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PubMed1981

Isolation and characterization of linear deoxyribonucleic acid plasmids from Kluyveromyces lactis and the plasmid-associated killer character.

Two linear deoxyribonucleic acid plasmids, designated pGK11 and pGK12, were isolated from the yeast Kluyveromyces lactis IFO 1267. pGK11 and pGK12 had molecular weights of 5.4 X 10(6) and 8.4 X 10(6), respectively. Both plasmids possessed the same density of 1.687 g/cm3, lighter than the densities of mitochondrial (1.692 g/cm3) and nuclear (1.699 g/cm3) deoxyribonucleic acids. A restriction map of pGK11 was constructed from digestions by EcoRI, HindIII, PstI, and BamHI. pGK12 was cleaved by EcoRI into seven fragments and by BamHI into two fragments K. lactis IFO 1267 killed Saccharomyces cerevisiae sensitive and killer strains and certain strains of Saccharomyces italicus, K. lactis, Kluyveromyces thermotolerans, and K. vanudenii. All K. lactis strains lacking the pGK1 plasmids were nonkillers. A hybrid was constructed between K. lactis IFO 1267 and a nonkiller K. lactis strain lacking the plasmids and subjected to tetrad analysis after sporulation. The killer character was extrachromosomally transmitted in all tetrads in association with the pGK1 plasmids. The double-stranded ribonucleic acid killer plasmid could not be detected in any K. lactis killer strains. It is thus highly probable that the killer character is mediated by the linear deoxyribonucleic acid plasmids. A single chromosomal gene was found which was responsible for the resistance to the K. lactis killer.

Ascomycota↗

PubMed1982

The incompatibility product of IncFII R plasmid NR1 controls gene expression in the plasmid replication region.

The incompatibility properties of IncFII R plasmid NR1 were compared with those of two of its copy number mutants, pRR12 and pRR21. pRR12 produced an altered incompatibility product and also had an altered incompatibility target site. The target site appeared to be located within the incompatibility gene, which is located more than 1,200 base pairs from the plasmid origin of replication. The incompatibility properties of pRR21 were indistinguishable from those of NR1. Lambda phages have been constructed which contain the incompatibility region of NR1 or of one of its copy mutants fused to the lacZ gene. In lysogens constructed with these phages, beta-galactosidase was produced under the control of a promoter located within the plasmid incompatibility region. Lysogens containing prophages with the incompatibility regions from pRR12 and pRR21 produced higher levels of beta-galactosidase than did lysogens containing prophages with the incompatibility region from the wild-type NR1. The introduction into these inc-lac lysogens of pBR322 plasmids carrying the incompatibility regions of the wild-type or mutant plasmids resulted in decreased levels of beta-galactosidase production. For a given lysogen, the decrease was greater when the pBR322 derivative expressed a stronger incompatibility toward the plasmid from which the fragment in the prophage was derived. This suggested that the incompatibility product acts on its target to repress gene expression in the plasmid replication region.

Bacteriophage lambda↗

PubMed1983

Streptococcus plasmid pAM alpha 1 is a composite of two separable replicons, one of which is closely related to Bacillus plasmid pBC16.

A tetracycline resistance plasmid of Streptococcus faecalis, pAM alpha 1, is shown to contain two independent sets of replication functions, separated from each other on either side by short (300- to 400-base-pair) sequences of homology. The homologous sequences are oriented as direct repeats and therefore permit the dissociation of pAM alpha 1 into its component replicons, referred to here as pAM alpha 1 delta 1 and pAM alpha 1 delta 2, as the reciprocal products of a simple intramolecular recombination. pAM alpha 1 delta 1 is a 4.6-kilobase plasmid which carries the tet gene, and pAM alpha 1 delta 2 is a 5.1-kilobase plasmid which carries no known selectable marker. pAM alpha 1 delta 1 is shown to replicate efficiently in Bacillus subtilis and to confer tetracycline resistance on Bacillus hosts. We demonstrate by restriction mapping analysis that pAM alpha 1 delta 1 is virtually identical to a 4.6-kilobase tetracycline resistance plasmid of Bacillus cereus, pBC16, which is known to show extensive homology to plasmid isolates from Staphylococcus species (such as pUB110), as well as from other Bacillus species. The pAM alpha 1 delta 1-pBC16-pUB110 replicon thus exists naturally in at least three different gram-positive genera, indicating that these plasmids have a high degree of interspecific functional adaptability and supporting the view that plasmid DNA is commonly exchanged among many species of gram-positive bacteria in their natural environments.

