The sensitivity of phage DNA and plasmid DNA for a restriction enzyme from Staphylococcus aureus

In Staphylococcus aureus transduction of different tetracycline and chloramphenicol plasmids with a group I/III modification was possible to group I and III strains. Group II strains, containing a restriction endonuclease, had a restriction both for the phage and the plasmids: two restriction-deficient group II strains were good acceptors for these plasmids.     

Construction and restriction endonuclease mapping of hybrid plasmids containing Saccharomyces cerevisiae ribosomal DNA.

Summary Fragments produced by partial digestion of Saccharomyces cerevisiae ribosomal DNA (rDNA) with the restriction endonuclease EcoRI were ligated in vitro to the bacterial plasmid RSF2124. The resulting hybrid plasmids were cloned in Escherichia coli. Three hybrid plasmids which contain at least one intact repetitive unit of the multiple, tandem sequences of the yeast rDNA genes have been further characterized. These plasmids have been used to construct a map of the EcoRI, SmaI, HindII and HindIII restriction sites in the individual repetitive units of yeast rDNA.     

Study of plasmids isolated from saccharolytic Clostridia: Construction of hybrid plasmids and transfer toEscherichia coli andBacillus subtilis

Summary Small plasmids ofClostridium acetobutylicum and related strains were isolated and studied. Their restriction maps were established and different hybrid plasmids were constructed by ligation with plasmid pHV33.     

Isolation and characterization of megaplasmid DNA from lithoautotrophic bacteria

A method is described for the preparative isolation of megaplasmids ranging in size from 340 to 700 kb. These plamids were isolated from chemolithoautotrophic bacteria including the species Alcaligenes, Pseudomonas, and Paracoccus. The procedure was based on alkaline sodium dodecyl sulfate lysis of the cells, followed by heat treatment, salt precipitation, several phenol extractions, dialysis steps, and proteinase and RNase treatment. The various parameters were evaluated and controlled. Hydrogen-oxidizing-ability (Hox) encoding plasmids were compared by EcoRI restriction enzyme analysis. pHG plasmids from Alcaligenes eutrophus wild-type strains appeared to be closely related; plasmids derived from the type strain TF93 and from A. hydrogenophilus exhibited major differences in restriction sites. Two cryptic plasmids harbored by Pseudomonas facilis and Paracoccus denitrificans showed scarcely detectable similarity to the plasmid species of Alcaligenes.     

Bacteriophage lambda-E. coli K12 vector-host system for gene cloning and expression under lactose promoter control. II. DNA fragment insertion at the vicinity of the lac UV5 promoter.

Summary Recombinant plasmids containing the entire 16S RNA gene from the rrn B cistron of E. coli inserted in Col E1 and pBR322 plasmid vectors have been constructed. These plasmids have been mapped using several restriction endonucleases as well as by DNA-RNA hybridization. These maps reveal previously undetected restriction sites in the rrn B cistron and in Col E1 plasmid DNA.     

Plasmids in methanotrophic bacteria: isolation,characterization and DNA hybridization analysis

Ten strains of obligate methanotrophs were screened for the presence of plasmid DNA using a variety of methods. Plasmids were detected in all strains except Methylococcus capsulatus Bath. No significant similarity between plasmids was observed with respect to size or restriction digest patterns except for three strains of Methylosinus trichosporium, which appeared to contain the same three plasmids. Nitrocellulose filter hybridization revealed that the plasmid DNA from the M. trichosporium strains shared a small region of homology with the plasmid DNA from Methylosinus sporium 5. All of the plasmids remain cryptic. As the first step in characterization, a restriction digest map of the 55 kb plasmid found in Methylomonas albus BG8 was constructed.Abbreviations kb kilobases Formerly Mary L. O'Connor     

Incompatibility and molecular relationships between small bacteriocinogenic plasmids of Staphylococcus aureus

The sequence relations between small bacteriocinogenic plasmids (pRJ6, pRJ9, pRJ10 and pRJ11) of Staphylococcus aureus were investigated by comparing restriction maps and by hybridization. Plasmids pRJ10 and pRJ11 showed identical restriction maps, similar to that of pRJ9. The restriction map of pRJ6 differed from those of pRJ9 and pRJ10/pRJ11. Both groups of plasmids were shown to share a region of homology of at least 2.6 kb. The incompatibility relationships between them were also investigated by using plasmid derivatives tagged with transposon Tn551. Plasmids pRJ6 and pRJ9 proved to belong to different incompatibility groups.     

