The linear mitochondrial plasmid pClK1 of the phytopathogenic fungus Claviceps purpurea may code for a DNA polymerase and an RNA polymerase

Summary Plasmid pClK1, a linear mitochondrial plasmid of Claviceps purpurea, was completely sequenced. The sequence contains two long open reading frames (ORF1, 3291 bp; ORF2, 2910 bp), and at least four smaller ORFs. The potential polypeptide derived from ORF1 shows homology to the family B type DNA polymerases. The product of ORF2 has significant homology to the mitochondrial RNA polymerase of yeast and RNA polymerases from bacteriophages. ORF1 and ORF2 show homology to URF3 and URF1 of the maize plasmids S1 and S2, respectively. No homology to any published protein sequence was found for the smaller ORFs. The origin of the terminal protein attached to the 5 ends of pClK1 remains open; several alternatives for its origin are discussed. The sequence data as a whole confirm the virus-like character of pClK1 already postulated from structural properties. Thus pClK1 together with S plasmids of maize and several other linear plasmids make up a distinct class of DNA species of plants and fungi probably derived from a common virus-like ancestor.     

Novel Plasmids from Alkaliphilic Halomonads

Seventeen alkaliphilic halomonads were examined for the presence of plasmids. Of these, eight strains harbored one or more from 5.3 to 33 kb in size, the first plasmids to be identified from an alkaliphilic halomonad source. Restriction and hybridization analysis revealed three strains that maintained an identical 5.9-kb plasmid which we named pAH1, two that had an identical 33-kb plasmid, and three others, of which one carried two plasmids of 5.3 and 15 kb, the former being designated pAH2. The two final strains maintained plasmids of 15 and 20.5 kb. Restriction mapping of both pAH1 and pAH2 indicated that they have a number of unique restriction sites and are of a small enough size to make them suitable for vector construction.     

Plasmids of serogroup 1 strains ofLegionella pneumophila

Twelve strains ofLegionella pneumophila were tested for the presence of plasmid DNA. Three strains, belonging to serogroup 1, had large plasmids of 83.8×10⁶ daltons, as determined by electron microscopy. A fourth strain, also from serogroup 1, had a similar large plasmid in addition to a smaller plasmid. Restriction analysis of plasmid DNA isolated from the strains with a single size plasmid indicated that the plasmids were structurally very similar. The biologic functions of these plasmids are yet to be determined.     

Thermosensitive plasmid replication, temperature-sensitive host growth, and chromosomal plasmid integration conferred by Lactococcus lactis subsp. cremoris lactose plasmids in Lactococcus lactis subsp. lactis.

Evidence is presented that lactose-fermenting ability (Lac+) in Lactococcus lactis subsp. cremoris AM1, SK11, and ML1 is associated with plasmid DNA, even though these strains are difficult to cure of Lac plasmids. When the Lac plasmids from these strains were introduced into L. lactis subsp. lactis LM0230, they appeared to replicate in a thermosensitive manner; inheritance of the plasmid was less efficient at 32 to 40 degrees C than at 22 degrees C. The stability of the L. lactis subsp. cremoris Lac plasmids in lactococci appeared to be a combination of both host and plasmid functions. Stabilized variants were isolated by growing the cultures at 32 to 40 degrees C; these variants contained the Lac plasmids integrated into the L. lactis subsp. lactis LM0230 chromosome. In addition, the presence of the L. lactis subsp. cremoris Lac plasmids in L. lactis subsp. lactis resulted in a temperature-sensitive growth response; growth of L. lactis subsp. lactis transformants was significantly inhibited at 38 to 40 degrees C, thereby resembling some L. lactis subsp. cremoris strains with respect to temperature sensitivity of growth.     

