Exceptionally high copy numbers of a staphylococcal plasmid in Bacillus subtilis revealed by a sandwich hybridization technique

Abstract The copy number of a pUB110 derivative, pKTH10, containing the α-amylase gene from Bacillus amyloliquefaciens , was determined, using an assay based on a sandwich hybridization technique. In this method, a known gene on the plasmid is hybridized between two non-overlapping fragments of that same gene, cloned into separate vectors. One fragment is used as a radiolabelled probe and the other bound to a filter, forming a three-component, 'sandwich' hybrid when the relevant gene is present in the sample. Since the hybridization can only take place in the presence of the relevant gene, the amount of radioactivity binding to the filters will be proportional to the concentration of this gene in the sample. We utilized the α-amylase gene on the plasmid to form the sandwich hybrid. The copy number was of a totally different magnitude from what has previously been reported, and ranged from 2500 copies/viable cell in early logrithimic growth phase to about 500 in late stationary phase.     

Regulation of copy number and stability of phage λ derived pTCλ1 plasmid in the light of the dimer/multimer catastrophe hypothesis

The dimer catastrophe hypothesis has been proposed previously to explain instability of multicopy plasmids whose partitioning is random, contrary to low copy number plasmids which are stably maintained and actively partitioned. Until now, this hypothesis has been investigated using multicopy ColE1 plasmids. However, for more detailed testing of the dimer/multimer catastrophe hypothesis, one should use a plasmid which can be maintained at either low or high copy number and still possesses the same mechanism of replication regulation. Here we used a modified lambda plasmid, pTC lambda 1. The advantage of this plasmid is that it can be maintained at different copy numbers depending on the concentration of an inducer which stimulates the initiation of plasmid replication. Results obtained with this plasmid in recombination proficient and deficient cells generally support the dimer/multimer catastrophe hypothesis, but also suggest some modification in the model.     

The effect of the location of the proteic post-segregational stability system within the replicon of plasmid pTF-FC2 on the fine regulation of plasmid replication

The broad host-range IncQ-2 family plasmid, pTF-FC2, is a mobilizable, medium copy number plasmid that lacks an active partitioning system. Plasmid stability is enhanced by a toxin–antitoxin (TA) system known as pas (plasmid addiction system) that is located within the replicon between the repB (primase) and the repA (helicase) and repC (DNA-binding) genes. The discovery of a closely related IncQ-2 plasmid, pRAS3, with a completely different TA system located between the repB and repAC genes raised the question of whether the location of pas within the replicon had an effect on the plasmid in addition to its ability to act as a TA system. In this work we demonstrate that the presence of the strongly expressed, autoregulated pas operon within the replicon resulted in an increase in the expression of the downstream repAC genes when autoregulation was relieved. While deletion of the pas module did not affect the average plasmid copy number, a pas-containing plasmid exhibited increased stability compared with a pas deletion plasmid even when the TA system was neutralized. It is proposed that the location of a strongly expressed, autoregulated operon within the replicon results in a rapid, but transient, expression of the repAC genes that enables the plasmid to rapidly restore its normal copy number should it fall below a threshold.     

Isolation of chromosomal mutations that affect carotenoid production in Escherichia coli: mutations alter copy number of ColE1-type plasmids

Chromosomal mutants were isolated in Escherichia coli that altered carotenoid production from transformed carotenoid biosynthesis genes on a pACYC-derived plasmid (pPCB15). The mutations were mapped by sequencing. One group of mutations appeared to affect the cell metabolism without changing the copy number of the carotenoid synthesis plasmid. The other group of mutations either increased or decreased the copy number of the pPCB15 plasmid as determined by real-time PCR. The copy number change in most mutants was likely specific for ColE1-type plasmids for which copy number is controlled by a small antisense RNA. This collection of host strains would be useful for fine tuning expression of proteins and adjusting production of desired molecules without recloning to different vectors.     

Low copy number plasmid vectors for gene cloning and for monitoring gene expression

Abstract Derivatives of an IncW incompatibility plasmid with a low copy number are described which can be used for gene cloning or for analysing gene expression in conditions similar to those found in the host chromosome. Gene expression can be monitored after construction of operon or protein fusions with the lacZYA operon and measurement in Escherichia coli of the β-galactosidase activity.     

Loss of secreted hemolysin activity in the mutant strain Hsb. 1 is due to a lesion in a plasmid copy number locus

Further studies have been carried out on mutation hsb which was previously suggested to block hemolysin secretion (Mu?oa et al., 1988, FEMS Microbiol. Lett. 56: 167-172). We show that the reported reduction in the extracellular hemolytic activity of mutant Hsb. 1 is due to lower hemolysin synthesis and that this is itself a consequence of a decrease in plasmid copy number. We suggest that the hsb is identical to the pcnB lesion located at minute 3.6 of the chromosome.     

