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PubMed1981

Identification and characterization of Col plasmids from classical colicin E-producing strains.

A series of transformants have been derived which carry the Col plasmids from 11 E-colicin-producing strains isolated by Fredericq or Hamon. The ColE plasmids identified included four of type E1, five of type E2, and two of a type designated E4 by Horak (Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 233:58-63, 1975). Strain K317 was shown to carry a ColE7 plasmid and a 2.7-megadalton (Md) ColE2imm plasmid which confers immunity to colicin E2 but not the ability to produce colicin. Other plasmids identified in the colicinogenic isolates were a 3.4-Md ColN plasmid and a 1.3-Md Col plasmid of unknown type. The ColE1 plasmids all continued replicating in cultures treated with chloramphenicol. Strains carrying the ColE4 or ColE3-CA38 plasmid exhibited partial sensitivity to their own colicins and exhibited an unusual clearing-zone morphology when overlaid on stabs of colicin E2- or E7-producing strains. Except for ColE1-K53, ColE1-K47, ColE2-CA42, and ColE7-K317, all of the ColE plasmids were found to be about 4.3 Md in size. ColE2-CA42 and ColE7-K317 are both about 3.9 Md and are the only two plasmids of the E2, E3, E4, and E7 types that did not yield a 0.39-Md deoxyribonucleic acid fragment as an EcoRI digestion product. The comparisons of the ColE plasmids suggest several structural and functional relationships among them.

Bacteriocin Plasmids↗
PubMed1983

Characterization of conjugative plasmid EDP208.

EDP208 is a conjugative plasmid belonging to incompatibility group IncF0 lac, A restriction endonuclease map of this plasmid was constructed using five restriction enzymes: BamHI, HindIII, PvuI, SstI, and XhoI. On the basis of these mapping studies, the plasmid was found to be 90 kilobases in length. Clones were constructed from four large HindIII fragments of plasmid EDP208. One fragment, HindIII-20.5, was found to contain the lac genes and the origin of vegetative replication (oriV). Another fragment, HindIII-27.5, was found to contain all of the genes necessary for sex pilus formation, but it was nontransmissible. However, when used to complement a plasmid carrying an adjacent fragment, HindIII-23, the transfer of the latter occurred, suggesting that HindIII-23 contains the origin of transfer (oriT). The further localization of genes concerned with pilus biosynthesis was achieved by transposon mutagenesis. Six EDP208::Tn1 and thirty-seven EDP208::Tn5 mutants were isolated on the basis of their resistance to f1, a filamentous phage which adheres to intact pilus tips. The positions of the inserted transposons were determined on the restriction map and a 16.5-kilobase region was found to be required for pilus synthesis.

Conjugation, Genetic↗
PubMed1984

Conjugal transfer and characterization of bacteriocin plasmids in group N (lactic acid) streptococci.

Thirteen bacteriocin-producing strains of group N (lactic acid) streptococci were screened for their potential to transfer this property by conjugation to Streptococcus lactis subsp. diacetylactis Bu2-60. Bacteriocin production in three strains was plasmid encoded as shown by conjugal transfer and by analysis of cured, bacteriocin-negative derivatives of the donor strains and the transconjugants. With Streptococcus cremoris strains 9B4 and 4G6 and S. lactis subsp. diacetylactis 6F7 as donors, bacteriocin-producing transconjugants were isolated with frequencies ranging from ca. 2 X 10(-2) to 2 X 10(-1) per recipient cell. Bacteriocin-producing transconjugants had acquired a 39.6-megadalton plasmid from the donor strains 9B4 and 4G6, and a 75-megadalton plasmid from the donor strain 6F7. As shown by restriction endonuclease analysis, the plasmids from strains 9B4 and 4G6 were almost identical. The plasmid from strain 6F7 yielded some additional fragments not present in the two other plasmids. In hybridization experiments any of the three plasmids strongly hybridized with each other and with some other bacteriocin but nontransmissible plasmids from other S. cremoris strains. Homology was also detected to a variety of cryptic plasmids in lactic acid streptococci.

Bacteriocins↗
PubMed1984

Characterization of interspecific plasmid transfer mediated by Bacillus subtilis temperate bacteriophage SP02.