Thermosensitive plasmid replication, temperature-sensitive host growth, and chromosomal plasmid integration conferred by Lactococcus lactis subsp. cremoris lactose plasmids in Lactococcus lactis subsp. lactis

Evidence is presented that lactose-fermenting ability (Lac+) in Lactococcus lactis subsp. cremoris AM1, SK11, and ML1 is associated with plasmid DNA, even though these strains are difficult to cure of Lac plasmids. When the Lac plasmids from these strains were introduced into L. lactis subsp. lactis LM0230, they appeared to replicate in a thermosensitive manner; inheritance of the plasmid was less efficient at 32 to 40 degrees C than at 22 degrees C. The stability of the L. lactis subsp. cremoris Lac plasmids in lactococci appeared to be a combination of both host and plasmid functions. Stabilized variants were isolated by growing the cultures at 32 to 40 degrees C; these variants contained the Lac plasmids integrated into the L. lactis subsp. lactis LM0230 chromosome. In addition, the presence of the L. lactis subsp. cremoris Lac plasmids in L. lactis subsp. lactis resulted in a temperature-sensitive growth response; growth of L. lactis subsp. lactis transformants was significantly inhibited at 38 to 40 degrees C, thereby resembling some L. lactis subsp. cremoris strains with respect to temperature sensitivity of growth.     

Acquisition of mitochondrial DNA by a transformation vector for Ustilago violacea

Plasmid pUCH1 is a 5.2-kb pUC18 construct bearing the hygB gene fused to a promoter from Cochliobolus heterostrophus. Haploid cells of the basidiomycete, Ustilago violacea, were transformed with this plasmid. In addition to multiple integrations of plasmid sequences into U. violacea nuclear DNA, vector sequences independent of the nuclear genome were indicated by Southern-blot analysis using all or part of pUCH1 as a probe. Hybridization also revealed intact pUCH1 and several larger derivatives in satellite bands from CsCl-bis-benzamide gradients of whole cellular DNA and in DNA from purified mitochondria [mitochondrial (mt) DNA preparations] of transformed U. violacea; circular DNAs consistent with the sizes of DNAs in these satellite bands were seen in electron microscope analyses of the same mt DNA preparations as well. The plasmids could be detected in mt DNA preparations even after 30 generations of transformant growth under selective pressure. Transformation of Escherichia coli by these mt DNA preparations produced bacterial transformants bearing intact pUCH1, as well as several pUCH1 derivatives, including pUCH2, an approx. 8.0-kb plasmid. A 2.5-kb EcoRI fragment from pUCH2 showed only weak hybridization with pUCH1. This unique fragment did hybridize strongly with mt DNA from untransformed U. violacea. This derivative thus appears to have acquired mt sequences from U. violacea.     

Restriction Fragment Length Polymorphism Analyses of the Plasmid DNAs in Strains of Xanthomonas campestris pv. vesicatoria from Different Geographic Areas

Restriction fragment length polymorphisms (RFLPs) of plasmid DNAs in Xanthomonas campestris pv. vesicatoria were analysed using 77 strains from the United states, Argentina, Australia, Taiwan, and Korea. One or more plasmids were detected in all tested strains, irrespective of geographic origin, host plant from which isolated, or chemical resistance. All Korean strains contained a few plasmids of similar high molecular weight, whereas some small plasmids occured only in strains from the United States, Argentina, and Taiwan. After digesting total plasmid DNAs with each of four restriction endonucleases, 18 fragments with sizes from about 1 to 23 kb were visualized. Seventy-seven strains of diverse geographic origins, with different levels of resistance to streptomycin and copper, were classified into the 14 RFLP groups based on the restriction endonuclease digestion patterns of their plasmid DNAs. Strains belonging to each group shared DNA fragments of identical size, suggesting the possible presence of similar plasmids in these strains. A 5.8-kb EcoRI plasmid DNA probe prepared from the United States strain 81-23 hybridized to EcoRI plasmid digests from all tested strains. Other plasmid DNA fragments of the strain81-2,3 used as probes had no homology to plasmid DNA fragments from several strains around the world. The variation in hybridization profiles of plasmid DNA was very similar to the results obtained by RFLP analysis of plasmid DNA digested by four restriction enzymes. Most of the Korean strains tested were highly sensitive to streptomycin and copper, whereas most strains from other geographic areas showed a high level of resistance to one or two of the chemicals. Cluster analysis of genetic distance between the strains based on the data obtained generated the dendrograms that separated all Korean strains from the other strains, suggesting that plasmid DNA of the Korean strains may be genetically very different from those of the others.     