Cloning and restriction analysis of the hexokinase PII gene of the yeast Saccharomyces cerevisiae

Summary Carbon catabolite repression in yeast depends on catalytic active hexokinase isoenzyme PII (Entian 1980a). A yeast strain lacking hexokinase isoenzymes PI and PII was transformed, using a recombinant pool with inserts of yeast nuclear DNA up to 10 kbp in length. One hundred transformants for hexokinase were obtained. All selected plasmids coded for hexokinase isoenzyme PII, none for hexokinase isoenzyme PI, and carbon catabolite repression was restored in the transformants. Thirty-five independently isolated stable plasmids were investigated further. Analysis with the restriction enzyme EcoRI showed that these plasmids fell into two classes with different restriction behaviour. One representative of each class was amplified in Escherichia coli and transferred back into the yeast hexokinase-deficient strain with concomitant complementation of the nuclear mutation. The two types of insert were analysed in detail with 16 restriction enzymes, having 0–3 cleavage sites on transformant vector YRp7. The plasmids differed from each other by the orientation of the yeast insert in the vector. After yeast transformation with fragments of one plasmid the hexokinase PII gene was localised within a region of 1.65 kbp.     

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.     

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.     

Characterization of a cryptic plasmid (p0M1) inButyrivibrio fibrisolvens by restriction endonuclease analysis and its cloning inEscherichia coli

A small cryptic plasmid has been identified in a strain of the ruminal bacteriumButyrivibrio fibrisolvens. This plasmid has been isolated and purified. It is approximately 2.8 kbp in length and contains restriction sites for a number of common endonucleases including single sites for EcoRI, PvuII, and PstI. A map of the plasmid restriction sites has been constructed. This plasmid, designated p0M1, has been ligated to pBR325, pAT153, and pHV33 and transformed intoEscherichia coli, and the resulting hybrid plasmids have been mapped. The possible uses of such hybrid plasmids for gene cloning inB. fibrisolvens are discussed.     

Transformation of Staphylococcus aureus by heterologous plasmids

Plasmids isolated from Bacillus subtilis and Staphylococcus epidermidis were transformed into Staphylococcus aureus. Heterologous transformation was susceptible to restriction in S. aureus but could be performed in restriction-negative mutants or in heat-treated host bacteria. Three plasmids isolated from S. epidermidis were transformed into S. aureus with this technique and characterized. Two of them, pTE109 and pCE109, appear to be similar to two tet and cml plasmids previously isolated from S. aureus. The third, pPE109, carries penicillin and cadmium resistance and shows a restriction enzyme pattern which differs from known penicillinase plasmids in S. aureus.     

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.     

A novel plasmid-mediated DNA restriction-modification system in E. coli

R plasmids from 101 clinical isolates were transferred to E. coli J62 by conjugation and tested for the presence of R plasmid-mediated restriction-modification DNA systems. Thirty R plasmids were found to inhibit phage λ. vir development. Ten plasmids determined restriction modification system; nine of them proved identical with R.M. EcoRII. One transconjugant, E. coli J62 pLG74, was shown to have a restriction-modification system different from all the known R plasmid-mediated systems. Site-specific endonuclease has been isolated from E. coli J62 pLG74 which differed from all the known restriction endonucleases in the number of cleavage sites on phages λ, φX 174, virus SV40, plasmid pBR322 DNA molecules.     