Bacillus↗

PubMed1988

Mini-P1 plasmid partitioning: excess ParB protein destabilizes plasmids containing the centromere parS.

The partition system of the unit-copy plasmid P1 consists of two proteins, the parA and parB gene products, and a cis-acting site, parS. Production of high levels of the P1 ParB protein, from an external promoter on a high-copy-number vector, inhibits the propagation of lambda-mini-P1 prophages and destabilizes other P1-derived plasmids. The interference by ParB protein depends on the parS site, or centromere, of the P1 partition region; plasmids lacking parS are unaffected. The defect is more severe than the defect due to mutations that simply eliminate par function. In the presence of excess ParB protein, plasmids carrying parS are more unstable than would be predicted from a random distribution at cell division. The destabilization is a segregation defect, as the copy number of parS-bearing plasmids is not decreased under these conditions. Thus, it appears that ParB protein binds to parS; if too much protein is present, it sequesters such plasmids so they cannot be properly, or even randomly, partitioned. This suggests that under normal conditions, ParB protein recognizes and binds to parS and may be the protein responsible for pairing plasmids during the process of partitioning at cell division.

Bacterial Proteins↗

PubMed1988

Insertion of foreign DNA into plasmids from gram-positive bacteria induces formation of high-molecular-weight plasmid multimers.

Plasmids pUB110, pC194, pE194, and pT181 are commonly used as cloning vectors in both Bacillus subtilis and Staphylococcus aureus. We report that insertion of foreign DNA into any of these plasmids results in the generation of high-molecular-weight plasmid multimers (HMW) of the recombinant, present as tandem head-to-tail copies. HMW was detected in wild-type B. subtilis and S. aureus strains. The production of HMW depended on the nature of the DNA insertion. Inserts of Escherichia coli DNA, e.g., pBR322 or pUC18, resulted in large amounts of HMW, whereas some inserts of S. aureus DNA of the same size had no effect on plasmid profile. The generation of HMW depended on the mode of plasmid replication; plasmids which replicate via a single-stranded DNA intermediate produced HMW upon foreign DNA insertion, whereas plasmid pAM beta 1, which does not generate single-stranded DNA, did not generate HMW. We propose that HMW is a product of imparied termination of rolling-circle replication and that the impairment is due to the nature of the DNA insertion.

Bacillus subtilis↗

PubMed1989

Involvement of Tn4430 in transfer of Bacillus anthracis plasmids mediated by Bacillus thuringiensis plasmid pXO12.

The self-transmissible plasmid pXO12 (112.5 kilobases [kb]), originally isolated from strain 4042A of Bacillus thuringiensis subsp. thuringiensis, codes for production of the insecticidal crystal protein (Cry+). The mechanism of pXO12-mediated plasmid transfer was investigated by monitoring the cotransfer of the tetracycline resistance plasmid pBC16 (4.2 kb) and the Bacillus anthracis toxin and capsule plasmids, pXO1 (168 kb) and pXO2 (85.6 kb), respectively. In matings of B. anthracis donors with B. anthracis and Bacillus cereus recipients, the number of Tcr transcipients ranged from 4.8 x 10(4) to 3.9 x 10(6)/ml (frequencies ranged from 1.6 x 10(-4) to 7.1 x 10(-2), and 0.3 to 0.4% of them simultaneously inherited pXO1 or pXO2. Physical analysis of the transferred plasmids suggested that pBC16 was transferred by the process of donation and that the large B. anthracis plasmids were transferred by the process of conduction. The transfer of pXO1 and pXO2 involved the transposition of Tn4430 from pXO12 onto these plasmids. DNA-DNA hybridization experiments demonstrated that Tn4430 was located on a 16.0-kb AvaI fragment of pXO12. Examination of Tra- and Cry- derivatives of pXO12 showed that this fragment also harbored information involved in crystal formation and was adjacent to a restriction fragment containing DNA sequences carrying information required for conjugal transfer.

Bacillus anthracis↗

PubMed1997

Plasmid maintenance functions of the large virulence plasmid of Shigella flexneri.

