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PubMed1981

Physical characterization of the plasmid pON5300.

The antibiotic resistance plasmid R1drd19Km- which has spontaneously lost its kanamycin resistance marker, and its derivative pON5300, were analysed using the restriction endonucleases SalI, BamHI, HindIII and EcoRI. The fragment patterns were compared with that of the R1drd19 and the fragments responsible for kanamycin resistance were found to be missing in R1drd19Km- and pON5300. In these plasmids a 7 Mg/mol EcoRI fragment was observed instead of the D (6.3 Mg/mol) fragment of R1drd19. Further a new 6 Mg/mol EcoRI restriction fragment was observed in pON5300. Using double digestions it was shown that the new fragment does not carry restriction sites for HindIII, BamHI and SalI endonucleases. The non-homology of the analysed plasmid was proved electron microscopically by heteroduplex techniques. The possibility of amplification in the regulatory region for the expression of R-determinants in pON5300 is discussed.

DNA Restriction Enzymes↗

PubMed1993

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 pFV1 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 pFV1-specific element FR-I were detected in the M. thermoformicicum strains CSM3, FF1, FF3 and M. thermoautotrophicum delta H. Sequence analysis showed that one FR-I element had been integrated in almost identical sequence contexts into the chromosomes of the strains CSM3 and delta H. Comparison of the FR-I elements from these strains with that from pFV1 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 pFV1 and pFZ1, 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.

Amino Acid Sequence↗

PubMed1989

Nucleotide sequence and molecular characterization of plasmid-like DNAs from mitochondria of cytoplasmic male-sterile rice.

Two plasmid-like DNAs, B2 and B3, were isolated from mitochondria of the cytoplasmic male-sterile rice, A-58 CMS. Molecular clones having their complete sequences were constructed and used as probes of mitochondrial and nuclear genomes by Southern hybridization. No evidence was found that integrated copies of either one exist in the main mitochondrial genome, but sequences homologous to both were present in the nuclear genome. The complete nucleotide sequences of B2 and B3 were established and compared to those of rice B1 and B4 and to the 1.9- and the 1.4-kbp plasmid-like DNAs of maize. Many of the sequences were common to both plant species.

Base Sequence↗

PubMed1981

Isolation and physical characterization of streptomycete plasmids.

Covalently closed circular DNA was isolated from a strain of Streptomyces coelicolor ATCC 10147 and from a strain of Streptomyces coelicolor subspecies flavus ATCC 19894, using two different methods. The two plasmids were of uniform monomer size: 8.9 kb for pS 10147, the plasmid from S. coelicolor ATCC 10147, and around 125 kb for the plasmid from S. coelicolor ATCC 19894. A restriction enzyme map was constructed for pS 10147, using seven enzymes. Four of the enzymes, (BamHI, Bgl,II, PvuII, and XhoI) cut pS 10147 once while PstI made two cuts. The GC content of this plasmid was calculated to be 72%. The possible utilisation of pS 10147 as a cloning vector in Streptomyces is discussed.

DNA Restriction Enzymes↗

PubMed1991

Characterization of new plasmids from methylotrophic bacteria.

Several tens of methanol-utilizing bacterial strains isolated from soil were screened for the presence of plasmids. From the obligate methylotroph Methylomonas sp. strain R103a plasmid pIH36 (36 kb) was isolated and its restriction map was constructed. In pink-pigmented facultative methylotrophs (PPFM), belonging to the genus Methylobacterium four plasmids were detected: plasmids pIB200 (200 kb) and pIB14 (14 kb) in the strain R15d and plasmids pWU14 (14 kb) and pWU7 (7.8 kb) in the strain M17. Because of the small size and the presence of several unique REN sites (HindIII, EcoRI, NcoI), plasmid pWU7 was chosen for the construction of a vector for cloning in methylotrophs. Cointegrates pKWU7A and pKWU7B were formed between pWU7 and the E. coli plasmid pK19 Kmr, which were checked for conjugative transfer from E. coli into the methylotrophic host.

Anti-Bacterial Agents↗

PubMed1976

Isolation and characterization of Co1E2 plasmid mutants unable to kill colicin-sensitive cells.

