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PubMed1977

[Incidence and mobility of R plasmids, Col plasmids and Hly plasmids in E. coli isolated from healthy calves and calves with diarrhea].

Within the set of 200 strains of E. coli isolated from healthy calves and 60 strains of E. coli isolated from calves suffering from diarrhoea we compared the incidence and transfer of determinants of antibioticoresistance, colicinogenesis and hemolytical activity. A significant difference in the incidence and independent mobility of the agents under examination in favour of E. coli from calves suffering from diarrhoea was determined in the case of resistance to chloramphenicol. The increased incidence and independent mobility of the chloramphenicol element in the antibioticoresistent strains of E. coli isolated from calves suffering from diarrhoea can be explained by the three to five-day therapy with a chloramphenicol product.

Animals↗

PubMed1984

Plasmids in different strains of Streptomyces ambofaciens: free and integrated form of plasmid pSAM2.

Five strains of Streptomyces ambofaciens were examined for their plasmid content. Among these strains, four belong to the same lineage (strains B) and the other was isolated independently (strain A). A large plasmid (ca. 80 kb), called pSAM1 in this paper and already described, was present in all B strains, and absent in strain A. A second plasmid, not described before, was found as covalently closed circular DNA in two of the four B strains. This plasmid with a size of 11.1 kb was called pSAM2. A restriction map for 14 enzymes was established. Hybridization experiments showed that a unique sequence homologous to this plasmid is integrated in a larger replicon, which is not pSAM1 and is probably the chromosome, in all B strains and not in strain A. It seems probable that the integrated sequence is the origin of the free plasmid found in two strains of the B family. It is noteworthy that the integrated form and the free plasmid may be found together. Transformation experiments proved that pSAM2 may be maintained autonomously in S. ambofaciens strain A and in S. lividans. pSAM2 is a self-transmissible plasmid, able to elicit the lethal zygosis reaction. pSAM2 was compared to the plasmids SLP1, pIJ110 and pIJ408, which all come from integrated sequences in three Streptomyces species and are found as autonomous plasmids after transfer to S. lividans. If pSAM2 resembles these plasmids in its origin, it does not appear to be related directly to them. Concerning their plasmid content, the two isolates of S. ambofaciens are very different.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA, Bacterial↗

PubMed2003

Clustering versus random segregation of plasmids lacking a partitioning function: a plasmid paradox?

Plasmids lacking a functional partition system are randomly distributed to the daughter cells; plasmid-free daughter cells are formed with a frequency of (1/2)2n per cell and cell generation where 2n is the (average) copy number at cell division. Hence, the unit of segregation is one plasmid copy. However, plasmids form clusters in the cells. A putative solution to this potential paradox is presented: one plasmid copy at a time is recruited from the plasmid clusters to the replication factories that are located in the cell centres. Hence, replication offers the means of declustering that is necessary in a growing host population. The daughter copies diffuse freely and each copy may with equal probability end up in either of the two cell halves. In this way, the random segregation of the plasmids is coupled to replication and occurs continuously during the cell cycle, and is not linked to cell division. The unit of segregation is the plasmid copy and not the plasmid clusters. In contrast, the two daughters of a Par+ plasmid are directed in opposite directions by the plasmid-encoded partition system, thereby assuring that each daughter cell receives the plasmid.

Bacterial Proteins↗

PubMed1996

Comparison of plasmids of strains of Yersinia enterocolitica biovar 1A with the virulence plasmid of a pathogenic Y. enterocolitica strain.

The plasmid content of apathogenic Y. enterocolitica biovar 1A strains was determined and the plasmids were compared with the virulence plasmid of a pathogenic Y. enterocolitica strain. About 38% of the selected biovar 1A strains contained plasmids of different sizes ranging from 2.7 kb to more than 70 kb. Some of the larger plasmids had a size similar to that of the virulence plasmid of a pathogenic reference strain. The restriction patterns of these plasmids were different from the restriction pattern of the virulence plasmid of the pathogenic reference strain. Differences were also observed in hybridization studies with the virulence plasmid. The plasmids from 15 out of 16 biovar 1A strains showed no homology, whereas the plasmid of one biovar 1A strain partially hybridized to the virulence plasmid.