Plasmid copy number and stability determination in Clostridium perfringens transformants

The copy number of the streptococcal plasmid pAM beta 1 (26.5 kb), and its deletion derivatives, pVA1 (11 kb) and pVA677 (7.6 kb) contained in Clostridium perfringens 3624A transformants was determined by incorporation of [methyl-3H]thymidine (4 muCi/ml) into chromosomal and plasmid DNA and sizing of the C. perfringens genome using transverse alternating field electrophoresis. Plasmids pAM beta 1, pVA1, and pVA677 were found to be present at 1.0, 97, and 216 copies/cell, respectively. 10.2, 54, and 96% of the initial pAM beta 1-, pVA1- and pVA677-containing transformants, respectively, remained resistant to erythromycin over 220 generations of growth. The results indicate a size-dependent relationship between plasmid stability and plasmid copy number in C. perfringens.     

质粒拷贝数的测定方法

在研究生物工程重组蛋白产量的过程中 ,质粒的拷贝数是一个需要重点考虑的参数 ,对它进行精确定量至关重要。本文概述了几种主要的测定方法的原理和特点。     

pSAR1, a natural plasmid from Streptomyces arenae, shows rapid increase and decrease of copy numbers on changes of growth media

A natural plasmid, pSAR1, was isolated from the antibiotic producer Streptomyces arenae TU469. Its size is estimated to approx. 80 kbp by restriction analysis. pSAR1 occurs in two copy-number states differing by a factor of at least 10, depending on culture conditions. The high copy-number state is strongly correlated with the production of the antibiotic pentalenolactone. The decrease of copy numbers after change of culture conditions is completed within 1 h. These unusually rapid kinetics and the occurrence of degradational intermediates suggest the participation of specific catalytic mechanisms in copy number regulation.     

New pSC101-derivative cloning vectors with elevated copy numbers

Mutations that increase the copy number of the pSC101 replicon have been used for construction of new cloning vectors. Replacement of glutamate at position 93 in RepA yields plasmids that replicate at medium (27 copies/cell) and high (approximately 240 copies/cell) copy numbers. Based on the crystal structure of RepE, a structurally similar replication initiator protein from the F factor, the pSC101 repA mutants are predicted to be defective in dimerization. The cloning vectors permit increased expression of gene products along with the advantages of pSC101-derivative plasmids, including stable maintenance and compatibility with ColE1 plasmids. The plasmids also allow blue/white screening for DNA inserts and impart resistance to ampicillin, chloramphenicol and kanamycin. The vectors were used in a genetic assay to suppress temperature-sensitive mutants of ffh, encoding the protein component of the Escherichia coli signal recognition particle, by overproduction of 4.5S RNA. While expression of 4.5S RNA from a wild type pSC101-derivative plasmid was not sufficient for suppression, use of the new vectors did suppress the temperature-sensitive phenotype.     

Isolation and characterization of novel cold-sensitive dnaA mutants of Escherichia coli

We developed an efficient method for isolation of novel dnaA mutations based on PCR mutagenesis in the presence of manganese ion and shuffling of dnaA-carrying plasmids in a dnaA deletion host bacterium. Using this system, we obtained 30 cold-sensitive mutants from 4000 clones carrying plasmids with a mutagenized dnaA gene. All 27 cold-sensitive mutants analyzed were defective in DNA replication; none had a DnaAcos (over-initiation) phenotype. Nucleotide sequencing revealed that novel 15 alleles (mutations in 14 amino acid residues) are responsible for the cold-sensitive phenotype and are all located in the carboxy-terminal half of the DnaA protein.     

Isolation and characterization of Escherichia coli chromosomal mutants affecting plasmid copy number.

We have isolated chromosomal mutants of an Escherchia coli K-12 strain that maintain higher levels of an F' plasmid. The mutants are designated as plasmid copy number (pcn) mutants. They were detected by selecting for increased lactose fermentation in bacteria deleted for the lac operon but harboring an F'lacI,P pro+ plasmid. When examined for the amount of F' plasmid deoxyribonucleic acid (DNA) by the dye-CsCl isopycnic technique, the mutants show two to seven times as much covalently closed, circular (CCC) DNA as does the parental strain. The increased plasmid level in one mutant strain (pcn-24) was confirmed by DNA-DNA hybridization; however, this latter technique indicated about a twofold lower increase when compared with the increase measured for pcn-24 by the dye-CsCl technique. In mutant pcn-24 the increased amount of F' DNA reflects a proportional increase in monomeric-size plasmid molecules because oligomeric forms are not found. Also, in mutant pcn-24 the extra CCC plasmid copies do not seem to be randomly distributed throughout the cell's cytoplasm but appear complexed in situ with their host's folded chromosome. In all pcn mutants examined to date, the classical sex factor F is maintained at normal levels, whereas the viral plasmid Pl CM is maintained at two to three times the normal level. In all 17 pcn mutants isolated, the pcn mutation maps on the chromosome and not on the plasmid. Finally, the absolute amount of CCC F' DNA detectable in lysates of the six different pcn mutants examined decreased 50 to 90% upon incubation of the lysate at 37 C. In contrast, no loss of CCC DNA occurs when lysates of the parental F' strain are incubated at 37 C.     

