, in the Xenopus embryo, promoters for which transcription factors are available are demethylated by a replication-dependent, possibly passive mechanism.

DNA bending of pSC101 ori induced by Rep, a replication initiator protein and IHF.

Purified Rep (or RepA) protein, a replication initiator of plasmid pSC101, is present almost solely in the dimer form, and its binding activity for the directly repeated sequences (iterons) in the replication origin (ori) is very low. When Rep protein was treated with guanidine hydrochloride followed by renaturation, it was shown to bind to the iterons with very high efficiency. A gel shift experiment suggested that guanidine-treated Rep bound to iterons as a monomer form. The Rep monomer bound noncooperatively to the three iterons and induced bending of the DNA helix axis in the same direction (about 100 degrees ). The configuration of the IHF box that is a binding site of another DNA bending protein IHF, the three iterons and an AT-rich region between these sequences was important for efficient bending of the ori region. Furthermore, a mutant Rep protein (Rep(IHF)) which can support the plasmid replication in IHF-deficient host cells was purified, and it was found that affinity of the Rep(IHF) monomer for iterons was similar to that of wild-type Rep and bent DNA only 14 degrees more strongly than did the wild-type Rep. Rep(IHF)-dependent plasmid replication, however, required both enhancer regions, par and IR-1, in addition to "core ori" as a minimal essential ori, whereas only one of these two enhancers was necessary for wild-type Rep-dependent replication. How Rep(IHF) can support plasmid replication in the absence of IHF is discussed.

Initiation of DNA replication at cloned origins of bacteriophage T7

Bacteriophage T7 DNA replication is initiated at a site 15% of the distance from the genetic left end of the chromosome. This primary origin contains two tandem T7 RNA polymerase promoters (φ1.1A and φ1.1B) followed by an A + T-rich region. When the primary origin region is deleted replication initiates at secondary origins. We have analyzed the ability of plasmids containing cloned fragments of T7 to replicate after infection of Escherichia coli with bacteriophage T7. All cloned T7 fragments that support plasmid replication contain a T7 promoter but a T7 promoter alone is not sufficient for replication. Replication of plasmids containing the primary origin is dependent on T7 DNA polymerase and gene 4 protein (helicase/primase) and a portion of the A + T-rich region. The other T7 fragments that support plasmid replication after T7 infection are promoter regions φOR, φ13 and φ6.5 (secondary origins). When both the primary and secondary origins are present simultaneously on compatible plasmids, replication of each is temporally regulated. Such regulation may play a role during T7 DNA replication.

Autonomous replication of plasmids bearing monkey DNA origin-enriched sequences.

Twelve clones of origin-enriched sequences (ORS) isolated from early replicating monkey (CV-1) DNA were examined for transient episomal replication in transfected CV-1, COS-7, and HeLa cells. Plasmid DNA was isolated at time intervals after transfection and screened by the Dpn I resistance assay or by the bromodeoxyuridine substitution assay to differentiate between input and replicated DNA. We have identified four monkey ORS (ORS3, -8, -9, and -12) that can support plasmid replication in mammalian cells. This replication is carried out in a controlled and semiconservative manner characteristic of mammalian replicons. ORS replication was most efficient in HeLa cells. Electron microscopy showed ORS8 and ORS12 plasmids of the correct size with replication bubbles. Using a unique restriction site in ORS12, we have mapped the replication bubble within the monkey DNA sequence.

Trans activation of an Epstein-Barr viral transcriptional enhancer by the Epstein-Barr viral nuclear antigen 1.