Animals↗

PubMedDate not supplied

Sterilizing filtration of plasmid DNA: effects of plasmid concentration, molecular weight, and conformation.

Plasmid DNA purification development has been driven by the increased need for large quantities of highly purified, sterile plasmid DNA for clinical studies. Detailed characterization and development of the terminal sterile filtration process step is often limited due to time constraints and the scarcity of sufficient quantities of purified plasmid. However, the large size of the plasmid molecule and variations in conformation can lead to significant yield losses if this process step is not optimized. In this work, the gradual pore-plugging model of flow decay was found to be valid for plasmid DNA by using an ultra scaledown apparatus (1-4 cm(2) filter area). Filtration capacity was found to be insensitive to pressure. Multiple filter types were screened and both source and composition of materials were found to affect filter capacity dramatically. The filter capacity for plasmid was improved by increasing plasmid concentrations as well as by modifying buffer conditions to reduce the apparent size of the plasmid. Filtration capacities varied over a greater than 2 log range when plasmids with sizes ranging from 5.5 to 11 kb and supercoiled plasmid content of 55-95% were explored. Larger plasmids and feeds with lower supercoiled contents led to reduced capacities. These results can be used to define conditions for scale-up of plasmid sterile filtration, as evidenced by processing a 30 g lot using a filtration area of 1,000 cm(2), with a 96% yield, based on filtration capacity data from 4 cm(2) test filters.

Buffers↗

PubMed2006

Growth studies of plasmid bearing and plasmid cured Yersinia enterocolitica GER O:3 in the presence of cefsulodin, irgasan and novobiocin at 25 and 37 degrees C.

AIM: In this study, the growth characteristics of Yersinia enterocolitica biotype 4, GER O:3 plasmid bearing (P+) and plasmid cured (P-) strain types were evaluated in brain heart infusion broth supplemented with cefsulodin, irgasan, and novobiocin alone or in combination. METHODS AND RESULTS: Growth curves were obtained for the two strain types in broth supplemented with selective agents at 25 or 37 degrees C for 32 h to obtain data on the lag phase durations and growth rates of the strains. Generally, the lag times and growth rates of the P+ and P- strains were similar for cultures incubated at 25 degrees C regardless of the selective agent added and where plasmid replication and expression were not under any significant burden. However, where the lag times and growth rates of the strains were examined at 37 degrees C, significant differences were observed in the lag phase durations of the plasmid bearing strain type compared the plasmid cured strain, an effect that was due to the burden of the plasmid and the influence of selective agents. Generally, when two or more agents were present, lag phase durations were longer for the plasmid bearing strain. Some exceptions noted where in the presence of irgasan or full selective agent (CIN) the opposite case was observed. When growth rates were compared, the plasmidless strain type was typically faster than the plasmid bearing strain in the presence of most selective agents at 37 degrees C and the growth rates of both strain types at 25 degrees C were similar where the temperature appeared to negate the effects of plasmid. CONCLUSIONS: The data obtained in these studies suggest that selective agents (in particular irgasan) and incubation temperature play a significant role in influencing the growth characteristics of plasmid bearing and plasmid cured strains of Y. enterocolitica. SIGNIFICANCE AND IMPACT OF THE STUDY: This data presented in this study has significant implications for enrichment methods used in the detection or recovery of plasmid bearing Y. enterocolitica strains from food, environmental or clinical samples.

Anti-Bacterial Agents↗

PubMed1990

Two plasmids other than the nodulation plasmid are necessary for formation of nitrogen-fixing nodules by Rhizobium leguminosarum.

