Isolation of plasmids present in thermophilic strains from hot springs in Jordan

The plasmid profile of two thermophilic bacterial strains isolated from recreation thermal springs in Jordan has been investigated. These strains are Streptococcus thermophilus and Bacillus sp1, which have been isolated from Zerka – Maeen and Himma hot springs respectively. Supercoiled and circular plasmid forms were detected, explaining the effect of DNA conformation on the mobility of the plasmid in the agarose gel electrophoresis. Two plasmids have been isolated and characterized by restriction endonucleases to facilitate their use as cloning vectors in thermophilic strains. The sizes of the plasmids were approximately 3 kb (from Streptococcus thermophilus) and 7 kb (from Bacillus spl). These plasmids were then digested with three different restriction enzymes (EcoRI, Bam HI, and HindIII), one of which was found to possess a single site for both plasmids, this enzyme is EcoRI.     

Plasmids from bacterial endosymbionts of hump-killer paramecia

Six isolates ofCaedibacter taeniospiralis, collected from four continents, were screened for plasmid DNA. Plasmid DNA species containing between 41.5 and 49.5 kilobase pairs (kb) were observed in all strains. Physical maps of plasmids were constructed by determining relative positions of the restriction endonuclease (BamHI,SalI,XhoI,SacI,PstI,AvaI, andEcoRI) recognition sequences in each plasmid. The physical map of the smallest plasmid (41.5 kb), pKAP30, is reflected in each of the plasmids isolated from the other strains ofC. taeniospiralis. Plasmid DNA from three of the isolates (strains 51 and 116 both from Indiana and strain 169 from Japan) each contain 43 kb, where 41.5 kb appear to be identical to pKAP30 (obtained from the Australian strain, A30). The extra 1.5 kb present in pKAP51, pKAP116, and pKAP169 is included as a single polynucleotide sequence. The 1.5-kb inclusion is located at apparently identical positions in pKAP116 and pKAP169 and at a totally different position in pKAP51. The two remaining plasmids, pKAP47 (from California strain 47) and pKAP298 (from Panama strain 298), both contain 49 kb to include a continuous 41.5-kb sequence that is apparently identical to pKAP30. The results indicate that the polynucleotide sequences of these plasmids are highly conserved and that the observed variations among them may be accounted for by transposable elements.     

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.     

Restriction enzyme digestion patterns ofFrankia plasmids

Summary After the initial screening of more than 200Frankia strains, the plasmid DNA observed in eight Frankiae was analyzed.In situ lysis was performed to obtain an estimate of their copy number and molecular weight. Four plasmid classes were distinguished, 7–9, 18–20, 30–35 and 50–55 kb. Twelve plasmids were thus analysed with restriction enzymes to determine their plasmid restriction patterns.While someFrankia plasmids with comparable molecular weights were found to be heterologous in their restriction enzyme pattern, an 8 kb plasmid found in bothFrankia sp. ArI3, isolated fromAlnus rubra andFrankia sp. CpI1 isolated fromComptonia peregrina showed undistinguishable fingerprints. Furthermore, an 18 kb plasmid found in the same two strains, also showed homologous restriction enzyme patterns. However, the copy numbers of the two ArI3 plasmids were higher than those of the CpI1 plasmids.Similarly, strains ACN1^AG, , isolated fromAlnus crispa all contained a 50 kb plasmid, and the three plasmids were found upon restriction analysis to be undistinguishable.In one strain, ARgX17c isolated fromAlnus rugosa, it was found through restriction enzyme analysis that two plasmids of a similar molecular weight were in fact heterologous.The possible origin of the homologous plasmids and their potential as specificFrankia markers to be used in ecological studies are discussed.     

Evidence for the presence of plasmids in four therapeutically important strains of Lactobacillus acidophilus

Four therapeutically important strains of Lactobacillus acidophilus designated as R, 301, 1899 and NCFM were screened for the presence of plasmids. Two lysis methods were used for the isolation of plasmid DNA: an alkaline method and a more gentle technique. It was found that the gentle lysis method yielded better plasmid DNA both quantitatively and qualitatively. All four strains studied apparently possess plasmids. The strains 301 and NCFM possessed one plasmid each, with a size of 4.2 kb, whereas R possessed three plasmids (3.5, 2.4 and 2.1 kb) and 1899 possessed two plasmids (4.1 and 4.2 kb). Restriction analysis revealed that the plasmid DNA from strain R was cleaved by Bam HI but not by Hin d III and Eco RI. The plasmid DNA from the remaining three strains was cleaved by all three restriction enzymes used.     