Plasmid pPL1010 is a 7.0-kilobase derivative of plasmid pUB110 that harbors the cohesive end site of the bacteriophage SP02 genome. Plasmid pPL1017 is a 6.8-kilobase derivative of plasmid pC194 that contains the immunity region of bacteriophage phi 105 and the cohesive end site of bacteriophage SP02. These plasmids are transducible by bacteriophage SP02 at a frequency of 10(-2) transductants per PFU among mutant derivatives of Bacillus subtilis 168 and have been transferred to other strains of B. subtilis and B. amyloliquefaciens by means of bacteriophage SP02-mediated transduction, with frequencies ranging from 10(-5) to 10(-7) transductants per PFU. The introduced plasmids were stably maintained in nearly all new hosts in the absence of selective pressure. An exception was found in B. subtilis DSM704, which also harbored three cryptic plasmids. Plasmids pPL1010 and pPL1017 were incompatible with a 7.9-kilobase replicon native to strain DSM704. Furthermore, plasmid pPL1017 was processed by strain DSM704 into a approximately 5.3-kilobase replicon that was compatible with the resident plasmid content of strain DSM704. The use of bacteriophage SP02-mediated plasmid transduction has allowed the identification of Bacillus strains that are susceptible to bacteriophage SP02-mediated genetic transfer but cannot support bacteriophage SP02 lytic infection.

Bacillus↗
PubMed2003

Molecular epidemiology and characterization of plasmid-encoded beta-lactamases produced by Tunisian clinical isolates of Salmonella enterica serotype Mbandaka resistant to broad-spectrum cephalosporins.

We studied 31 clinical isolates of Salmonella enterica serotype Mbandaka resistant to broad-spectrum cephalosporins and recovered in Tunisia over a 5-year period. The transferability of this resistance was demonstrated by conjugation experiments. Thirty of the 31 isolates were positive in the double-disk synergy test. By isoelectric focusing analysis, all of the isolates were found to produce a band of beta-lactamase activity with a pI of 5.9. Three of these isolates produced an additional band with a pI of 7.6. PCR and DNA sequencing identified these beta-lactamases as TEM-4 and SHV-2a, respectively. The remaining isolate, highly resistant to ceftazidime but susceptible to cefepime, produced a beta-lactamase that focused at pI 7.8. No synergy was detected by the double-disk synergy test. Sequence analysis of the bla gene amplified by PCR showed that the plasmid-mediated AmpC-type enzyme was ACC-1a. Fingerprinting analysis by repetitive-element PCR and enterobacterial repeat intergenic consensus-PCR suggested that 29 of the 31 Salmonella serotype Mbandaka isolates belonged to the same clonal population.

Anti-Bacterial Agents↗
PubMed2005

Characterization of virulence plasmids and serotyping of rhodococcus equi isolates from submaxillary lymph nodes of pigs in Hungary.

The plasmid types and serotypes of 164 Rhodococcus equi strains obtained from submaxillary lymph nodes of swine from different piggeries in 28 villages and towns located throughout the country were examined. The strains were tested by PCR for the presence of 15- to 17-kDa virulence-associated protein antigen (VapA) and 20-kDa virulence-associated protein antigen (VapB) genes. Plasmid DNAs were isolated and analyzed by digestion with restriction endonucleases to estimate size and compare their polymorphism characteristics. None of the 164 isolates contained the vapA gene, and 44 (26.8%) isolates were positive for the vapB gene, showing a product of the expected 827-bp size in the PCR amplification. The 44 isolates of intermediate virulence contained virulence plasmids that were identified as types 1 (3 isolates), 4 (1 isolate), 5 (36 isolates), 6 (1 isolate), and 7 (2 isolates) and as a new variant (1 isolate). On the basis of restriction digestion patterns of plasmid DNAs, we tentatively designated the variant as type 17. Use of the serotyping method of Prescott showed that 110 (67.1%) out of the 164 isolates were typeable and that serotype 2 predominated (83 isolates [50.6%]), followed by serotype 1 (26 strains [15.9%]). Only one isolate belonged to serotype 3. A total of 54 (32.9%) isolates were untypeable in Prescott's system. The prevalence of R. equi strains of intermediate virulence among the isolates that came from the submaxillary lymph nodes of swine in Hungary was lower than that seen with isolates obtained elsewhere.

Animals↗
PubMed1986

Characterization of a plasmid in isolates of Corynebacterium sepedonicum.

Lysis of mid-log phase cells of the Gram-positive bacterium, Corynebacterium sepedonicum, by a combination of lysozyme treatment and incubation with alkaline sodium dodecyl sulfate at 56 degrees C led to the recovery of a single plasmid. The plasmid was purified in CsCl density gradients, and its molecular weight estimated to be 31 megadaltons (46 kilobases), as determined from its relative mobility in agarose gels, from its contour dimensions in electron micrographs, and from the size of the fragments generated when it was cleaved with various restriction endonucleases. Thirteen widely divergent isolates of C. sepedonicum were screened for the presence of plasmid, and of these, 11 were shown to harbour a single plasmid at a level of about 30 copies per cell. Cleavage of the plasmid with PstI gave an identical banding pattern in agarose gels for the fragments from all of the plasmid-carrying isolates. The relevance of plasmid incidence and distribution in C. sepedonicum is discussed in relation to the possibility of developing a test for the detection of bacterial ring rot by using plasmid DNA as a hybridization probe.