A system which allows direct selection for curing of plasmids in Gram-negative bacteria was used to generate derivatives of Rhizobium leguminosarum VF39 cured of each of six plasmids present in this strain. Phenotypes could be correlated with the absence of five of the six plasmids. The smallest plasmid, pRleVF39a, carries genes for the production of a melanin-like pigment as has been previously reported. Plasmid pRleVF39d carries nodulation and nitrogen fixation genes. Curing of the plasmids pRleVF39c and pRleVF39e gave rise to strains which formed Fix- nodules on peas, lentils, and faba beans. The nodules formed by the strains cured of pRleVF39c contained few, if any, bacteria. Analysis of washed cells by SDS-PAGE showed that this strain is defective in lipopolysaccharide (LPS) production; the defect could be complemented by introducing plasmids from several other R. leguminosarum strains, and by the R. leguminosarum biovar phaseoli LPS gene clones pCos126 and pDel27. The nodules formed by the strain cured of pRleVF39e had a reduced symbiotic zone, an enlarged senescence zone, and an abundance of starch granules. This strain grew at a much slower rate than the wild type, was unable to grow on minimal medium, and no longer produced melanin. These defects could be complemented by at least one other Rhizobium plasmid, pRle336e, a plasmid of strain 336 which is distinct from the nodulation plasmid (pRle336c) and the plasmid (pRle336d) which could complement the LPS defect associated with the loss of pRleVF39c. This demonstrates that genes necessary for symbiosis can be carried on at least three different plasmids in R. leguminosarum.

DNA Transposable Elements↗

PubMed1993

Identification of plasmids in Actinobacillus actinomycetemcomitans and construction of intergeneric shuttle plasmids.

A collection of 39 isolates of Actinobacillus actinomycetemcomitans, obtained from laboratories located in 5 different geographical regions of the United States, was examined for the presence of plasmid DNA. Only 2 of the strains examined, designated VT736 and VT745, harbored detectable plasmids. Strain VT736 contained a 1.9 kb plasmid species (pVT736-1) and a larger ( > 30 kb) species (pVT736-2). Both plasmids were detected in the covalently closed circular DNA fraction of dye buoyant density gradients. However, only the smaller plasmid was observed in agarose gels containing plasmid-enriched cell lysates prepared by a rapid screening procedure. Strain VT745 contained a single, 24 kb, plasmid (pVT745) that was observed consistently in plasmid-enriched lysates, as well as in the plasmid band of dye buoyant density gradients. A restriction endonuclease map of pVT736-1 was constructed. The plasmid contained one site each for the enzymes HincII, KpnI and XhoI, located 600 to 700 bp from each other on the pVT736-1 map. HincII-digested pVT736-1 DNA could not be cloned in Escherichia coli. However, intact pVT736-1 digested with KpnI or XhoI could be cloned in E. coli on pUC19 or pGEM7Zf(-), respectively. KpnI-digested pVT736-1 was cloned in both orientations on pUC19, but XhoI-digested pVT736-1 was clonable in only one orientation on pGEM7Zf(-). Each of the 3 types of chimeric plasmid constructs provided a potential A. actinomycetemcomitans/E. coli shuttle plasmid for the development of a genetic transfer system in A. actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans↗

PubMed1998

Rolling-circle plasmids from Bacillus subtilis: complete nucleotide sequences and analyses of genes of pTA1015, pTA1040, pTA1050 and pTA1060, and comparisons with related plasmids from gram-positive bacteria.

Most small plasmids of Gram-positive bacteria use the rolling-circle mechanism of replication and several of these have been studied in considerable detail at the DNA level and for the function of their genes. Although most of the common laboratory Bacillus subtilis 168 strains do not contain plasmids, several industrial strains and natural soil isolates do contain rolling-circle replicating (RCR) plasmids. So far, knowledge about these plasmids was mainly limited to: (i) a classification into seven groups, based on size and restriction patterns; and (ii) DNA sequences of the replication region of a limited number of them. To increase the knowledge, also with respect to other functions specified by these plasmids, we have determined the complete DNA sequence of four plasmids, representing different groups, and performed computer-assisted and experimental analyses on the possible function of their genes. The plasmids analyzed are pTA1015 (5.8 kbp), pTA1040 (7.8 kbp), pTA1050 (8.4 kbp), and pTA1060 (8.7 kbp). These plasmids have a structural organization similar to most other known RCR plasmids. They contain highly related replication functions, both for leading and lagging strand synthesis. pTA1015 and pTA1060 contain a mobilization gene enabling their conjugative transfer. Strikingly, in addition to the conserved replication modules, these plasmids contain unique module(s) with genes which are not present on known RCR plasmids of other Gram-positive bacteria. Examples are genes encoding a type I signal peptidase and genes encoding proteins belonging to the family of response regulator aspartate phosphatases. The latter are likely to be involved in the regulation of post-exponential phase processes. The presence of these modules on plasmids may reflect an adaptation to the special conditions to which the host cells were exposed.