Genetic organization and restriction enzyme cleavage map of the ksgA-pdxA region of the Escherichia coli chromosome

Summary Small multicopy plasmids carrying the Escherichia coli genes ksgA and pdxA were constructed by ligation in vitro of an EcoRI restriction fragment from ksg10 (Andrésson and Davies, 1980a) into the EcoRI sites of the ColE1 plasmids RSF2124 and pVH51. Cleavage maps of the plasmids were determined for 21 different restriction enzymes. The ksgA gene was located in a 750 basepairs (bp) region 1,450 bp clockwise of the EcoRI site in folA; pdxA is in a 2,040 bp region immediately clockwise of ksgA.     

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.     

ColV plasmids pColV-B188 and pColV-K30: Genetic maps according to restriction enzyme sites and landmark phenotypic characteristics

The ColV plasmids are large virulence plasmids of the incompatibility group IncFI. We have obtained the genetic maps of two of the most studied ColV plasmids, pColV-B188 and pColV-K30, according to restriction enzyme sites and landmark phenotypic characteristics such as colicin V, the aerobactin iron uptake system, the transfer region, replication regions, and repeated sequences. Although the two plasmids differ in size (pColV-B188 is 80 kb and pColV-K30 is 144 kb), the maps reveal many regions which are apparently identical or very similar.     

Construction ofClostridium butyricum hybrid plasmids and transfer toBacillus subtilis

Summary Small plasmids were isolated from type strains ofClostridium butyricum. Strain NCIB 7423 carries one plasmid (pCBU1) of 6.4 kb, whereas strain NCTC 7423 carries two unrelated plasmids of 6.3 kb (pCBU2) and 8.4 kb (pCBU3). Cleavage sites for 18 restriction endonucleases have been mapped on these plasmids and detailed physical maps are presented. For the purpose of developing vector plasmids for gene cloning in saccharolytic clostridia these crypticC. butyricum plasmids were joined to a selectable marker that will likely be expressed in clostridia. This was achieved by cloning the clostridial plasmids into theE. coli vector pBR322 carrying the chloramphenicol acetyltransferase (CAT) gene from theStaphylococcus aureus plasmid pC194. The recombinant plasmids were tested for their ability to confer chloramphenicol resistance toBacillus subtilis. Hybrid plasmids (pHL105, pHL1051) derived from pCBU2 were identified, which are capable of replication and expression of theS. aureus drug resistance marker in bothE. coli andB. subtilis. No structural instability was detected upon retransformation of pHL105 fromB. subtilis intoE. coli. The recombinant plasmids might thus be useful as shuttle vectors for the gene transfer betweenE. coli and a wide range of bacilli and clostridia.     

Identification of Agrobacterium strains by PCR-RFLP analysis of pTi and chromosomal regions

Chromosomes and Ti plasmids of 41 Agrobacterium strains, belonging to biovars 1, 2, 3, and Agrobacterium rubi species were characterized by the restriction fragment length polymorphism of PCR-amplified DNAs. Profiles that were obtained by the analysis of the amplified 16S rDNA confirmed the grouping of the strains according to their species. Higher polymorphism was detected in the intergenic spacer between the 16S rDNA and 23S rDNA genes, allowing efficient discrimination of strains. Identification of most strains was possible, and the genetic relatednesses of Agrobacterium strains could be estimated. The analysis of the plasmid Ti encoded regions between the tmr and nos genes, and the virA and virB₂ genes, allowed fingerprinting of Ti plasmids. Genomic typing by the rapid PCR-RFLP method is thus shown to be useful for an independant identification of strains and of the conjugative Ti plasmids.Abbreviations PCR polymerase chain reaction - RFLP restriction fragment length polymorphism - IGS intergenic spacer Funded by Institut National de la Recherche Agronomique     

A rapid method for the screening of plasmids in transformed yeast strains

A method for the rapid screening of plasmids in yeast cells has been developed. The method is an adaptation of the currently used alkaline lysis methods forEscherichia coli plasmids. Following the conditions described, several dozen ofSaccharomyces cerevisiae-transformed clones can be analyzed for their plasmid content in less than 2 h. The plasmids obtained by this procedure are suitable for restriction analysis or forE. coli andS. cerevisiae transformation.