Plasmids controlling synthesis of hemolysin inEscherichia coli

Summary Plasmids of three different sizes, designated as plasmid A (mw: 65×10⁶), plasmid B (mw: 41×10⁶) and plasmid C (mw: 32×10⁶) respectively, have been isolated from various hemolytic wild-type strains ofE. coli. DNA-DNA hybridization was performed to determine their relationship. The wild-type strain, PM167a, harbours plasmids of all three sizes. Hybridization studies indicate that all three plasmids share extented sequence homologies but that plasmid A is not composed of plasmids B and C. Hybridization between plasmids of the donor strain and those of appropriate transconjugants demonstrates that in some cases plasmids with identical size are not longer completely homologous in their nucleotide sequences. This indicates that despite their defined sizes these plasmids are not stable genetic entities, but rather they undergo frequently recombination and dissociation during conjugation. In one particular transconjugant strain, K12-PM152/1, a plasmid D was found which is a stable recombined molecule of plasmids B and C of the original strain. Plasmids of size B found as the only extrachromosomal elements in a hemolytic wild-type strain (P224) and two transconjugant strains (e.g. K12-CM20 and K12-PM167/1) share extended nucleotide sequence homologies but are not identical. Little sequence homology was observed between two different hemolytic plasmids and the F and the Col Ib plasmids suggesting that the former do not belong to either the F-like or the I-like group of plasmids. Another hemolytic plasmid is F-like based on its sequence homologies with the F factor.     

Electrotransformation of industrial strains of Streptococcus thermophilus

A standard electroporation procedure was utilized to introduce a range of Gram-positive plasmid vectors into nine industrial strains of Streptococcus thermophilus. All the strains were transformable with at least two of the plasmids assessed, but electrotransformation frequencies depended on both the strain and the nature of transforming DNA. In general, small rolling circle (RC) plasmids could be electroporated at high frequency into a wide range of strains with efficiencies of 10(2)-10(5) transformants microgram-1 of transforming DNA. The presence of these plasmids did not influence doubling times during growth in broth, and they were generally extremely stable in slow milk acidifying strains, with 85-100% of transformants retaining the selective markers over 105 generations. Vectors were less stable in fast-growing cultures. Of the three theta-type plasmids assessed, only one, pIL253, could be electroporated at low frequency into some slow growing strains. The presence of this plasmid caused a 40% increase in doubling time and it was lost from cells at a rate of 3% per generation. Attempts to alter the proteolytic status of slow acidifying strains of Strep. thermophilus by the introduction of heterologous proteinase genes are also described.     

A novel family of linear plasmids with homology to plasmid pAL2-1 of Podospora anserina

Three recently isolated wild-type strains of the ascomycete Podospora anserina were analyzed for the presence of linear mitochondrial plasmids. In one of these strains, designated Wa6, at least 12 distinct plasmid-like elements were identified. From molecular analyses a minimum number of 78 individual linear molecules with proteins bound to their 5′ ends was estimated. In addition, the different members of this family of typical linear plasmids were shown to possess a common central region and terminal sequences which differ from one plasmid to another due to the presence of different numbers of a 2.4 kb sequence module. Finally, the pWa6 plasmids share a high degree of sequence similarity with pAL2-1, a linear plasmid previously identified in mitochondria of a long-lived mutant of P.anserina. A mechanism is proposed which explains the generation of these distinct, closely related extrachromosomal genetic traits.     