Identification and characterization of novel ColE1-type, high-copy number plasmid mutants in Legionella pneumophila

ColE1-type plasmids are commonly used in bacterial genetics research, and replication of these plasmids is regulated by interaction of RNA I and RNA II. Although these plasmids are narrow-host-range, they can be maintained in Legionella pneumophila under antibiotic selection, with low-copy number and instability. Here, we have described the isolation of two novel spontaneous mutants of pBC(gfp)Pmip, pBG307 and pBG309, which are able to mark the L. pneumophila with strong green fluorescence when exposed to visible light. One of the mutants, pBG307, has a single CG-->TA mutation in RNA II promoter located 2-bases upstream the - 10 region. Another one, pBG309, has the same mutation, as well as an additional CG-->AT mutation in the 76th nucleotide of RNA I, or in the 6th nucleotide of RNA II. A plasmid with the single mutation in RNA I, pBG308, was also constructed. Characterization of these plasmids carrying the enhanced green fluorescent protein (gfpmut2) gene revealed that the green fluorescence intensities of these plasmids were 2- to 30-fold higher than that of the wild type and both of the mutations contribute to increase the plasmid copy number and/or plasmid stability. The mutation located in RNA II promoter played a more dominant role in elevating the copy number, compared to the mutation in RNA I. We also tested the mutant plasmids for replication in Escherichia coli, and found that their copy number and stability were dramatically decreased, except pBG307. Our data suggest that these plasmids might be useful and convenient in genetic studies in L. pneumophila.     

Number matters: control of mammalian mitochondrial DNA copy number

Regulation of mitochondrial biogenesis is essential for proper cellular functioning. Mitochondrial DNA （mtDNA） depletion and the resulting mitochondrial malfunction have been implicated in cancer, neurodegeneration, diabetes, aging, and many other human diseases. Although it is known that the dynamics of the mammalian mitochondrial genome are not linked with that of the nuclear genome, very little is known about the mechanism of mtDNA propagation. Nevertheless, our understanding of the mode of mtDNA replication has ad- vanced in recent years, though not without some controversies. This review summarizes our current knowledge of mtDNA copy number control in mammalian cells, while focusing on both mtDNA replication and turnover. Although mtDNA copy number is seemingly in excess, we reason that mtDNA copy number control is an important aspect of mitochondrial genetics and biogenesis and is essential for normal cellular function.     

In vivo definition of the functional origin of leading strand replication on the lactococcal plasmid pFX2

The lactococcal plasmid pFX2 belongs to a family of plasmids, whose prototype is the streptococcal plasmid pMV158, that replicates by the rolling circle mechanism. Determination of the nucleotide sequence of the repX gene of pFX2 allowed us to make some minor corrections in the published sequence, and to show that the repX gene is identical to the rep gene of plasmid pWV01. We have established pFX2 in Escherichia coli and in Streptococcus pneumoniae. In the latter host, we have defined in vivo the nick site introduced by the RepX protein. Plasmid pFX2 and the pMV158 derivative pLS1 exhibit a moderate degree of incompatibility in S. pneumoniae. Cloning of the double strand origin (dso) of pFX2 into a high-copy-number plasmid that is compatible with the pMV158 replicon led to an increase in incompatibility toward pLS1. Plasmids pFX2 and pLS1 exhibit homologies in their Rep proteins and in their dso sequences, but not in their negative control elements. Thus, the observed incompatibility indicates that cross-recognition of Rep proteins and dso takes place. Received: 25 May 1998 / Accepted: 8 July 1998     

Construction of small plasmid vectors for use in genetic improvement of the extremely acidophilic Acidithiobacillus caldus

The genetic improvement of biomining bacteria including Acidithiobacillus caldus could facilitate the bioleaching process of sulfur-containing minerals. However, the available vectors for use in A. caldus are very scanty and limited to relatively large broad-host-range IncQ plasmids. In this study, a set of small, mobilizable plasmid vectors (pBBR1MCS-6, pMSD1 and pMSD2) were constructed based on plasmid pBBR1MCS-2, which does not belong to the IncQ, IncW, or IncP groups. The function of the tac promoter on 5.8-kb pMSD2 was determined by inserting a kanamycin-resistant reporter gene. The resulting recombinant pMSD2-Km was successfully transferred by conjugation into A. caldus MTH-04 with transfer frequency of 1.38 ± 0.64 × 10^?5. The stability and plasmid copy number of pMSD2-Km in A. caldus MTH-04 were 75 ± 2.7% and 5–6 copies per cell, respectively. By inserting an arsABC operon into pMSD2, an arsenic-resistant recombinant pMSD2-As was constructed and transferred into A. caldus MTH-04 by conjugation. The arsenic tolerance of A. caldus MTH-04 containing pMSD2-As was obviously increased up to 45 mM of NaAsO₂. These vectors could be applied in genetic improvement of A. caldus as well as other bioleaching bacteria.