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PubMed1989

Characterization of a plasmid coding for resistance to broad-spectrum cephalosporins in Salmonella typhimurium.

Five strains of Salmonella typhimurium isolated from faeces of infants hospitalized at Trousseau Hospital in Paris have been found to be resistant to third-generation cephalosporins. This resistance is by a transferable plasmid-mediated mechanism. The beta-lactamase gene which encodes for an enzyme similar to SHV-2 has been cloned into plasmid pBR322 and expressed in Escherichia coli RR1.

Cefotaxime↗
PubMed1989

Genetic and molecular characterization of pR351 plasmid from enteropathogenic Escherichia coli strain.

A pR351 plasmid (Tc Ap Cb) conferring drug resistance of naturally occurring E. coli strain was examined. Conjugation and transduction experiments have indicated that this plasmid is R plasmid aggregate consisting of three independent plasmids: a) conjugative plasmid pR351 A (SuTc) fi- (F) belonging to incompatibility group L, b) conjugative plasmid pR351 B (SuApCb) fi- (F) belonging to incompatibility O, and c) non-conjugative plasmid pR351 C (ApCb). The existance of these plasmids in pR351 plasmid aggregate was confirmed by the agarose gel analysis of plasmid DNA isolated from the lysates of E. coli J53 transconjugants carrying pR351 A or pR351 B plasmids and from E. coli K12 C600 transductant carrying pR351 C plasmid. Molecular mass of these plasmids was found to be 55.60 and 3 Mdal respectively. The non-conjugative pR351 C plasmid could be mobilized by Col B and F factors. Our findings have indicated that two fi- (F) R plasmids can stably coexist in R plasmid aggregate.

Drug Resistance, Microbial↗
PubMed1989

Characterization of the plasmids of Moraxella bovis.

Restriction endonuclease digestions were performed on plasmids purified from Moraxella bovis isolates GRS, Newport, and IBH64. It was determined from single and double digestions of plasmid DNA that GRS and Newport isolates carried 3 large plasmids having molecular sizes of 43.8, 41.3, and 32.8 kilobases (kb). Digestion of the 3 large plasmids and restriction endonucleases Hae III, HindIII, Nde I, and Ava I strongly indicated that these isolates shared structurally identical large plasmids. Timed single digestions with Ava I revealed that the IBH64 isolate carried 2 large plasmids having molecular sizes of 45 and 32.8 kb. The 32.8-kb plasmid was the only large plasmid that appeared to be shared by all 3 M bovis isolates. Two isolates, Newport and IBH64, carried small plasmids in addition to the large plasmids. Restriction maps were constructed for the 43.8-, 41.3-, and 32.8-kb plasmids.

Animals↗
PubMed1983

[Detection and characterization of the plasmids of the plague microbe which determine the synthesis of pesticin I, fraction I antigen and "mouse" toxin exotoxin].

Plasmid DNA was isolated from Yersinia pestis strains containing pesticin I or fraction I antigen and "mouse" toxin determinants. Specificity of DNA preparations was studied by using them for transformation of plague agent strains carrying no plasmids. pPstI plasmid (molecular weight 7,0-7,8 MD) encoded pesticin I, fibrinolysin and plasmacoagulase synthesis. Fraction I antigen and "mouse" toxin production determinants were borne on pFraI/Tox plasmid (molecular weight about 50 MD). The observation that some Y. pestis cultures, having lost the ability to synthesize one of pFraI/Tox products, still retained this plasmid in their cells, is regarded as an evidence for a complicated regulation of pFraI/Tox function.

Antigens, Bacterial↗
PubMedDate not supplied

[Characterization of R plasmids in "Salmonella typhi" and "S. paratyphi B" from Algeria (author's transl)].

The distribution of R plasmids in six drug-resistant Salmonella typhi strains and four S. paratyphi B strains isolated from human in Algeria was studied. All strains but one S. typhi harbored one conjugative R plasmid. Plasmids belonging to the incompatibility groups FIme, B, W or P were found in the S. typhi strains and those belonging to groups FIme or B were found in the S. paratyphi B strains. The epidemiological surveillance of R plasmids shows that the drug resistance of these strains is not the result of the epidemic spread of particular strains of Salmonella.

Algeria↗
PubMed1994

Characterization of the plasmid pVS809 from Bifidobacterium globosum.

A plasmid from a B. globosum strain was cut with 38 restriction enzymes and a physical map was constructed. Out of a total of 121 clones from curing experiments, plasmid was lost in 58% and 100% for acridine orange and ethidium bromide curing agent respectively. The plasmid does not exist as a chromosomal integrated form. An attempt to determine phenotypic characters encoded by the plasmid was made by electrophoretic analyses of the total proteins.