The evolution of a conjugative plasmid and its ability to increase bacterial fitness

Conjugative plasmids are extra-chromosomal DNA elements that are capable of horizontal transmission and are found in many natural isolated bacteria. Although plasmids may carry beneficial genes to their bacterial host, they may also cause a fitness cost. In this work, we studied the evolution of the R1 plasmid and we found that, in spite of the R1 plasmid conferring an initial cost to its host, after 420 generations the cost disappeared in all five independent evolution experiments. In fact, in two of these five experiments evolved conjugative plasmids actually conferred a fitness advantage to their hosts. Furthermore, the relative fitness of the ancestral clone bearing one of the evolved plasmids is significantly higher than both the plasmid-free ancestral cells and the evolved cells carrying the evolved plasmid. Given that the R1 plasmid may spread among different species of enterobacteria, we wondered what the effect of the evolved plasmid would be inside Salmonella enterica cells. We found that the evolved plasmid is also able to dramatically increase the relative fitness of these cells. Our results suggest that even if general usage of antibiotics is halted, conjugative plasmids that have been selected with antibiotics in previous years can still persist among bacterial populations or even invade new strains.     

Evidence for the existence of distinct populations of Vibrio anguillarum serogroup O1 based on plasmid contents and ribotypes.

A total of 103 Vibrio anguillarum serogroup O1 strains displaying 15 different plasmid profiles were characterized with respect to biochemical properties and ribotypes. The results confirmed that V. anguillarum O1 is a biochemically homogeneous group. The 103 strains could be allocated to three main clusters with high similarity coefficients. None of the biochemical properties were connected with the presence of plasmids. In total, 12 different ribotypes were demonstrated, with HindIII being used as the restriction enzyme. Forty of the strains were isolated from the same Danish fish farm, some from the kidneys of diseased fish and some from the environment, and some strains were isolated from the mucus, gills, and feces of healthy fish. Nineteen of these isolates possessed the 67-kb virulence plasmid alone or in combination with other plasmids, while 21 had no plasmids. All strains isolated from the kidneys of diseased fish on this farm had plasmids. Irrespective of their origin (kidneys, gills, or mucus), all 19 strains carrying the 67-kb virulence plasmid had the same ribotype, profile 1, while isolates without plasmids belonged to five different profiles, all different from profile 1. These results suggest that pathogenic V. anguillarum O1 strains possessing a virulence plasmid and nonpathogenic strains without plasmids from a small geographical area and even from the same fish may constitute two essentially distinct populations. Thus, it may be suggested that an exchange of virulence plasmids among strains is unlikely to occur in vivo.     

Plasmids in toxic and nontoxic strains of the cyanobacteriumMicrocystis aeruginosa

The plasmid content and toxicity of nine different strains ofMicrocystis aeruginosa have been analyzed. The two toxic strains of the HUB Culture Collection were found to carry each two plasmids, pMA1 and pMA2, of 2.9 kb and 8.5 kb, respectively. In strains PCC 7813 and PCC 7820, also toxic, two different plasmids of 2.6 kb and 16 kb were detected. Hybridization experiments showed that there exists no sequence homology between the pMA plasmids and the plasmids found in the PCC strains; but the pMA plasmids hybridized to chromosomal DNA of the toxic strains PCC 7820, PCC 7813, HUB 063, and the nontoxic strain HUB 5-3. In nontoxic strains no or at most one plasmid of unstable occurrence could be detected. Only one of the toxic strains investigated, SAG 14.85 (NRC-1), contained no plasmid.     