The large virulence plasmid pMYSH6000 of Shigella flexneri contains a replicon and a plasmid maintenance stability determinant (Stb) on adjacent SalI fragments. The presence of a RepFIIA replicon on the SalI C fragment was confirmed, and the complete sequence of the adjacent SalI O fragment was determined. It shows homology to part of the transfer (tra) operon of the F plasmid. Stb stabilizes a partition-defective P1 miniplasmid in Escherichia coli. A 1.1-kb region containing a homolog of the F trbH gene was sufficient to confer stability. However, the trbH open reading frame could be interrupted without impairing stability. Deletion analysis implicated the involvement of two small open reading frames, STBORF1 and STBORF2, that fully overlap trbH in the opposite direction. These open reading frames are closely related to the vagC and vagD genes of the Salmonella dublin virulence plasmid and to open reading frame pairs in the F trbH region and in the chromosomes of Dichelobacter nodosus and Haemophilus influenzae. Stb appears to promote better-than-random distribution of plasmid copies and is a plasmid incompatibility determinant. The F homolog does not itself confer stability but exerts incompatibility against the activity of the Stb system. Stb is likely to encode either an active partition system or a postsegregational killing system. It shows little similarity to previously studied plasmid stability loci, but the genetic organization of STBORF1 and STBORF2 resembles that of postsegregational killing mechanisms.

Amino Acid Sequence↗

PubMed2001

Mobilization function of the pBHR1 plasmid, a derivative of the broad-host-range plasmid pBBR1.

The pBHR1 plasmid is a derivative of the small (2.6-kb), mobilizable broad-host-range plasmid pBBR1, which was isolated from the gram-negative bacterium Bordetella bronchiseptica (R. Antoine and C. Locht, Mol. Microbiol. 6:1785-1799, 1992). Plasmid pBBR1 consists of two functional cassettes and presents sequence similarities with the transfer origins of several plasmids and mobilizable transposons from gram-positive bacteria. We show that the Mob protein specifically recognizes a 52-bp sequence which contains, in addition to the transfer origin, the promoter of the mob gene. We demonstrate that this gene is autoregulated. The binding of the Mob protein to the 52-bp sequence could thus allow the formation of a protein-DNA complex with a double function: relaxosome formation and mob gene regulation. We show that the Mob protein is a relaxase, and we located the nic site position in vitro. After sequence alignment, the position of the nic site of pBBR1 corresponds with those of the nick sites of the Bacteroides mobilizable transposon Tn4555 and the streptococcal plasmid pMV158. The oriT of the latter is characteristic of a family of mobilizable plasmids that are found in gram-positive bacteria and that replicate by the rolling-circle mechanism. Plasmid pBBR1 thus appears to be a new member of this group, even though it resides in gram-negative bacteria and does not replicate via a rolling-circle mechanism. In addition, we identified two amino acids of the Mob protein necessary for its activity, and we discuss their involvement in the mobilization mechanism.

Amino Acid Sequence↗

PubMed2004

Mutations in a partitioning protein and altered chromatin structure at the partitioning locus prevent cohesin recruitment by the Saccharomyces cerevisiae plasmid and cause plasmid missegregation.

The 2 microm circle is a highly persistent "selfish" DNA element resident in the Saccharomyces cerevisiae nucleus whose stability approaches that of the chromosomes. The plasmid partitioning system, consisting of two plasmid-encoded proteins, Rep1p and Rep2p, and a cis-acting locus, STB, apparently feeds into the chromosome segregation pathway. The Rep proteins assist the recruitment of the yeast cohesin complex to STB during the S phase, presumably to apportion the replicated plasmid molecules equally to daughter cells. The DNA-protein and protein-protein interactions of the partitioning system, as well as the chromatin organization at STB, are important for cohesin recruitment. Rep1p variants that are incompetent in binding to Rep2p, STB, or both fail to assist the assembly of the cohesin complex at STB and are nonfunctional in plasmid maintenance. Preventing the cohesin-STB association without impeding Rep1p-Rep2p-STB interactions also causes plasmid missegregation. During the yeast cell cycle, the Rep1p and Rep2p proteins are expelled from STB during a short interval between the late G(1) and early S phases. This dissociation and reassociation event ensures that cohesin loading at STB is replication dependent and is coordinated with chromosomal cohesin recruitment. In an rsc2 Delta yeast strain lacking a specific chromatin remodeling complex and exhibiting a high degree of plasmid loss, neither Rep1p nor the cohesin complex can be recruited to STB. The phenotypes of the Rep1p mutations and of the rsc2 Delta mutant are consistent with the role of cohesin in plasmid partitioning being analogous to that in chromosome partitioning.