Animals↗
PubMed1993

Characterization of R plasmids from Shigella sonnei in Taiwan.

Plasmids of 70MDa from 6 Shigella sonnei isolates originating in Kaohsiung, Taiwan were found. All of them encoded resistance to chloramphenicol and tetracycline. These plasmids belonged to the H II incompatibility group. The fertility inhibition property of plasmids was the fi- type. Restriction endonuclease analysis revealed the six plasmids all had identical restriction patterns with BamH I, Hind III and Nru I. All six plasmids carried the type I chloramphenicol acetyltransferase gene and the class B tetracycline resistance gene as determined by Southern hybridization with DNA probes of various antimicrobial resistance genes. The results demonstrated that S. sonnei isolates harboured the same R plasmid.

Chloramphenicol O-Acetyltransferase↗
PubMed1993

Characterization of a plasmid codifying the synthesis of a beta-lactamase produced by Shigella flexneri.

The resistance to beta-lactam antibiotics shown by a strain of Shigella flexneri was plasmid-coded. This plasmid, pMAM-1, when transferred to Escherichia coli K-12 by conjugation, presented the same molecular weight (100 kbp) and conferred the same high level of resistance to ampicillin in the transconjugant as in the wild type strains (MIC, 2048-4096). Restriction analysis of the plasmid in transconjugants revealed various restrictive sites to some endonucleases (i.e. Bam HI, Eco RI, Pst I, Nco I, Cla I, Sf I and Sau 3AI, Nhe and Hin dIII), and no restrictive sites at all for other endonucleases (such as Xho I, Dra I, Kpn I, and Sal I). Some restricted DNA fragments were appropriate for cloning and isolation of the beta-lactamase gene present in Shigella flexneri UCSF 129. This work provides the first step in this direction.

Ampicillin Resistance↗
PubMed1989

Phenoxyacetic acid degradation by the 2,4-dichlorophenoxyacetic acid (TFD) pathway of plasmid pJP4: mapping and characterization of the TFD regulatory gene, tfdR.

Plasmid pJP4 enables Alcaligenes eutrophus JMP134 to degrade 3-chlorobenzoate and 2,4-dichlorophenoxyacetic acid (TFD). Plasmid pRO101 is a derivative of pJP4 obtained by insertion of Tn1721 into a nonessential region of pJP4. Plasmid pRO101 was transferred by conjugation to several Pseudomonas strains and to A. eutrophus AEO106, a cured isolate of JMP134. AEO106(pRO101) and some Pseudomonas transconjugants grew on TFD. Transconjugants with a chromosomally encoded phenol hydroxylase also degraded phenoxyacetic acid (PAA) in the presence of an inducer of the TFD pathway, namely, TFD or 3-chlorobenzoate. A mutant of one such phenol-degrading strain, Pseudomonas putida PPO300(pRO101), grew on PAA as the sole carbon source in the absence of inducer. This isolate carried a mutant plasmid, designated pRO103, derived from pRO101 through the deletion of a 3.9-kilobase DNA fragment. Plasmid pRO103 constitutively expressed the TFD pathway, and this allowed the metabolism of PAA in the absence of the inducer, TFD. Complementation of pRO103 in trans by a DNA fragment corresponding to the fragment deleted in pRO101 indicates that a negative control-regulatory gene (tfdR) is located on the BamHI E fragment of pRO101. Other subcloning experiments resulted in the cloning of the tfdA monooxygenase gene on a 3.5-kilobase fragment derived from pRO101. This subclone, in the absence of other pRO101 DNA, constitutively expressed the tfdA gene and allowed PPO300 to grow on PAA. Preliminary evidence suggests that the monooxygenase activity encoded by this DNA fragment is feedback-inhibited by phenols.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1980

Characterization of different plasmid-borne dihydropteroate synthases mediating bacterial resistance to sulfonamides.

Plasmid-borne resistance to sulfonamides was studied in both newly isolated and earlier characterized R plasmids. Two different classes of drug-resistant dihydropteroate synthases were found to be responsible for most cases of plasmid-mediated sulfonamide resistance. The plasmid-coded enzymes could be completely separated from their chromosomal counterpart and also showed differences in heat stability and molecular size. The resistant and chromosomal enzymes could bind the normal substrate, p-aminobenzoic acid, with equal efficiency. In contrast, sulfonamide binding was about 10,000 times lower with the plasmid-coded enzymes than with the chromosomal enzyme. Another substrate analog, p-aminosalicylic acid, on the other hand, inhibited chromosomal and plasmid-mediated enzymes to a similar extent. Evidence was also found for the existence of a plasmid-borne resistance mechanism independent of drug-insensitive enzymes.