Bacillus subtilis↗

PubMed1976

Plasmids in Streptococcus lactis: evidence that lactose metabolism and proteinase activity are plasmid linked.

Populations of lactose positive (Lac+) and proteinase positive (Prt+) cells from Streptococcus lactis M18, C10, and ML3 grown at 39 degrees C gave rise to increasing proportions of Lac- Prt- clones. The deficiencies did not appear until after a number of generations at the elevated temperature, and the rate depended on the strain.Lac- Prt+ and Lac+ Prt- mutants were isolated after treatment with ethidium bromide. Plasmid deoxyribonucleic acid was isolated by cesium chloride-ethidium bromide equilibrium density gradient centrifugation from the parent cultures as well as from their Lac- Prt-, Lac- Prt+, and Lac+ Prt- mutants. Five distinct plasmid sizes of approximate molecular weights of 2,4, 8, 21, and 27 million were found in S. lactis C10, whereas the Lac- Prt- derivative lacked the 8- and 21-million-dalton plasmids, but the 8-million-dalton plasmid was present in the Lac-Att mutant. In S. lactis m18 five plasmids possessing molecular weights of about 2, 4, 10, 18 and 27 million were observed. The 10- and 18-million-dalton plasmids were not detected in the Lac- Prt- mutants, whereas the Lac- Prt+ derivative lacked only the 18-million-dalton plasmid and the Lac+ Prt- mutant lacked only the 10-million-dalton plasmid. In S. lactis ML3 five distinct plasmids, with approximate molecular weights of 2, 4, 8, 22, and 30 million, were present. The 8- and 22-million-dalton plasmids were not detected in the Lac- Prt- derivative, but the 8-million-dalton plasmid was present in the Lac- Prt+ mutant. The evidence suggests that lactose-fermenting ability and proteinase activity in these organisms are mediated through two distinct plasmids having molecular weights of 8 x 10(6) to 10 x 10(6) for proteinase activity and 18 x 10(6) to 22 x 10(6) for lactose metabolism.

DNA, Bacterial↗

PubMed1988

BamHI restriction endonuclease analysis of Yersinia ruckeri plasmids and their relatedness to the genus Yersinia 42- to 47-megadalton plasmid.

The plasmid profile and BamHI restriction pattern of 17 sorbitol-negative and 1 sorbitol-positive French Yersinia ruckeri strain of the American type strain were studied. The 17 sorbitol-negative strains and the American strain harbored a 62-megadalton (MDa) plasmid with an identical BamHI restriction pattern. Southern hybridization indicated that this 62-MDa plasmid is common among these various strains. The sorbitol-positive strain had four plasmid bands (70, 62, 32, and 25 MDa), and there was no comigration of the DNA fragments of these cleaved plasmids with the fragments of the 62-MDa plasmid. Hybridization of these restricted plasmids with the common 62-MDa plasmid showed a weak DNA homology. The Y. ruckeri plasmid (62 MDa) had a different molecular weight than the virulence plasmid (42 to 47 MDa) of the genus Yersinia, and they had different BamHI restriction patterns. Furthermore, no sequence of the Y. ruckeri plasmid DNA was recognized after Southern hybridization when the 47-MDa plasmid of Y. enterocolitica was used as a probe.