Plasmid content of salt stress-tolerant <Emphasis Type="Italic">Rhizobium</Emphasis> strains from Egyptian soils nodulating common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Plasmid profile analysis is useful to characterize Rhizobium strains within the same species. Among the 16 Rhizobium strains examined, 14 had distinct plasmid profiles. The size of plasmids ranged from 40 to 650 kb, and three plasmids of 650, 510 and 390 kb were common to several strains. Plasmid analysis revealed that Rhizobium etli contained a mega-plasmid, similar in size to Rhizobium tropici. All the salt-tolerant strains examined had a plasmid of 250 kb, except for strain EBRI 29. This suggests that this plasmid may play an important adaptive role under salt stress conditions.     

Distribution and homology of plasmids in several strains of Cyanothece

The plasmid distribution of several clonal isolates of the unicellular, diazotrophic, cyanobacterium Cyanothece sp. has been analyzed. The Cyanothece isolates contain three to four plasmids ranging in size from 4.8 kb to 40 kb. The plasmid profiles of three Cyanothece strains (BH63, BH68, BH93) indicated that strains BH68 and BH93 were closely related and that strain BH63 may be more distantly related. A small 4.8-kb plasmid (pSE480), from the clonal isolate Cyanothece sp. strain BH68F, has been subcloned and restriction mapped. Ten restriction sites have been mapped, five of which are unique and suitable for further subcloning. Southern hybridization revealed that this plasmid was present in two out of five clonal isolates of strain BH68 and in one isolate of strain BH93. A 10-kb plasmid from strain BH68F (pSE1000) was found in all of the BH68 isolates and was absent in the BH93 isolate, Cyanothece sp. strain BH93A. No notable physiological changes were observed in the absence of either the 4.8-kb or 10-kb plasmids. Therefore, these plasmids remain cryptic. Further analysis of these plasmids may provide insight into the function of these plasmids and will allow the construction of shuttle vectors for gene transfer experiments.     

Plasmids of Pseudomonas cepacia strains of diverse origins.

Thirty-seven strains of Pseudomonas cepacia from clinical, pharmaceutical-industrial, and environmental origins were analyzed for the presence of plasmid DNA by a modification of the rapid alkaline extraction method of Birnboim (H. C. Birnboim, Methods Enzymol. 100:243-255, 1983). Plasmids were present in 31 strains (84%) from all sources, with no one source showing less than 75% plasmid carriage among its strains. The plasmid profiles indicated that the presence of large plasmids (146 to 222 kb) was the norm. Those strains with greater antibiotic resistance were mainly in the clinical and pharmaceutical groups and carried large plasmids (222 kb) that appeared essentially identical by restriction digest analysis. The ability for conjugative transfer was shown with the broad-host-range plasmid R751 carrying the gene for resistance to trimethoprim, one of the few antimicrobial agents effective against P. cepacia. The plasmid was transferred from Pseudomonas aeruginosa to P. cepacia strains as well as from P. cepacia transconjugants to other P. cepacia strains.     

An integrative plasmid and multiple-sized plasmids of Pseudomonas syringae pv. phaseolicola have extensive homology

Summary Plasmids isolated from five strains of the bean pathogen Pseudomonas syringae pv. phaseolicola were characterized by restriction endonuclease and filter hybridization analyses. BamHI and EcoRI restriction patterns revealed that total plasmid DNA from each strain had a high level of sequence homology with pMC7105, a 148 kbp integrative plasmid found in a sixth strain. Only six BamHI fragments from the eight plasmids in these strains failed to hybridize with pMC7105 probe. Four of these fragments, three from pPP6520 and one from pPP6525 of strain PP652, hybridized strongly to plasmid DNA from a closely-related pathovar, P. syringae pv. glycinea. BamHI fragment 8, which is involved in the integration of pMC7105 into the host chromosome, contains a repeat sequence that was present on all the plasmids except pPP6120 (6.8 kbp), pPP6310 (40 kbp) and pPP6520 (45 kbp). Every plasmid but pPP6520 had fragments that showed weak hybridization to the small plasmid, pPP6120. This homology suggests that a second repetitive sequence is common to these plasmids. The large plasmids (148 to 151 kbp) were essentially identical to pMC7105. The intermediate plasmids (122 to 128 kbp) appeared to be derived mainly from pMC7105 or a related plasmid, whereas the smaller plasmids (6.8 to 45 kbp) appear to have been derived in part from sequences not present in pMC7105.     