Cell Cycle↗

PubMed1984

Transformation of Neurospora crassa with recombinant plasmids containing the cloned glutamate dehydrogenase (am) gene: evidence for autonomous replication of the transforming plasmid.

We have characterized Neurospora crassa transformants obtained with plasmid pJR2, which consists of the Neurospora glutamate dehydrogenase (am) gene cloned in pUC8 and an am132 host strain which contains a deletion encompassing the cloned fragment. Every one of 33 transformants tested showed extreme meiotic instability: less than 1 or 2% am+ progeny were obtained in initial or successive backcrosses between am+ transformants and am132 or in intercrosses between am+ progeny. Furthermore, am+ progeny from backcrosses gave a high proportion of auxotrophic (am) mitotic segregants during vegetative growth. These results indicate that the am+ character is not stably integrated into chromosomal DNA in any of the transformants tested. Nuclear DNAs from six transformants were analyzed by Southern hybridization. All six transformants contained sequences homologous to pJR2. Four showed restriction fragments expected for unmodified pJR2, but most showed additional bands. Southern blots of undigested DNAs showed that the plasmid sequences are present predominantly in high-molecular-weight form (larger than 20 kilobases). Southern blots showed that auxotrophic (am) progeny from a backcross to am132 had lost restriction bands corresponding to free plasmid but retained additional bands, apparently integrated into chromosomal DNA in a nonfunctional manner. Considered together, these results are most reasonably interpreted as follows: recombinant plasmids containing the am+ gene can replicate autonomously in N. crassa, the free plasmids are present in oligomeric or modified form or both, and plasmid sequences also integrate at multiple sites in the deletion host but in a nonfunctional manner. An alternate interpretation--that tandem repeats of the plasmid are integrated into chromosomal DNA but eliminated during meiosis--cannot be completely excluded. However, stable integration of the am gene can be obtained under a variety of other conditions, viz., using the am gene cloned in a phage lambda vector (J. A. Kinsey and J. A. Rambosek, Mol. Cell. Biol. 4:117-122, 1984), using derivatives of pJR2, or using pJR2 to transform a frameshift mutant.

DNA Replication↗

PubMed2002

A novel plasmid curing method using incompatibility of plant pathogenic Ti plasmids in Agrobacterium tumefaciens.

Ti (Tumor inducing) plasmids in Agrobacterium tumefaciens can transfer their T-DNA region into dicotyledonous plants, in which the expression of T-DNA genes causes plant tumors and the production of bacterial nutrients, e.g., opines such as nopaline. Naturally occurring Ti plasmids (pTi) are difficult to cure by conventional curing methods because of their high stability. Here, we developed a novel curing method based on plasmid incompatibility. For this, a curing plasmid, pMGTrep1, was newly constructed and subsequently introduced into A. tumefaciens strains harboring pTi by conjugation with Escherichia coli harboring pMGTrep1. The conjugation yielded 32-99% nopaline non-utilizing agrobacterial transconjugants in which pMGTrep1 replaced pTi due to incompatibility. Then, pMGTrep1-less derivatives of the transconjugants are easily selected in the presence of sucrose because pMGTrep1 contains a sucrose-sensitive sacB gene. This efficient method is directly applicable for curing plasmids with the same incompatibility group and shoud also applicable to other types of plasmids in Agrobacterium groups, including A. rhizogenes, by replacing the rep gene region of the curing plasmid with that of the corresponding incompatibility.

Agrobacterium tumefaciens↗

PubMedDate not supplied

[The study of heterogeneity of plasmid-bearing and plasmid-f ree populations of Bacillus subtilis under different environmental conditions].