Animals↗

PubMed1988

Transfer of conjugative plasmids and mobilization of a nonconjugative plasmid between Streptomyces strains on agar and in soil.

The conjugative plasmid pIJ101 and its conjugative nondeletion derivatives pIJ303 and pIJ211 were tested for their transferability between strains of Streptomyces on laboratory media and in the soil environment. Their roles in the mobilization of the cloning vector plasmid pIJ702, a nonconjugative deletion derivative of pIJ101, were also examined. Biparental and triparental crosses were performed on agar slants and in sterile soil between the plasmid donor Streptomyces lividans and several recipient Streptomyces strains previously isolated from soil. Conjugative plasmids were transferred to seven recipients in slant crosses and to three recipients in soil. Plasmids isolated from recipients showed restriction fragment patterns identical to that of the original plasmid in S. lividans. Plasmid pIJ303 was transferred less frequently in soil than on slants, and the frequency of transfer was higher at 30 degrees C than at the other temperatures examined. Transconjugant Streptomyces strains differed in their ability to maintain pIJ303. The nonconjugative plasmid pIJ702 was mobilized on agar slants into S. coelicolor 2708, which already contains a self-transmissible plasmid. Plasmid pIJ702 was also mobilized into S. flavovirens, Streptomyces sp. strain 87A, and S. parvulus on slants and in sterile soil after triparental crosses with two donors, one containing pIJ702 and the other containing either pIJ101 or pIJ211. The presence of a conjugative plasmid donor was required for the transfer of pIJ702 to S. parvulus 1234, S. flavovirens 28, and Streptomyces sp. strain 87A. Plasmid pIJ702 was always transferred in its normal, autonomous form. Chromosomal recombination also occurred in transconjugants after the transfer of pIJ702. This is the first report of gene transfer between Streptomyces strains in soil.

Conjugation, Genetic↗

PubMed1993

Isolation, characterization, and sequence analysis of cryptic plasmids from Acinetobacter calcoaceticus and their use in the construction of Escherichia coli shuttle plasmids.

Three cryptic plasmids have been discovered in Acinetobacter calcoaceticus BD413. These three plasmids, designated pWM10 (7.4 kb), pWM11 (2.4 kb), and pWM12 (2.2 kb), exhibited extensive homology to one another, as shown by Southern blot hybridization and restriction site analysis data, and also hybridized with three plasmids having slightly different sizes detected in a second strain, A. calcoaceticus BD4. Plasmid pWM11 and a fragment of pWM10 were each subcloned into pUC19, yielding plasmids pWM4 and pWM6, respectively, and were used in a series of inter- and intraspecies transformation experiments. Both plasmids replicated as high-copy-number plasmids in A. calcoaceticus BD413, as well as in strains of Escherichia coli. However, when transformed into the oil-degrading strain Acinetobacter lwoffii RAG-1, both plasmids were maintained at low copy numbers. No modification of the plasmids was detected after repeated transfers between hosts. An analysis of a series of deletions demonstrated that (i) a 185-bp fragment of pWM11 was sufficient to permit replication of the shuttle plasmid in A. calcoaceticus BD413, (ii) the efficiency of transformation of A. calcoaceticus BD413 decreased according to the size of the deletion in the insert by up to 4 orders of magnitude, and (iii) the entire insert was required for transformation and replication in A. lwoffii RAG-1. The sequence of pWM11 contained several small (150- to 300-bp) open reading frames, none of which exhibited any homology to known DNA or protein sequences. In addition, a number of inverted and direct repeats, as well as six copies of the consensus sequence AAAAAAATA previously described for a cryptic plasmid from A. lwoffii (M. Hunger, R. Schmucker, V. Kishan, and W. Hillen, Gene 87:45-51, 1990), were detected. Cloning and expression of the alcohol dehydrogenase regulon from A. lwoffii RAG-1 were accomplished by using the Acinetobacter shuttle plasmid.

