Thermostability and superhelicity of plasmid DNA in Bacillus stearothermophilus

Summary The thermostability of the staphylococcal plasmids pC194 and pUB110 and their antibiotic-resistance determinants was examined upon transfer to Bacillus stearothermophilus CU21. Plasmid pGS13, a pUB110 derivative carrying the chloramphenicol acetyltransferase (CAT) gene of pC194, could be maintained up to the maximum growth temperature (68° C) by selection for chloramphenicol resistance. In the absence of selective pressure, pGS13 was lost at temperatures above 60° C. Segregational instability of pGS13 was accompanied by a progressive loss of negative superhelicity at elevated temperatures. Thermostable mutants of pGS13 were isolated by screening for expression of the antibiotic-resistance determinants after growth under non-selective conditions. These mutants were found to contain an insertion of a 1.7 kb DNA sequence derived from the cryptic B. stearothermophilus plasmid pBS02. Increased thermostability correlated with preservation of plasmid superhelicity at elevated temperatures.     

Senescence associated with the over-replication of a mitochondrial retroplasmid in Neurospora crassa

Mitochondrial DNA rearrangements and deletions are a prevailing feature of filamentous fungal cultures that undergo senescence. In Neurospora spp., strains containing the Mauriceville and Varkud mitochondrial retroplasmids routinely senesce at elevated temperatures, a process that is initiated by the integration of variant forms of the plasmids into the mitochondrial genome. Here, we describe a strain that is phenotypically distinguishable from previously characterized senescent strains and show that senescence can occur in the absence of plasmid integration and associated alterations in mitochondrial DNA. The MS4416 strain contains a unique variant of the Mauriceville retroplasmid, and undergoes senescence at highly predictable frequencies at 37°, 25° and 18 °C. Decline in vegetative growth rate correlates with increased levels of the variant plasmid and alterations in the synthesis of mitochondrially encoded proteins, suggesting that plasmid over-replication interferes with mitochondrial translation. We also report the isolation of a mutant strain that escapes senescence yet still maintains high levels of the variant plasmid. Its ability to tolerate a growth-suppressive retroplasmid suggests that there are mechanisms in Neurospora which compensate for the deleterious effects that plasmid over-replication has on mitochondrial function. Received: 12 July 1999 / Accepted: 17 December 1999     

Plasmid pSC101 replication in integratively suppressed cells requires dnaA function.

Summary Maintenance of plasmid pKC17, a derivative of plasmid pSC101, in E. coli requires a functional dnaA gene product. This was demonstrated by segregation experiments using an E. coli dnaA46 mutant, at various temperatures. Growth of this mutant at elevated temperature was allowed by the presence of a P2sig5 prophage. rpoB mutations which suppress the temperature sensitivity of a dnaA46 mutant permit efficient maintenance of plasmid pKC17 at temperatures up to 40°, conditions which normally inactivate the dnaA46 product.     

Mutagenesis of plasmid DNA with hydroxylamine: Isolation of mutants of multi-copy plasmids

Summary An investigation of in vitro mutagenesis of plasmid DNA with hydroxylamine is described. The treated plasmid DNA was used to transform Escherichia coli K12. Mutants of the plasmid NTP3, which codes for resistance to ampicillin and sulphonamides, were isolated and characterised. They were classified according to the reduction in level of their -lactamase activity. Hydroxylamine-induced mutants of NTP14 were also isolated. This plasmid codes for ampicillin resistance, synthesis of colicin E1, and the EcoRI restriction and modification enzymes. One class of mutants is lethal to the host strain at temperatures above 33° C, but carrier strains grow well at 28° C. There is evidence that these mutants code for a temperature-sensitive EcoRI modification activity: the lethal effect probably results from the cleavage of the host-cell DNA by the restriction enzyme at non-permissive temperatures. The possible genetic uses of the mutant plasmids for the production of hybrid plasmids in the bacterial cell are discussed.     