The population heterogeneity of recombinant and plasmid-free Bacillus subtilis strains introduced into aquatic microcosms was studied. After introduction, the population of the plasmid-free strain B. subtilis 2335 in microcosms has long been represented by both vegetative cells and spores, whereas, already ten days after introduction, the population of the recombinant strain B. subtilis 2335/105 (Km[symbol: see text]nf+) was represented only by spores. The number of plasmid copies in the spore isolates of the recombinant strain was the same as before introduction, but the plasmid abundance in the vegetative isolates of this strain decreased. The isolates of B. subtilis 2335/105 obtained from microcosms and the variants of this strain obtained by ten successive subcultures on M9 and 0.1 x M9 media with and without kanamycin (Km) differed in the number of plasmid copies, Km resistance, and maximum biomass yield during batch cultivation. Irrespective of the presence of Km, more than 50% of the variants subcultured on M9 medium showed reduced plasmid abundance. At the same time, about 70% of the variants subcultured on 0.1 x M9 medium with Km and 90% of the variants subcultured on the same medium without Km retained the initial number of plasmid copies. The variants subcultured on media with Km retained the initial biomass level. In more than 70% of the variants isolated from media without Km, the biomass yield increased.

Bacillus subtilis↗

PubMed1989

Plasmid transfer and plasmid-mediated genetic exchange in Brucella abortus.

Naturally-occurring plasmids and gene transfer mechanisms have not yet been reported in brucellae. Here we show that Brucella abortus is capable of maintaining and transferring the broad-host-range plasmids pTH10 (IncP), pSa (IncW) and R751 (IncP), and describe pTH10-mediated transfer of B. abortus chromosomal genes to Escherichia coli. All three plasmids transferred by conjugation from E. coli to B. abortus S19, and from B. abortus S19 to B. abortus 292 (biovar 4). They were stably maintained with no effect on biotyping characteristics. Plasmid pTH10 is a Tn1-containing derivative of RP4. It confers temperature-sensitive resistance to kanamycin, tetracycline and ampicillin to E. coli, but its tetracycline resistance and temperature sensitivity were poorly expressed in B. abortus. Plasmids pTH10 and pSa both transferred from B. abortus to E. coli DP50, a strain that is auxotrophic for diaminopimelic acid (DAP) Plasmid pTH10 (but not pSa) mobilized Brucella chromosomal gene(s) for DAP synthesis to DP50, yielding non-DAP-requiring (NDR) transconjugants. Neither plasmid transferred the NDR marker from their original E. coli host strains, nor did pTH10 transfer it from NDR transconjugants. Escherichia coli NDR transconjugant EP8.11 was cured of pTH10 by passage at the nonpermissive temperature, but retained the NDR marker and the Tn1-encoded resistance to ampicillin, indicating Tn1-mediated integration of Brucella chromosomal DNA into the E. coli chromosome.

Brucella abortus↗

PubMed1989

Induced bending of plasmid pLS1 DNA by the plasmid-encoded protein RepA.

The broad host range streptococcal plasmid pLS1 encodes for a 5.1-kDa repressor protein, RepA. This protein has affinity for DNA (linear or supercoiled) and is translated from the same mRNA as the replication initiator protein RepB. By gel retardation assays, we observed that RepA shows specificity for binding to the plasmid HinfID fragment, which includes the target of the protein. The target of RepA within the plasmid DNA molecule has been located around the plasmid single site ApaLI. This site is included in a region that contains the promoter for the repA and repB genes and is contiguous to the plasmid ori(+). A complex sequence-directed DNA curvature is observed in this region of pLS1. Upon addition of RepA to plasmid linear DNA or to circularly permuted restriction fragments, this intrinsic curvature was greatly enhanced. Thus, a strong RepA-induced bending could be located in the vicinity of the ApaLI site. Visualization of the bent DNA was achieved by electron microscopy of complexes between RepA and plasmid DNA fragments containing the RepA target.

Bacterial Proteins↗

PubMed1986

[Mobilization of plasmid determinants of Yersinia pestis using the plasmid pKA7].

Yersinia pestis plasmids having inserted Tn-movable elements and determining pesticinogenicity, Ca2+-dependence, production of mice toxin and fraction I have been mobilized for conjugal transfer into Y. pestis, Y. pseudotuberculosis or E. coli cells by plasmid pKA7. Plasmid determinants have been transferred with the frequencies 10-4-10-7 per donor cell. The plasmid DNA of high molecular mass has been identified in transconjugant cells. Plasmid mobilization in Yersinia pestis is supposed to be due to cointegrate formation of yersinia plasmids with R-plasmids.