Acinetobacter calcoaceticus↗

PubMed1999

Role of the 85-kilobase plasmid and plasmid-encoded virulence-associated protein A in intracellular survival and virulence of Rhodococcus equi.

Rhodococcus equi is a facultative intracellular pathogen of macrophages and a cause of pneumonia in young horses (foals) and immunocompromised people. Isolates of R. equi from pneumonic foals typically contain large, 85- or 90-kb plasmids encoding a highly immunogenic virulence-associated protein (VapA). The objective of this study was to determine the role of the 85-kb plasmid and VapA in the intracellular survival and virulence of R. equi. Clinical isolates containing the plasmid and expressing VapA efficiently replicated within mouse macrophages in vitro, while plasmid-cured derivatives of these organisms did not multiply intracellularly. An isolate harboring the large plasmid also replicated in the tissues of experimentally infected mice, whereas its plasmid-cured derivative was rapidly cleared. All foals experimentally infected with a plasmid-containing clinical isolate developed severe bronchopneumonia, whereas the foals infected with its plasmid-cured derivative remained asymptomatic and free of visible lung lesions. By day 14 postinfection, lung bacterial burdens had increased considerably in foals challenged with the plasmid-containing clinical isolate. In contrast, bacteria could no longer be cultured from the lungs of foals challenged with the isogenic plasmid-cured derivative. A recombinant, plasmid-cured derivative expressing wild-type levels of VapA failed to replicate in macrophages and remained avirulent for both mice and foals. These results show that the 85-kb plasmid of R. equi is essential for intracellular replication within macrophages and for development of disease in the native host, the foal. However, expression of VapA alone is not sufficient to restore the virulence phenotype.

Actinomycetales Infections↗

PubMed1983

Plasmid transformation in Agmenellum quadruplicatum PR-6: construction of biphasic plasmids and characterization of their transformation properties.

Biphasic, chimeric plasmids for the transformation of Agmenellum quadruplicatum PR-6 (Synechococcus sp. strain 7002) were constructed by splicing the 3.0-megadalton cryptic plasmid from strain PR-6 into plasmids pBR322 and pBR325 from Escherichia coli. Transformants of either E. coli or strain PR-6 by these plasmids could be detected on the basis of the drug resistance marker(s) carried by the chimeric plasmids. Plasmid DNA isolated from a PR-6 transformant transformed PR-6 much more efficiently than plasmid DNA prepared from E. coli. Plasmids from which the AvaI recognition site was deleted (AvaI is an isoschizomer of the AquI restriction endonuclease of strain PR-6) also transformed strain PR-6 much more efficiently than did plasmids containing the AvaI recognition site. These and other results suggest that AquI strongly effects plasmid transformation when the donor plasmid contains an unmodified AquI recognition site. Multimeric forms of the chimeric plasmids are also much more efficient at transforming strain PR-6 than are the analogous monomeric forms.

Chimera↗

PubMed1992

Distinct plasmid profiles of Pasteurella haemolytica serotypes and the characterization and amplification in Escherichia coli of ampicillin-resistance plasmids encoding ROB-1 beta-lactamase.

Thirty-five isolates of Pasteurella haemolytica from cattle or sheep were screened for the presence of plasmids and for resistance to a range of antibiotics. Eight strains (four of serotype A1, three of serotype A2 and one untypable) contained plasmid DNA and isolates of the same serotype had similar plasmid profiles, which were different from those of the other serotypes. All but one of the plasmid-bearing strains were isolated from pneumonic animals or from animals in contact with pneumonic cattle or sheep. In A2 and untypable strains, there was no obvious correlation between antibiotic resistance and the presence of a specific plasmid. In contrast, all plasmid-bearing A1 strains exhibited ampicillin resistance (ApR), which was shown by transfer studies to be plasmid-mediated. Plasmid DNA prepared from E. coli transformants was not routinely detected on ethidium-bromide-stained agarose gels, but could be amplified to detectable levels by treatment of cultures with chloramphenicol (Cm) or by modifying the growth conditions. The ApR plasmids from P. haemolytica were identical by restriction enzyme analysis. Restriction analysis and hybridization data indicated that these plasmids were closely related to the prototype ROB-1 beta-lactamase-encoding plasmid, originally isolated from Haemophilus influenzae. From substrate profiles and isoelectric focusing data, the beta-lactamases encoded by the P. haemolytica plasmids were indistinguishable from the ROB-1 beta-lactamase.