Ligation mediated PCR performed at low denaturation temperatures—PCR melting profiles

We show that using low denaturation temperatures (80–88°C) during ligation mediated PCR (LM PCR) of bacterial DNA leads to the amplification of limited sets of the less stable DNA fragments. A set of electrophoretic patterns of such fragments obtained at different denaturation temperatures forms the PCR melting profile (PCR MP). A single pattern obtained for a given temperature and a set of patterns arising after application of several denaturation temperatures (PCR MP) are very specific for the given bacterial genome and may be used for strain characterisation and differentiation. The method may also be used for amplification and isolation of the less stable DNA fragments in a genome.     

Conditions and mutations affecting the maintenance and replication of plasmid DNA in Escherichia coli 15

Summary An Escherichia coli 15 strain has been constructed which contains, in addition to the plasmids inherent to E. coli 15 (P 1-like DNA and minicircular DNA), the colicinogenic factor E₁ (Col E₁). Whereas the P 1-like DNA of E. coli 15 is unaffected by the uptake of the colicin plasmid, the number of copies of minicircular DNA of E. coli 15 decreases and an equivalent amount of Col E₁ DNA becomes established in the cell. The ratio between these two small plasmids is dependent on the growth temperature. The mode of replication of minicircular DNA and Col E₁ DNA is very similar, but is different in various respects from that of the P 1-like plasmid: 1. Both small plasmids continue to replicate in the presence of chloramphenicol, whereas the replication of P 1-like DNA stops like the chromosomal DNA. 2. Rifampicin inhibits the synthesis of both small plasmids rather rapidly. The replication of P 1-like DNA continues during the remaining replication cycle of the chromosome in the presence of rifampicin. 3. The replication of Col E₁ DNA and of the minicircular DNA still proceeds at elevated temperatures (45–50°C), whereas little or no incorporation of ³H-thymidine into P 1-like DNA is observed at these temperatures. 4. Mutants have been obtained, which show altered properties in the maintenance and replication of the plasmids without being affected in the replication of the chromosomal DNA. In all these mutants the replication and (or) maintenance of the minicircular DNA of E. coli 15 and Col E₁ DNA is affected in the same way, but not that of the P 1-like plasmid.     

Plasmid amplification-promoting sequences from the origin region of Chinese hamster dihydrofolate reductase gene do not promote position-independent chromosomal gene amplification

Initiation of DNA synthesis occurs with high frequency at oriß, a region of DNA from the amplified dihydrofolate reductase (DHFR) domain of Chinese hamster CHOC 400 cells that contains an origin of bidirectional DNA replication (OBR). Recently, sequences from DHFR oriß/OBR were shown to stimulate amplification of cis-linked plasmid DNA when transfected into murine cells. To test the role of oriß/OBR in chromosomal gene amplification, linearized plasmids containing these sequences linked to a DHFR expression cassette were introduced into DHFR- CHO DUKX cells. After selection for expression of DHFR, cell lines that contain a single integrated, unrearranged copy of the linearized expression plasmid were identified and exposed to low levels of the folate analog, methotrexate (MTX). Of seven clonal cell lines containing the vector control, three gained resistance to MTX by 5 to 15-fold amplification of the integrated marker gene. Of 16 clonal cell lines that contained oriß/OBR linked to a DHFR mini-gene, only 6 gained resistance to MTX by gene amplification. Hence, sequences from the DHFR origin region that stimulate plasmid DNA amplification do not promote amplification of an integrated marker gene in all chromosomal contexts. In addition to showing that chromosomal position has a strong influence on the frequency of gene amplification, these studies suggest that the mechanism that mediates the experiment of episomal plasmid DNA does not contribute to the early steps of chromosomal gene amplification.     

DNA assay for recombinant pharmaceutical substances using the real-time PCR technique

A real-time PCR procedure is proposed for assaying E. coli residual DNA in the pharmaceutical substance of human recombinant insulin. For the quantitative analysis of the DNA content, an amplification of fragments of the bla gene plasmid DNA and E. coli genomic DNA of the 16S RNA gene were used. The contents of plasmid and genomic DNA were detected both in intermediates at various stages of the insulin purification process and in the finished product.     