Distribution and characterization of plasmid-related sequences in the chromosomal DNA of different thermophilic Methanobacterium strains

The genomes of several thermophilic members of the genus Methanobacterium were analyzed for homology to the related restriction-modification plasmids pFVI and pFZ1 from M. thermoformicicum strains THF and Z-245, respectively. Two plasmid regions, designated FR-I and FR-II, could be identified with chromosomal counterparts in six Methanobacterium strains. Multiple copies of the pFVI-specific element FR-I were detected in the M. thermoformicicum strains CSM3, FF1, FF3 and M. thermoautotrophicum ΔH. Sequence analysis showed that one FR-I element had been integrated in almost identical sequence contexts into the chromosomes of the strains CSM3 and AH. Comparison of the FR-I elements from these strains with that from pFVI revealed that they consisted of two subfragments, boxI (1118 bp) and boxII (383 bp), the order of which is variable. Each subfragment was identical on the sequence level with the corresponding plasmid-borne element and was flanked by terminal direct repeats with the consensus sequence A(A/T)ATTT. These results suggest that FR-I represents a mobile element. FR-II was located on both plasmids pFVI and pFZI, and on the chromosome of M. thermoformicicum strains THF, CSM3 and HN4. Comparison of the nucleotide sequences of the two plasmid FR-II copies and that from the chromosome of strain CSM3 showed that the FR-II segments were approximately 2.5–3.0 kb in size and contained large open reading frames (ORFs) that may encode highly related proteins with an as yet unknown function.     

Sym plasmid transfer to various symbiotic mutants of Rhizobium trifolii, R. leguminosarum, and R. meliloti.

Two self-transmissible Sym(biosis) plasmids, one encoding pea-specific nodulation and nitrogen-fixation functions (plasmid pJB5JI) and the other encoding clover-specific nodulation and nitrogen-fixation functions (plasmid pBR1AN) were used to determine whether the symbiotic genes encoded on these plasmids are expressed in various members of the Rhizobiaceae. The host specificity of Rhizobium trifolii and R. leguminosarum Sym plasmid-cured strains could be directly determined by the transfer to these strains of the appropriate Sym plasmid. The nodulation of white clovers was restored by either plasmid pJB5JI or pBR1AN when these plasmids were transferred to two transposon Tn5-induced hair-curling (Hac-) R. trifolii mutants. In addition, lucerne nodulation was restored to a Hac- R. meliloti mutant when either plasmid pBR1AN or pJB5JI was transferred to this strain. The phenotype of nonmucoid (Muc-) Rhizobium mutants, which had altered cell surfaces, was not influenced by the transfer to these strains of plasmid pBR1AN or plasmid pJB5JI.     

Plasmid contents of lactic acid bacteria isolated from different types of sour doughs

The plasmid contents of 13 lactic acid bacteria isolated from different types of sour doughs were examined and compared with the plasmid contents of 11 culture collection strains and one commercial pure starter culture for sour doughs. In addition, plasmid analysis was used as a tool to study the stability of a starter culture during sour dough fermentation in a bakery.The tested strains varied in plasmid content from no plasmid up to six plasmids, with molecular weights from 1.5 to 43 MDal. In most cases, the wild-type strains contained a higher number of plasmids than the culture collection strains. The ability of the strains to ferment different carbohydrates was also investigated, but no obvious correlations between the fermentation patterns and the plasmid patterns could be observed. During the fermentation of the bakery sour dough, strains other than the inoculated starter culture gradually became dominant in the microflora. These new strains contained 1–3 plasmids, contrary to the plasmidless starter culture, and they also fermented more carbohydrates than the starter culture.     

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

Summary 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 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 se1uence 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. One of them contains neither pSAM1 not pSAM2. As this isolate produces spiramycin, these plasmids probably do not play an important role in spiramycin production. Apart from its intrinsic biological interest, pSAM2 may be useful in the construction of cloning vectors for S. ambofaciens. Very stable transformants might be obtained in certain strains of S. ambofaciens, because of the possibility of integration of the pSAM2 derivative vector.     