Corynebacterium↗
PubMed1998

Isolation and characterization of two plasmids that mediate macrolide resistance in Escherichia coli: transferability and molecular properties.

Escherichia coli BM2506 is highly resistant to macrolide antibiotics; it produces macrolide 2'-phosphotransferase II [MPH(2')II] which inactivates such drugs. We investigated the localization and the transfer of the macrolide-resistance determinant that encoded the mphB gene for MPH(2')II in strain BM2506. Although we detected no clear band of plasmid DNA after agarose gel electrophoresis, transformation analysis using satellite DNA that corresponded to plasmid DNA after CsCl-ethidium bromide gradient centrifugation and restriction analysis of plasmid DNA in transformants showed that strain BM2506 harbored two plasmids, pTZ3721 (84 kb) and pTZ3723 (24 kb), that specified resistance to macrolides, ampicillin, streptomycin, tetracycline and sulfonamide and to macrolides and ampicillin, respectively. Southern hybridization showed that the mphB gene hybridized to both plasmids. Furthermore, pTZ3721 was transferred by conjugation to another strain of E. coli and pTZ3723 was mobilized with a self-transferable plasmid RP1 to other strains of E. coli. Therefore, it appears that the mphB gene is located on two plasmids in BM2506 and can be transferred to other strains of E. coli by conjugation or mobilization.

Anti-Bacterial Agents↗
PubMed1996

Isolation and characterization of a plasmid from Lactobacillus helveticus CP53.

A plasmid, pCP53 (11.5 kilo base pairs long), was isolated from Lactobacillus helveticus CP53 and its restriction map was constructed. A chimeric plasmid, pCP53D (4.7 kb), which had a 2.0-kb HindIII fragment from pCP53 and a tetracycline resistance (Tc) gene from Streptococcus faecalis replicated and expressed tetracycline resistance in Lactobacillus casei CP680. Fifteen strains of L. helveticus were transformed with pCP53D by electroporation and selected for Tc resistance. Transformants were obtained from two out of 15 host strains, L. helveticus CP611 and CP791, with 5 x 10(3) and 50 transformants per microgram of DNA, respectively.

DNA, Bacterial↗
PubMed1994

Isolation and characterization of linear plasmids from lankacidin-producing Streptomyces species.

Streptomyces rochei 7434AN4, a producer of lankacidin and lankamycin contains three large linear plasmids, pSLA2-L (200 kb), M (100 kb), and S (17 kb). Studies on the mutants of 7434AN4 having a different plasmid profile showed a parallel relationship between the presence of pSLA2-L and the production of both lankacidin and lankamycin. When pSLA2-L was transferred by protoplast fusion to S. rochei 2-39, a non-antibiotic-producing mutant of 7434AN4 which contained no detectable plasmid, the fusants gained the capacity to produce both antibiotics. From the physical maps of pSLA2-L and pSLA2-L1, a deletion plasmid (160 kb) of pSLA2-L, the latter plasmid was determined to contain a symmetrical linear repeat composed of the right 80-kb part of pSLA2-L. Four other lankacidin-producing Streptomyces strains were also found to have distinctive large linear plasmids which hybridized with the pSLA2-L probe. These results support the involvement of pSLA2-L in the production of lankacidin and lankamycin in S. rochei 7434AN4.

Anti-Bacterial Agents↗
PubMedDate not supplied

[Isolation and preliminary characterization of cryptic plasmids from Erwinia carotovora].

Of the fifty-two Erwinia carotovora strains studied, sixteen were found to contain extrachromosomal DNA (plasmids) from 2.5 to 129 kbp in size. Some E. carotovora strains bore two to five different plasmids. Experiments showed that the cryptic plasmids of erwinia are not responsible for their resistance to antibiotics and are not involved in the synthesis of macromolecular colicin-like carotovoricins. At the same time, one of the E. carotovora strains, 13A, augmented the production of carotovoricin after curing from one of its plasmids, 47.7-kbp pCA 6-2. Three E. carotovora subsp. carotovora strains and one E. carotovora subsp. atroseptica strain contained large 129-kbp plasmids, which may play a role in the ecology of phytopathogenic pectinolytic erwinia.

DNA, Bacterial↗
PubMed2001

[Isolation and characterization of a plasmid pTS50, which encodes nisin resistance determinant in Lactococcus lactis TS1640].