Ampicillin Resistance↗

PubMed1998

Role of the parCBA operon of the broad-host-range plasmid RK2 in stable plasmid maintenance.

The par region of the stably maintained broad-host-range plasmid RK2 is organized as two divergent operons, parCBA and parDE, and a cis-acting site. parDE encodes a postsegregational killing system, and parCBA encodes a resolvase (ParA), a nuclease (ParB), and a protein of unknown function (ParC). The present study was undertaken to further delineate the role of the parCBA region in the stable maintenance of RK2 by first introducing precise deletions in the three genes and then assessing the abilities of the different constructs to stabilize RK2 in three strains of Escherichia coli and two strains of Pseudomonas aeruginosa. The intact parCBA operon was effective in stabilizing a conjugation-defective RK2 derivative in E. coli MC1061K and RR1 but was relatively ineffective in E. coli MV10Deltalac. In the two strains in which the parCBA operon was effective, deletions in parB, parC, or both parB and parC caused an approximately twofold reduction in the stabilizing ability of the operon, while a deletion in the parA gene resulted in a much greater loss of parCBA activity. For P. aeruginosa PAO1161Rifr, the parCBA operon provided little if any plasmid stability, but for P. aeruginosa PAC452Rifr, the RK2 plasmid was stabilized to a substantial extent by parCBA. With this latter strain, parA and res alone were sufficient for stabilization. The cer resolvase system of plasmid ColE1 and the loxP/Cre system of plasmid P1 were tested in comparison with the parCBA operon. We found that, not unlike what was previously observed with MC1061K, cer failed to stabilize the RK2 plasmid with par deletions in strain MV10Deltalac, but this multimer resolution system was effective in stabilizing the plasmid in strain RR1. The loxP/Cre system, on the other hand, was very effective in stabilizing the plasmid in all three E. coli strains. These observations indicate that the parA gene, along with its res site, exhibits a significant level of plasmid stabilization in the absence of the parC and parB genes but that in at least one E. coli strain, all three genes are required for maximum stabilization. It cannot be determined from these results whether or not the stabilization effects seen with parCBA or the cer and loxP/Cre systems are strictly due to a reduction in the level of RK2 dimers and an increase in the number of plasmid monomer units or if these systems play a role in a more complex process of plasmid stabilization that requires as an essential step the resolution of plasmid dimers.

Bacterial Proteins↗

PubMed1999

Linear DNA plasmid pPK2 of Pichia kluyveri: distinction between cytoplasmic and mitochondrial linear plasmids in yeasts.

The linear plasmids frequently found in plants and filamentous fungi are associated with mitochondria or chloroplasts. In contrast, all the linear plasmids known in yeasts are cytoplasmic elements. From a strain of the yeast Pichia kluyveri, we have isolated a new linear plasmid, pPK2, which was found to be associated with mitochondria. This 7.1 kilobase pairs-long DNA contained only two genes, which code for DNA and RNA polymerases, as judged from their nucleotide sequences translated by a mitochondrial genetic code. When we examined several recently isolated yeast plasmids for their subcellular localization, we found that two linear plasmids, pPH1 from Pichia heedii, as well as pPK1 from another strain of P. kluyveri, were also localized in mitochondria. These plasmids are the first examples of mitochondria-associated linear plasmids in yeast. All other linear plasmids we examined were of cytoplasmic origin. Whilst the cytoplasmic type linear plasmids were efficiently eliminated by ultraviolet irradiation of host cells, the mitochondria-associated plasmids were highly resistant. The mitochondrial pPK2 plasmid was rapidly lost by treatment of the host cells with ethidum bromide.

Amino Acid Sequence↗