Analysis of plasmid profiling as a method for rapid differentiation of food-associated Clostridium perfringens strains

Plasmid analysis of over 120 strains of Clostridium perfringens , isolated during food-poisoning incidents and from animal carcasses and food constituents with no association with food poisoning, showed the potential of plasmid profiling as a means of differentiating epidemiologically related strains. On average 65% of freshly isolated strains contained one or more plasmids which could be used in the analysis. Comparison of profiles of strains from unrelated sources or unrelated strains from the same source showed a particularly wide variety of plasmid profiles. Thus the possibility that epidemiologically-unrelated strains might possess similar profiles appears to be very low in this organism. Analysis of serologically-related strains from the same source revealed similar plasmid profiles in all the plasmid-bearing strains examined. A high proportion (71%) of fresh and well-characterized food-poisoning strains possessed plasmids of 6·2 kb in size (compared with 19% of non-food-poisoning strains). The possible role of these plasmids is discussed, since the structural gene encoding the enterotoxin type A was not present on any of the plasmids in the food-poisoning strains tested.     

Plasmid mutations affecting self-maintenance and host growth in Escherichia coli.

As reported in the accompanying paper, a number of mutants of the ColVBtrp plasmid that can not be maintained stably in the host cell of Escherichia coli have been isolated. Each of the mutated plasmids has been transferred to an isogenic Col minus strain, and the resulting Col+ strains were studied to examine the effects of plasmid mutations on some properties of the host bacteria. Many of the strains harboring a mutated plasmid were thus found to be temperature sensitive; they failed to grow and divide normally at high temperatures. Some of them formed "filaments" under these conditions. These abnormal growth characteristics were accompanied by an increased susceptibility to sodium deoxycholate and methylene blue, suggesting that the cytoplasmic membrane has been altered. Moreover, studies of temperature-independent revertants obtained from two of these temperature-sensitive Col+ strains suggested that a single mutation on the plasmid is responsible for the pleiotropic effects exerted on the host cell. The bearing of these findings on the mode of replication and segregated of stringent-type plasmids such as ColVBtrp in the host bacteria is discussed.     

Stability of recombinant plasmids containing the ars sequence of yeast extrachromosomal rDNA in several strains of Saccharomyces cerevisiae

The mitotic stabilities of hybrid plasmid Rcp21/11, which contains the replicator of yeast rDNA, have been compared for four yeast host strains of different origins. In two related strains, Saccharomyces cerevisiae A62-1G-P188 and 1A-P3812 from the Peterhof genetic stocks, the plasmid was much more stable than in strains DC5 and GRF18 from the USA stocks.The enhanced mitotic stability of Rcp21/11 in these two yeast strains is obviously attributable to a higher rate of integration of the plasmid into the chromosomal rDNA repeats of the hosts.The centromeric locus CEN3 was inserted into Rcp21/11 because it provides high mitotic and meiotic stability of plasmids with yeast replicators, due to an ordered distribution of plasmids throughout cell division. Using the new centromeric plasmid RcpCEN3, transformation of the four above-described yeast strains was carried out. It was found that, similarly to centromeric plasmids with other chromosomal replicators, RcpCEN3 remains in the cell as a single copy. In strains DC5, GRF18 and A62-1G-P188 the mitotic stability of RcpCEN3 was 20–50%, i.e., less than half that of plasmids containing locus CEN3 and other yeast repliiators, ars1, ars2 and the 2μ DNA replicator. The mitotic stability of RcpCEN3 in strains 1A-P3812 (from the Peterhof genetic stocks) for individual clones reached 85%, i.e. close to that of the other plasmids. Genetic analysis showed that the capacity of strain 1A-P3812 to stably retain RcpCEN3 has a recessive polygenic character. We suggest that the observed differences in mitotic stability of centromeric plasmid RcpCEN3 between various yeast strains reflects the differences in activity of rDNA replicator in these strains. The nature of extrachromosomal rDNA circles, found in some strains of S. cerevisiae, is discussed from the point of view of the data.