Plasmids of Pseudomonas cepacia strains of diverse origins.

Thirty-seven strains of Pseudomonas cepacia from clinical, pharmaceutical-industrial, and environmental origins were analyzed for the presence of plasmid DNA by a modification of the rapid alkaline extraction method of Birnboim (H. C. Birnboim, Methods Enzymol. 100:243-255, 1983). Plasmids were present in 31 strains (84%) from all sources, with no one source showing less than 75% plasmid carriage among its strains. The plasmid profiles indicated that the presence of large plasmids (146 to 222 kb) was the norm. Those strains with greater antibiotic resistance were mainly in the clinical and pharmaceutical groups and carried large plasmids (222 kb) that appeared essentially identical by restriction digest analysis. The ability for conjugative transfer was shown with the broad-host-range plasmid R751 carrying the gene for resistance to trimethoprim, one of the few antimicrobial agents effective against P. cepacia. The plasmid was transferred from Pseudomonas aeruginosa to P. cepacia strains as well as from P. cepacia transconjugants to other P. cepacia strains.     

Isolation and preliminary characterization of plasmids in Bacillus badius

The presence of urease and NADPH-specific glutamate dehydrogenase (GDH) coding plasmid in Bacillus badius was suggested by the loss of enzyme activites with a mitomycin C treatment and by the presence of 3 plasmids in urease-active strains compared to 2 plasmids in urease-negative strains. Furthermore two-dimensional protein gel electrophoresis showed that the urease-positive strains had some additional proteins compared to urease-negative strains. The two common plasmids had a size of 14.7 kb and 5.9 kb. The plasmid that was missing in the urease-negative strains had a size of 44.0 kb. The plasmids were purified and restriction map for AvaI, Bam HI and EcoRI was constructed for each plasmid. Hybridization tests showed that all three plasmids in Bacillus badius were independent entities.     

Two native plasmids of Pseudomonas syringae pathovar tomato strain PT23 share a large amount of repeated DNA, including replication sequences

Strain PT23 of Pseudomonas syringae pv, tomato contains four native plasmids, designated A, B, C, and D. By DNA hybridization of genomic and plasmid DNA digests from the wild type and a plasmid-cured strain, we determined that c. 61 kb (c. 74%) of pPT23B is repeated in pPT23A and only c. 17 kb (c. 21%) is in single copy in strain PT23. pPT23B also contains DNA repeated in the chromosome that occurs in three DNA fragments of 0.6, 4.6, and 9.6 kb that might be transposable elements. Additionally, the 9.6 kb fragment also shares sequences with the three other plasmids of strain PT23. By DNA hybridization with the origin of replication from a native plasmid of P. syringae pv. syringae and in vivo replication tests, we identified the origins of replication of plasmids A, B, and D and showed that they cross-hybridize. The putative par region from pPT23 A has also been identified and is not conserved in the other three native plasmids from strain PT23. By using the defined minimal origin of replication from pPT23 A as a probe, we showed that it is highly conserved in 14 strains belonging to nine different pathovars of P. syringae and that as many as five different native plasmids with closely related origins of replication coexist in the same cell. The duplication and reorganization of plasmids might therefore occur at high frequency and could be responsible for the existence of large numbers of native plasmids in P. syringae strains.     

Plasmid distribution and analyses in Rhodopseudomonas sphaeroides

Ten strains of Rhodopseudomonas sphaeroides were analyzed by agarose gel electrophoresis for plasmid DNA content and, by filter-hybridizations, for their molecular relationships. All strains examined contained at least one plasmid. Several strains carried as many as six different plasmid species with sizes ranging from 42 to 140 kilobases (kb). Those larger than 89 kb showed extensive homology with each other; the 42-kb plasmid of R. sphaeroides strain 2.4.1 was homologous to the smaller plasmid DNA of three other strains. A partial map of the 42-kb plasmid derived from R. sphaeroides 2.4.1 was prepared by analysis of restriction endonuclease digests. Cross-hybridization among the large plasmids indicated that those present in any one strain of R. sphaeroides showed homology to one or more of the large plasmids detected in strains L and 2.4.1.     