Genes, Bacterial↗

PubMed1985

[Replication of Streptomyces plasmids: the DNA nucleotide sequence of plasmid pSB 24.2].

The nucleotide sequence of DNA in plasmid pSB 24.2, a natural deletion derivative of plasmid pSB 24.1 isolated from S. cyanogenus was studied. The plasmid amounted by its size to 3706 nucleotide pairs. The G-C composition was equal to 73 per cent. The analysis of the DNA structure in plasmid pSB 24.2 revealed the protein-encoding sequence of DNA, the continuity of which was significant for replication of the plasmid containing more than 1300 nucleotide pairs. The analysis also revealed two A-T-rich areas of DNA, the G-C composition of which was less than 55 per cent and a DNA area with a branched pin structure. The results may be of value in investigation of plasmid replication in actinomycetes and experimental cloning of DNA with this plasmid as a vector.

Base Composition↗

PubMed1982

[Characteristics of hybrid plasmids carrying the genes of colicinogenic plasmid Collb-P9 responsible for colicin Ib synthesis and inhibition of phage T5 development].

The EcoRI and HindII restriction endonucleases and pBR325 vector plasmid were used to obtain a set of hybrid plasmids containing ColIb-P9 fragments carrying the characters for colicin Ib synthesis and immunity and the ability to inhibit T5 phage growth. The genes responsible for colicin synthesis and immunity are closely linked and localized in the EcoRI fragment with a molecular weight of 1.85 MD (pIV41) or in the HindII fragment of 2.4 MD (pIV1). The clones containing these plasmids show an increased level of both spontaneous and mitomycin C-induced colicin synthesis and an increased level of immunity due to a larger dosage of the genes. The genes controlling T5 growth inhibition are localized in other restriction fragments of ColIb DNA: the EcoRI fragment of 1.45 MD (pIV7) and the HindII fragment of 4.3 MD (pIV5). We have demonstrated by means of hybrid plasmids that T5 growth inhibition is not connected with the colicin Ib synthesized in infected cells and is controlled by other specific product(s) of the ColIb plasmid genes. T5 phage growth was as efficient in clones containing plasmids with cloned colicin Ib genes as in a strain without plasmids. An investigation of the expression of the genes inhibiting T5 phage growth in an in vitro protein synthesis system has revealed a protein with a molecular weight of 36 000 which seems to take part in the process.

Cloning, Molecular↗

PubMed1984

[Induced mutagenesis of plasmid and chromosomal genes inserted into plasmid DNA. I. The mutagenic action of radiation].

This paper describes the results of treating plasmid DNA in vitro with mutagens, to obtain mutations both in plasmid genes and chromosomal genes comprised within the plasmid, thus avoiding disorganization characteristic of in vivo mutagenesis. The model system is represented by DNA of RSF2124 responsible for colicine E1 synthesis and resistance to ampicillin. Col- mutants were looked for after exposure to UV- and gamma-irradiation. The lethal effect was estimated as inactivation of the ampicillin resistance marker. After reisolation from mutant transformant of the plasmid DNA, the novel character and resistance to ampicillin proved to retain in the course of subsequent transformations and passages of transformed colonies, suggesting the mutational nature of the changes. Exposure of RSF2124 to short-wave UV-irradiation (lambda = 254 nm) produced a pronounced mutagenic effect: the relative quantity of Col- mutants under optimal conditions of mutagenesis increased about 10 times. In the case of W-reactivation (additional UV-irradiation of C600 wild type cells) of lethal lesions, a 95% reliable increase in mutagenic effect was observed. Significant enhancement of mutagenesis (about 4-fold) was detected when only recipient cells were exposed to low doses of UV (the so-called indirect UV mutagenesis). Thus, with regard to W- and indirect UV mutagenesis, the plasmid DNA behaves like DNA of temperate phages which suggests their evolutionary relationship. Treatment of plasmid DNA with acridine orange prior to UV, only protected from lethal lesions. Gamma-irradiation (60Co) at the dose producing 100-fold inactivation, increased the yield of Col- mutants by one order of magnitude. The presence of RSF2124 plasmid in a cell does not affect its UV sensitivity.

Acridine Orange↗