Two regions of the Epstein-Barr virus (EBV) genome together make up an element, oriP, which acts in cis to support plasmid replication in cells that express the EBV nuclear antigen 1 (EBNA-1). The two components of oriP are a region containing a 65-base-pair (bp) dyad symmetry and a region containing 20 copies of a 30-bp direct repeat. Here we show that the 30-bp family of repeats of oriP can function as a transcriptional enhancer that is activated in trans by the EBNA-1 gene product. In either EBV-genome-positive cells or in cells that express EBNA-1, the 30-bp family of repeats, when positioned in either orientation upstream or downstream, enhances expression of the chloramphenicol acetyltransferase (CAT) gene expressed from either the simian virus 40 early promoter or the herpes simplex virus type 1 thymidine kinase promoter. The extent of transcriptional enhancement varies with the promoter and cell type. This enhanced CAT expression reflects an increased level of CAT mRNA and does not result from amplification of the plasmids expressing CAT. In addition, plasmids carrying the gene for resistance to hygromycin B and the 30-bp family of repeats yielded 10 to 100 times more hygromycin B-resistant colonies than the vector lacking the 30-bp family of repeats in both EBV-genome-positive cells and cells that express EBNA-1. EBNA-1 is known to bind to sequences within the 30-bp family of repeats (D. R. Rawlins, G. Milman, S. D. Hayward, and G. S. Hayward, Cell 42:859-868, 1985), and these trans- and cis-acting elements together have at least two functional roles: (i) they are required for DNA replication dependent upon oriP, and (ii) they can enhance expression of genes linked to the 30-bp family of repeats of oriP.

Trans activation of an Epstein-Barr viral transcriptional enhancer by the Epstein-Barr viral nuclear antigen 1.

Two regions of the Epstein-Barr virus (EBV) genome together make up an element, oriP, which acts in cis to support plasmid replication in cells that express the EBV nuclear antigen 1 (EBNA-1). The two components of oriP are a region containing a 65-base-pair (bp) dyad symmetry and a region containing 20 copies of a 30-bp direct repeat. Here we show that the 30-bp family of repeats of oriP can function as a transcriptional enhancer that is activated in trans by the EBNA-1 gene product. In either EBV-genome-positive cells or in cells that express EBNA-1, the 30-bp family of repeats, when positioned in either orientation upstream or downstream, enhances expression of the chloramphenicol acetyltransferase (CAT) gene expressed from either the simian virus 40 early promoter or the herpes simplex virus type 1 thymidine kinase promoter. The extent of transcriptional enhancement varies with the promoter and cell type. This enhanced CAT expression reflects an increased level of CAT mRNA and does not result from amplification of the plasmids expressing CAT. In addition, plasmids carrying the gene for resistance to hygromycin B and the 30-bp family of repeats yielded 10 to 100 times more hygromycin B-resistant colonies than the vector lacking the 30-bp family of repeats in both EBV-genome-positive cells and cells that express EBNA-1. EBNA-1 is known to bind to sequences within the 30-bp family of repeats (D. R. Rawlins, G. Milman, S. D. Hayward, and G. S. Hayward, Cell 42:859-868, 1985), and these trans- and cis-acting elements together have at least two functional roles: (i) they are required for DNA replication dependent upon oriP, and (ii) they can enhance expression of genes linked to the 30-bp family of repeats of oriP.

Human papillomavirus DNA replication mediated by simian virus 40 T antigen in trans.

The putative E1 gene product of papillomaviruses is thought to be involved in the initiation of viral replication, as large-T antigen (T antigen) is in the case of polyomaviruses. Mouse cell lines cloned after transformation by a plasmid consisting of the simian virus 40 (SV40) early region and the complete genome of human papillomavirus type 16 (HPV16) maintained episomal plasmid DNA. In contrast, the DNAs of either SV40 or HPV16, when employed separately in transfection experiments, were consistently integrated into the host DNA. To test the hypothesis that SV40 T antigen might be involved in the replication of the hybrid plasmids, HPV16 DNA was used in a transient replication assay for transfection of either CV-1 or COS-7 cells. The HPV16 DNA replicated to a high copy number in the T antigen-producing COS-7 cells, but failed to replicate in the CV-1 cells. To define the HPV16 sequences that were essential for the plasmid maintenance in SV40 T antigen-producing cells, restriction fragments of HPV16 were analysed for their replication capacity in COS-7 cells. Here it is reported that the presence of the coding region of the putative E1 gene product of HPV16 together with the 5' transcriptional control elements is essential and is sufficient to support plasmid replication mediated by SV40 T antigen in trans.