pIJ101, a multi-copy broad host-range Streptomyces plasmid: Functional analysis and development of DNA cloning vectors

Summary Streptomyces lividans ISP 5434 contains four small high copy number plasmids: pIJ101 (8.9 kb), pIJ102 (4.0 kb), pIJ103 (3.9 kb) and pIJ104 (4.9 kb). The three smaller species appear to be naturally occurring deletion variants of pIJ101. pIJ101 and its in vivo and in vitro derivatives were studied after transformation into S. lividans 66.pIJ101 was found to be self-transmissible by conjugation, to elicit lethal zygosis and to promote chromosomal recombination at high frequency in both S. lividans 66 and S. coelicolor A3(2). A restriction endonuclease cleavage map of pIJ101 was constructed for 11 endonucleases; sites for five others were lacking. Many variants of pIJ101 were constructed in vitro by inserting DNA fragments determining resistance to neomycin, thiostrepton or viomycin, and having BamHI termini, into MboI or BclI sites on the plasmid, sometimes with deletion of segments of plasmid DNA. The physical maps of these plasmids were related to their phenotypes in respect of lethal zygosis and transfer properties. In vivo recombination tests between pairs of variant plasmids were also done. These physical and genetic studies indicated that determinants of conjugal transfer occupy less than 2.1 kb of the plasmid. A second segment is required for spread of the plasmid within a plasmid-free culture to produce the normal lethal zygosis phenotype: insertion of foreign DNA in this region caused a marked reduction in the diameter of lethal zygosis zones. The minimum replicon was deduced to be 2.1 kb or less in size; adjacent to this region is a 0.5 kb segment which may be required for stable inheritance of the plasmid. The copy number of several derivatives of pIJ101 in S. lividans 66 was between 40 and 300 per chromosome and appeared to vary with the age or physiological state of the culture. pIJ101 derivatives have a wide host range within the genus Streptomyces: 13 out of 18 strains, of diverse species, were successfully transformed.Knowledge of dispensable DNA segments and the availability of restriction sites for the insertion of DNA, deduced from the properties of plasmids carrying the E. coli plasmid pACYC184 introduced at various sites, was used in the construction of several derivatives of pIJ101 suitable as DNA cloning vectors. These were mostly designed to be non-conjugative and to carry pairs of resistance genes for selection. They include a bifunctional shuttle vector for E. coli and Streptomyces; a Streptomyces viomycin resistance gene of this plasmid is expressed in both hosts.     

Overlapping sequences of Klebsiella pneumoniae nifDNA cloned and characterized.

Summary A HindIII (17.0 kb) and an EcoRl restriction fragment (6.9 kb) of Klebsiella pneumoniae nif DNA were cloned on two small amplifiable plasmids, pCM1 and pSA30 respectively. These plasmids between them carry 14 of the 15 known Klebsiella nif genes. The operon for the three structural genes for nitrogenase, nifpHDK, is carried on pSA30: four and five of the remaining six operons are on pCRA37 and pCM1 respectively. All of the nif genes were assigned to endonculease restriction fragments of DNA using the Southern blotting technique (Southern, 1975) with total DNA of nif insertion mutants and radioactive plasmid DNA which contained cloned nif DNA sequences. Their locations were consistent with the genetic map of nif genes. The estimated size of the nif gene cluster was 24 kb.     

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.     

Plasmid content and homology of 16 strains of filamentous,nonheterocystous cyanobacteria

We have developed several strain-specific, rapid, small-scale plasmid isolation procedures in order to characterize the plasmid profiles of 16 filamentous, nonheterocstous cyanobacteria. At least one distinct plasmid was found in eight strains, with seven of these containing two or more different plasmids. Eight strains were found to be without plasmid DNA. Both the large, 12.9 kb, and the small, 1.6 kb, plasmids fromPlectonema boryanum 581 were isolated, purified, and cloned. Southern blots of plasmid DNAs from the eight strains were probed with these cloned DNAs and also with ultra-pure plasmid DNA fromPhormidium liridum 426. Four strains ofP. boryanum (485, 581, 594, 1542) andP. luridum 426 have identical plasmid profiles, and plasmid homology is extensive.     