The Saccharomyces cerevisiae protein YJR043C (Pol32) interacts with the catalytic subunit of DNA polymerase α and is required for cell cycle progression in G2?/?M

We have analysed the YJR043c gene of Saccharomyces cerevisiae, previously identified by systematic sequencing. The deletion mutant (yjr043cΔ) shows slow growth at low temperature (15° C), while at 30° C and 37° C the growth rate of mutant cells is only moderately affected. At permissive and nonpermissive temperatures, mutant cells were larger and showed a high proportion of large-budded cells with a single duplicated nucleus at or beyond the bud neck and a short spindle. This phenotype was even more striking at low temperature, the mutant cells becoming dumbbell shaped. All these phenotypes suggest a role for YJR043C in cell cycle progression in G2/M phase. In two-hybrid assays, the YJR043c gene product specifically interacted with Poll, the catalytic subunit of DNA polymerase α. The pol1-1 /yjr043cΔ double mutant showed a more severe growth defect than the pol1-1 single mutant at permissive temperature. Centromeric plasmid loss rate elevated in yjr043cΔ. Analysis of the sequence upstream of the YJR043c ORF revealed the presence of an MluI motif (ACGCGT), a sequence associated with many genes involved in DNA replication in budding yeast. The cell cycle phenotype of the yjr043cΔ mutant, the evidence for genetic interaction with Pol1, the presence of an MluI motif upstream and the elevated rate of CEN plasmid loss in mutants all support a function for YJR043C in DNA replication. Received: 22 July 1998 / Accepted: 22 September 1998     

Low-copy-number plasmid-cloning vectors amplifiable by derepression of an inserted foreign promoter

By insertion of a DNA fragment, containing the phage lambda pR promoter and the pM-promoted cI857 allele of the lambda repressor gene, in plasmid R1 upstream of the replication control genes, cloning vectors have been constructed which are present in one copy per chromosome at temperatures below 37 degrees C, and which display uncontrolled replication at 42 degrees C. Derivatives have been made which carry the R1 par region, stabilizing the plasmid at low temperature when grown in the absence of selection pressure. Cells harbouring these plasmids stop growing after 1-2 h incubation at 42 degrees C, and at this time 50% of the total DNA in the cells is plasmid DNA corresponding to more than 1000 plasmid molecules per cell. Concomitant with plasmid amplification at the high temperature, synthesis of plasmid-coded gene products is amplified, and these vectors can therefore be utilized for obtaining greatly enhanced yields of gene products that may be detrimental to the host cell when present in large amounts.     

Characterization of putative circular plasmids in sponge‐associated bacterial communities using a selective multiply‐primed rolling circle amplification

Plasmid transfers among bacterial populations can directly influence the ecological adaptation of these populations and their interactions with host species and environment. In this study, we developed a selective multiply‐primed rolling circle amplification (smRCA) approach to enrich and characterize circular plasmid DNA from sponge microbial symbionts via high‐throughput sequencing (HTS). DNA (plasmid and total community DNA) obtained from sponge (Cinachyrella sp.) samples and a bacterial symbiont (Vibrio sp. CyArs1) isolated from the same sponge species (carrying unknown plasmids) were used to develop and validate our methodology. The smRCA was performed during 16 hr with 141 plasmid‐specific primers covering all known circular plasmid groups. The amplified products were purified and subjected to a reamplification with random hexamer primers (2 hr) and then sequenced using Illumina MiSeq. The developed method resulted in the successful amplification and characterization of the sponge plasmidome and allowed us to detect plasmids associated with the bacterial symbiont Vibrio sp. CyArs1 in the sponge host. In addition to this, a large number of small (<2 kbp) and cryptic plasmids were also amplified in sponge samples. Functional analysis identified proteins involved in the control of plasmid partitioning, maintenance and replication. However, most plasmids contained unknown genes, which could potentially serve as a resource of unknown genetic information and novel replication systems. Overall, our results indicate that the smRCA‐HTS approach developed here was able to selectively enrich and characterize plasmids from bacterial isolates and sponge host microbial communities, including plasmids larger than 20 kbp.     