After transfer from a mutagenized host, twenty one Co1E2 plasmid mutants were isolated after screening 10,000 clones for abnormal colicin production. Analysis by SDS polyacrylamide slab gel electrophoresis of proteins synthesized after mitomycin C-induction of mutant cultures, indicates that all but two of the mutations are in the structural gene from colicin E2. Of these, nine produce fragments of colicin in both whole cells and minicells and some are suppressed by nonsense suppressors. Studies with a nonsense mutant producing only a small colicin E2 fragment (Co1E2-421) suggest that colicin E2 in not involved in plasmid DNA replication, in the control of its own synthesis, or required for cell death when cells become committed to colicin production. The two plasmid mutants outside the colicin gene segregate plasmid-free cells at 33 degrees, 37 degrees and 43 degrees. One segregates fairly rapidly (about 4% per generation) though the colicin-producing cells make normal amounts of colicin, whilst the other segregates more slowly and the colicin-producing cells make much reduced amounts of colicin.

Colicins↗

PubMed1993

Genetic characterization of plasmid-encoded multiple antibiotic resistance in a strain of Listeria monocytogenes causing endocarditis.

One susceptible and two multiply resistant isolates of Listeria monocytogenes from a patient suffering from prosthetic valve endocarditis are described. They could not be distinguished by several typing methods. Two isolates were resistant to chloramphenicol, macrolide/lincosamide/streptogramin antibiotics and tetracycline. The resistance determinants were located on a 39 kb plasmid pWDB100 that was transferable by filter mating to several gram-positive bacteria. Evidence was obtained to support the hypothesis that the resistant variant had primarily infected the patient's blood and prosthetic valve, and later lost the resistance plasmid. The three resistance determinants showed homology to other known markers, cat221/cat223, ermB and tetM, which are frequently found in different gram-positive genera. Plasmid pWDB100 showed extensive homology to the Streptococcus agalactiae broad-host-range plasmid pIP501. It was also very similar to two listerial plasmids found in France. Thus, plasmid pWDB100 and the homologous plasmids from France, although isolated in geographically distant regions, may illustrate spread of a plasmid and its relatives.

Conjugation, Genetic↗

PubMedDate not supplied

Characterization of cryptic plasmids from marine cyanobacteria and construction of a hybrid plasmid potentially capable of transformation of marine cyanobacterium, Synechococcus sp., and its transformation.

Among forty strains of marine cyanobacteria isolated in our laboratory, five strains had 1-3 different plasmids. The unicellular marine cyanobacterium, Synechococcus sp. NKBG 042902, contains at least three plasmids (pSY09, pSY10, and pSY11). However, these plasmids are cryptic. Therefore, a hybrid plasmid pUSY02 containing the 1.4-kb HindIII fragment of pSY11 and Escherichia coli plasmid pUC18 was constructed. The plasmid pUSY02 transformed both marine Synechococcus sp. NKBG042902-YG1116, which is a cured strain, and fresh water Anacystis nidulans R2 by dark incubation or Ca2+ treatment. However, the plasmid pSG111 constructed from the plasmid DNA of A. nidulans R2 failed to transform marine Synechococcus sp. Electroporation was also applicable to transformation of marine Synechococcus sp. and fresh water A. nidulans R2. The plasmid pUSY02 was rapidly introduced into marine Synechococcus sp.

Aspergillus nidulans↗

PubMed2003

Characterization of pNI10 plasmid in Pseudomonas, and the construction of an improved Escherichia and Pseudomonas shuttle vector, pNUK73.

The complete nucleotide sequence of pNI10 (3.75 kb), from which pNI105 and pNI107 were constructed as medium-host-range vectors for Gram-negative bacteria, was determined. A fragment of about 2.1 kb of pNI10 was essential for replication in Escherichia coli and Pseudomonas fluorescens. This fragment encodes a putative origin of replication ( ori) and one putative replication-controlling protein (Rep). An improved version of the medium-host-range plasmid vector pNUK73 (5.13 kb) was constructed with the basic-replicon of pNI10 and pHSG298 (2.68 kb). We show that expression in pseudomonads of the bromoperoxidase gene ( bpo) of Pseudomonas putida, inserted downstream of the lac promoter in pNUK73, resulted in about 30% (13.6 U/l culture) of the enzyme level obtained in E. coli.

Amino Acid Sequence↗

PubMed1979