Molecular analyses of conjugative, gentamicin-resistance plasmids from staphylococcal clinical isolates

Gentamicin-resistant Staphylococcus aureus and Staphylococcus epidermidis strains which were isolated from infants with staphylococcal bacteremia were analyzed for the presence of self-transmissible gentamicin-resistance (Gmr) plasmids. Conjugative GMr plasmids of approximately 43.8-63 kilobases (kb) were found in all S. aureus strains. Inter- and intra-species transfer of Gmr plasmids by conjugation was observed from S. aureus to S. aureus and to S. epidermidis recipient strains. However, neither inter- nor intra-species transfer of gentamicin resistance by conjugation was observed with nine out of nine S. epidermidis donor strains which were mated with either S. epidermidis or S. aureus recipient strains. These conjugative Gmr plasmids were unable to comobilize a smaller (15-kb) plasmid present in all but two S. aureus clinical isolates. Many of the conjugative Gmr plasmids also carried genetic determinants for kanamycin, tobramycin, neomycin, and ethidium bromide resistance, and for beta-lactamase synthesis. EcoRI restriction endonuclease digests of the S. aureus Gmr conjugative plasmids revealed three different digestion patterns. Four EcoRI restriction endonuclease digestion fragments of 15, 11.4, 6.3, and 4.6 kb in size were common to all plasmids. These plasmids and conjugative Gmr staphylococcal plasmids from other geographical regions shared restriction digestion fragments of similar molecular weights. DNA hybridization with biotinylated S. aureus plasmid pIZ7814 DNA revealed a high degree of homology among these plasmids. A 50.9-kb plasmid from one of the nonconjugative S. epidermidis clinical isolates showed homology with the probe DNA but lacked a portion of a 6.3-kb fragment which was present in all conjugative plasmids and believed to carry much genetic information for conjugation.     

A quantitative analysis of shotgun cloning in Bacillus subtilis protoplasts

Summary We used the Escherichia coli-Bacillus subtilis shuttle vector pHP13, which carries the replication functions of the cryptic B. subtilis plasmid pTA1060, to study the effects of BsuM restriction, plasmid size and DNA concentration on the efficiency of shotgun cloning of heterologous E. coli DNA in B. subtilis protoplasts. In a restriction-deficient strain, clones were obtained with low frequency (19% of the transformants contained a recombinant plasmid) and large inserts (>6 kb) were relatively rare (12% of the clones contained inserts in the range of 6–9 kb). The efficiency of shotgun cloning was severely reduced in restricting protoplasts: the class of large inserts (>6 kb) was under-represented in the clone bank (4% of the clones contained inserts in the range of 6–6.1 kb). Furthermore, BsuM restriction caused structural instability of some recombinant plasmids. Transformation of protoplasts with individual recombinant plasmids showed that plasmid size and transforming activity were negatively correlated. The size effect was most extreme with cut and religated plasmid DNA. The yield of clones was independent of the DNA concentration during transformation. It is therefore unlikely that clones were not detected because of simultaneous uptake of more than one plasmid. It is concluded that shotgun cloning in B. subtilis protoplasts is inferior to that in competent cells.     

Evaluation of Plasmid Content and Tetracycline Resistance Conjugative Transfer in <Emphasis Type="Italic">Enterococcus italicus</Emphasis> Strains of Dairy Origin

Five Enterococcus italicus strains harbouring tet genes responsible for the tetracycline resistance were subjected to plasmid profile determination studies. For four strains tested the profiles showed between three and six plasmid bands, the size of which ranged between 1.6 and 18.5 kb. Southern hybridization experiments associated tetS and tetK genes with chromosomal DNA in all strains and tetM gene with plasmids of around the same size (18.5 kb) in two of the tested strains. The ability of the new species to transfer tetM gene was studied by transfer experiments with the tetracycline-susceptible recipient strains E. faecalis JH2-2 and OG1RF; mobilization experiments were performed with E. faecalis JH 2-2 harbouring the conjugative plasmid pIP501as helper plasmid. The results obtained show that the new enterococcal species was able to acquire antibiotic resistance by conjugation, but not to transfer its plasmids to other bacteria. Further PCR and hybridization experiments carried out to assess the presence of mobilization sequences also suggest that the tetM plasmid from E. italicus is a non-mobilizable plasmid.