Cooler temperatures slow the repair of DNA damage in tadpoles exposed to ultraviolet radiation: Implications for amphibian declines at high altitude

Ultraviolet B radiation (UVBR) damages the DNA of exposed cells, causing dimers to form between adjacent pyrimidine nucleotides. These dimers block DNA replication, causing mutations and apoptosis. Most organisms utilize biochemical or biophysical DNA repair strategies to restore DNA structure; however, as with most biological reactions, these processes are likely to be thermally sensitive. Tadpoles exposed to elevated UVBR at low environmental temperatures have significantly higher rates of mortality and developmental deformities compared with tadpoles exposed to the same levels of UVBR at higher environmental temperatures. We hypothesized that low environmental temperatures impair the primary enzymatic (photolyase) DNA repair pathway in amphibians, leading to the accumulation of DNA damage. To test this hypothesis, we compared DNA repair rates and photolyase gene expression patterns in Limnodynastes peronii. Tadpoles were acutely exposed to UVBR for 1 hr at either 20 or 30°C, and we measured DNA damage and photolyase expression levels at intervals following this exposure. Temperature had a significant effect on the rate of DNA repair, with repair at 30°C occurring twice as fast as repair at 20°C. Photolyase gene expression (6‐4 PP and CPD) was significantly upregulated by UVBR exposure, with expression levels increasing within 6 hr of UVBR exposure. CPD expression levels were not significantly affected by temperature, but 6‐4 PP expression was significantly higher in tadpoles in the 30°C treatment within 12 hr of UVBR exposure. These data support the hypothesis that DNA repair rates are thermally sensitive in tadpoles and may explain why enigmatic amphibian declines are higher in montane regions where UVBR levels are naturally elevated and environmental temperatures are lower.     

Isolation of Plasmid DNA rescued from single colonies of<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> by means of rolling circle amplification

We report a simple method to isolate plasmids from single colonies ofAgrobacterium tumefaciens by means of rolling circle amplification. The amplified DNA can be digested by restriction enzymes for plasmid verification and transformed intoEscherichia coli for plasmid rescue. Compared with conventional procedures, this method eliminates liquid culturing ofAgrobacterium cells and subsequent DNA isolation and enables large-scale plasmid analyses.     

Inactivation of recombinant plasmid DNA from a human erythropoietin-producing mouse cell line grown on a large scale

Summary Experiments were carried out to assess the survival of recombinant plasmid DNA during large-scale production of recombinant human erythropoietin (rhuEPO) in a fermentation pilot plant. The analyses revealed DNA-degrading activities in the fermentation broth and in the waste-water, leading to rapid destruction of plasmid DNA added to medium or waste-water. The capability of the plasmid-DNA-spiked samples to transform competent bacteria was drastically reduced. The DNA-degrading activity in the waste-waters could be blocked by addition of EDTA or by boiling, indicating the presence of DNA-degrading enzymes (DNases). No plasmid-specific DNA sequences were detected in waste-water samples by in-vitro amplification with Taqpolymerase. Genomic DNA preparations of cell debris collected from waste-water samples only contained degraded plasmid DNA. Furthermore, it was shown that intact plasmid DNA could be degraded to fragments of less than 1000 bp by incubation at 121°C for 20 min, leading to a decrease in the plasmid-specific transforming capacity by a factor of 10³ per minute. Thus, DNA from the rhuEPO production pilot plant was efficiently inactivated at three different levels: (i) in the fermentation medium (DNase), (ii) in the waste-water container (DNase), and (iii) by heat inactivation for 20 min at 120°C. These results indicate that the probability of delivery of recombinant DNA into the environment is extremely low in such biotechnological production processes. Offprint requests to: M. R. Fibi     

Horizontal transfer of a mitochondrial plasmid

Direct evidence for horizontal transfer of a mitochodnrial plasmid from the discomyceteAscobolus immersus to the pyrenomycetePodospora anserina is presented. Southern blot hybridisation analysis, polymerase chain reaction (PCR) amplification, and DNA sequencing demonstrate transmission of a linear plasmid upon hyphal contact. DNA extraction from isolated organelles indicates a mitochondrial localisation for the plasmid inP. anserina. This is the first report of horizontal gene transfer among unrelated fungi. These results have important evolutionary implications for plasmid propagation in fungi.     