Three nisin-resistant Lactococcus lactis strains were screened from 197 samples of fresh milk on a selective medium (M17) supplemented with nisin, lactose and bromocresol purple, and were confirmed to have the nisin resistance determinant (nsr) by PCR amplification. Physiological and biochemical tests as well as Lactococcus lactis specific 16S rDNA sequence analysis revealed that the three strains all belong to Lactococcus lactis subsp. lactis. A large plasmid, pTS50, was identified in L. lactis subsp. lactis TS1640 which encodes resistance to nisin following electroporation of the total plasmids DNA into L. lactis MG1363. The molecular weight of plasmid pTS50 was estimated to be 47 kb by restriction analysis of BamHI, EcoRI, HindIII, NcoI, PstI and nsr was localized on a 1.9 kb EcoRI fragment by Southern hybridization.

Animals↗
PubMed1992

[Construction and characterization of shuttle plasmid pBHG1 in Bacillus thuringiensis and Escherichia coli].

The recombinant plasmid pBHGA was constructed by ligating plasmid pHTA1030 from B. thuringiensis to plasmid pJH101 from E. coli. The recombinant plasmid pBHGA was digested and deleted by various restriction enzymes, generating 14 derived recombinant plasmids differing in molecular weight. The expressible analysis for the plasmid pBHG1 from among the derived plasmids in E. coli HB101 and B. subtilis 168, the results proved the recombinant plasmid pBHG1 is carring promotive region, original region and segregational stability region of B. thuringiensis plasmid pHTA1030. The recombinant plasmid pBHG1 can be high frequency transformation in to B. thuringiensis HD-1 Cry- B and high-expression in B. thuringiensis. HD-1 Cry- B. The plasmid pBHG1 is segregational stability in B. subtilis 168 at 45 degrees C. These plasmids may be useful tools for study of plasmid replication and expression in B. thuringiensis once a reproducible transformation system is available for this organism.

Bacillus thuringiensis↗
PubMed2003

[Study of susceptibility and genotype characterization of plasmid-mediated class I cephalosporinase in gram-negative bacteria].

OBJECTIVE: To investigate the susceptibility and genotype characteristics of gram-negative bacteria producing plasmid-mediated class I cephalosporinase (AmpC beta lactamase) epidemic in Southern China. METHODS: A total of 1,187 clinical isolates of nonrepetive gram-negative bacteria were collected from different cities in Southern China. AmpC beta lactamase producing isolates were identified by cefoxitin three-dimensional test, and antimicrobial susceptibility test was identified by Kirby-Bauer agar diffusion test; plasmid conjugation, plasmid extraction, universal PCR for gene amplication of corresponding group was done, and the PCR products were sequenced subsequently. RESULTS: The positive rate of cefoxitin three-dimensional test in gram-negative bacteria was 5.9% (70/1,187), and the prevalence of plasmid-mediated AmpC beta lactamase was: E. coli: 4.2% (19/451), Klebsiella: 4.7% (16/339), Enterobacter: 2.1% (4/190), Alcaligenes: 5.3% (1/19), Acinetobacter: 2.2% (1/45) and the total positive rate was: 3.5% (41/1,187). The susceptibility test showed that compared with the clinical isolates, the transconjugations remained resistance to cephamycins and ampicillin, and susceptible to cefepime and imipenem. PCR amplication and sequencing confirmed them to be bla(DHA-1) gene and bla(ACT-1) gene, and they were mainly distributed in Klebsiella and Escherichia. CONCLUSIONS: DHA-1 and ACT-1 were the most common genotypes in plasmid-mediated AmpC beta-lactamase produced by clinical isolates in Southern China. Fourth-generation cephalosporins and carbapenems could be better choices for the treatment of infection caused by AmpC betalactamase producers.

Bacterial Proteins↗
PubMed1989

[Molecular cloning of butirosin biosynthesis genes. I. Construction and characterization of recombinant plasmid DNA No. 2 and No. 11].

B. circulans NRRL-B 3312 is a butirosin-producing strain, butirosin being one of aminoglycoside antibiotics. We used shotgun strategy for molecular cloning of butirosin biosynthesis genes. EcoRI digests of B. circulans NRRL-B 3312 chromosome DNA were ligated to EcoRI digests of plasmid pUB110 with T4-DNA ligase. Recombinant DNA was transformed into competent cells of B. subtilis 168. Kanr transformants were selected on medium containing kanamycin (5 micrograms/ml). Results showed that kanamycin and neomycin resistance levels were higher than that of pUB110 for both No. 2 and No. 11 recombinant plasmid DNA. Restriction enzyme maps were constructed for two recombinant plasmid DNA No. 2 and No. 11 respectively. Southern hybridization showed that there were some homogenous DNA sequences between No. 2 and No. 11 recombinant plasmid DNA and B. circulans NRRL-B 3312 DNA.

Anti-Bacterial Agents↗