DNA synthesis in P1 infected E. coli mutants temperature-sensitive in DNA replication

Summary P1 DNA is synthesized in the E. coli ts dna mutants 165/70 (elongation defect) and 252 (initiation defect) at elevated temperatures. In strain 165/70, P1 infection at 41°C leads to phage production accompanied by a transient recovery of bacterial DNA synthesis. No phages are produced byt P1 DNA is still synthesized in strain 252 if infected after host DNA replication has come to a halt at 42°C.     

Delivery of plasmid DNA into intact plant cells by electroporation of plasmolyzed cells

This report describes the delivery of plasmid DNA containing either the β-glucuronidase (GUS) or the green fluorescent protein (GFP) reporter gene into intact plant cells of bamboo callus, lilium scales, and Nicotiana benthamiana suspension culture cells. By first plasmolyzing the tissues or cells with 0.4 m sucrose in the presence of plasmid DNA, electroporation effectively delivers plasmid DNA into the intact plant cells. Transient expression of the GUS gene, as revealed by histochemical assays, showed the presence of blue-staining areas in the electroporated tissues. A short exposure of cells to 2% DMSO (dimethyl sulfoxide) prior to plasmolysis elevated the level of transient GUS activity. When plasmid DNA containing a synthetic GFP gene was used, a strong green fluorescence was observed in N. benthamiana suspension culture cells that were subjected to plasmolysis and electroporation. These results suggest that plasmolysis brings the plasmid DNA into the void space that is in close vicinity to the plasmalemma, allowing electroporation to efficiently deliver the plasmid DNA into intact plant cells. Received: 15 June 1998 / Revision received: 18 August 1998 / Accepted: 28 August 1998     

Identification and characterization of CbeI,a novel thermostable restriction enzyme from <Emphasis Type="Italic">Caldicellulosiruptor bescii</Emphasis> DSM 6725 and a member of a new subfamily of HaeIII-like enzymes

Potent HaeIII-like DNA restriction activity was detected in cell-free extracts of Caldicellulosiruptor bescii DSM 6725 using plasmid DNA isolated from Escherichia coli as substrate. Incubation of the plasmid DNA in vitro with HaeIII methyltransferase protected it from cleavage by HaeIII nuclease as well as cell-free extracts of C. bescii. The gene encoding the putative restriction enzyme was cloned and expressed in E. coli with a His-tag at the C-terminus. The purified protein was 38 kDa as predicted by the 981-bp nucleic acid sequence, was optimally active at temperatures between 75°C and 85°C, and was stable for more than 1 week when stored at 35°C. The cleavage sequence was determined to be 5′-GG/CC-3′, indicating that CbeI is an isoschizomer of HaeIII. A search of the C. bescii genome sequence revealed the presence of both a HaeIII-like restriction endonuclease (Athe 2438) and DNA methyltransferase (Athe 2437). Preliminary analysis of other Caldicellulosiruptor species suggested that this restriction/modification activity is widespread in this genus. A phylogenetic analysis based on sequence alignment and conserved motif searches identified features of CbeI distinct from other members of this group and classified CbeI as a member of a novel subfamily of HaeIII-like enzymes.     

Regulation of replication of plasmid pBR322 in amino acid-starved Escherichia coli strains

The stringent response causes inhibition of replication of plasmid pBR322 in amino acid-starved Escherichia coli cells whereas in relaxed mutants the replication of this plasmid proceeds for several hours. On the basis of density shift experiments and pulse-labelling experiments we showed that most of the pBR322 molecules begin replication during the relaxed response and the rate of plasmid DNA synthesis in unstarved and isoleucine-starved relA ^–] bacteria is similar. We found that the Rom function plays a key role in the stringent control of plasmid pBR322 replication, as insertional inactivation of the rom gene causes amplification of pBR322rom ^– in both relA ^– and relA ⁺ strains during amino acid starvation. Moreover, pUC19, which is a pBR322-derived plasmid lacking the rom gene, behaves like pBR322rom ^–, whereas introduction of the rom gene into the pUC19 replicon drives it into the pBR322 mode of replication in amino acid-starved bacteria. A model for the regulation of pBR322 plasmid DNA replication by Rom protein in amino acid-starved Escherichia coli strains is proposed.