Aminosalicylic Acid↗
PubMed1998

Isolation, characterization, and transfer of cryptic gene-mobilizing plasmids in the wheat rhizosphere.

A set of self-transmissible plasmids with IncQ plasmid-mobilizing capacity was isolated by triparental exogenous isolation from the wheat rhizosphere with an Escherichia coli IncQ plasmid host and a Ralstonia eutropha recipient. Three plasmids of 38 to 45 kb, denoted pIPO1, pIPO2, and pIPO3, were selected for further study. No selectable traits (antibiotic or heavy-metal resistance) were identified in these plasmids. The plasmids were characterized by replicon typing via PCR and hybridization with replicon-specific probes and other hybridizations. pIPO1 and pIPO3 were similar to each other, whereas pIPO2 was different. None of these plasmids belonged to any known incompatibility group. pIPO2 was selected for further work, and a mini-Tn5-tet transposon was inserted to confer selectability. Plasmid pIPO2 had a broad IncQ plasmid mobilization and self-transfer range among the alpha, beta, and gamma subclasses of the Proteobacteria but did not show productive transfer to gram-positive bacteria. Plasmid pIPO2 mobilized IncQ plasmid pIE723 from Pseudomonas fluorescence to diverse indigenous proteobacteria in the rhizosphere of field-grown wheat. Transfer of pIE723 to indigenous bacteria was not observed in the absence of added pIPO2. A specific PCR primer system and a probe were developed for the detection of pIPO2-type plasmids in soil and rhizosphere. Analysis of soil DNA provided evidence for the presence of pIPO2 in inoculated wheat rhizosphere soil in the field study, as well as in the rhizosphere of uninoculated wheat plants growing in soil microcosms. The system failed to identify major reservoirs of pIPO2 in a variety of other soils.

Escherichia coli↗
PubMed1982

Characterization of two new plasmid DNAs found in mitochondria of wild-type Neurospora intermedia strains.

Mitochondria from two Neurospora intermedia strains (P4O5-Labelle and Fiji N6-6) were found to contain plasmid DNAs in addition to the standard mitochondrial DNA species. The plasmid DNAs consist of monomeric circles (4.1-4.3 kbp and 5.2-5.3 kbp for Labelle and Fiji, respectively) and oligomers in which monomers are organized as head-to-tail repeats. DNA-DNA hybridization experiments showed that the plasmids have no substantial sequence homology to mtDNA, to each other, or to a previously characterized mitochondrial plasmid from N. crassa strain Mauriceville-lc (Collins et al. Cell 24, 443-452, 1981). The intramitochondrial location of the plasmids was established by cell fractionation and nuclease protection experiments. In sexual crosses, the plasmids showed strict maternal inheritance, the same as Neurospora mitochondrial DNA. The plasmids may represent a novel class of mitochondrial genetic elements.

Base Composition↗
PubMed1998

A novel method of extracting plasmid DNA from Helicobacter species.

BACKGROUND: Plasmids are extra-chromosomal DNA that may encode products that aid in virulence, pathogenesis, and the spread of antibiotic resistance among a wide spectrum of bacteria. Plasmids have been detected in Helicobacter pylori, H. felis, H. fennelliae, and H. cinaedi. However, no function has been attributed to the Helicobacter plasmids studied to date. Moreover, the characterization of plasmids in other Helicobacter species is an as yet unexplored area of research. Several laboratories have reported difficulties in the extraction and isolation of plasmid DNA from H. pylori and H. felis isolates due to the presence of large amounts of DNase, necessitating cumbersome and time-consuming purification steps. The development of a method for extracting plasmid DNA from Helicobacter species would be useful for future systematic studies of plasmids in this important group of microorganisms. MATERIALS AND METHODS: Eight H. pylori isolates, including the Sydney Strain SS1, three H. felis isolates, and one isolate each of H. hepaticus, H. bilis, H. mustelae, and H. rodentium, were screened for plasmid DNA using a novel method that includes a potassium xanthogenate-sodium dodecyl sulfate-phenol (XSP) buffer. A specific PCR targeting a highly conserved plasmid replication protein gene, repA, was used to confirm the presence of plasmids in the H. pylori isolates examined. The PCR primers used were designed based on the sequence of the H. pylori plasmid pHPM180. To demonstrate the effectiveness of this method, plasmid DNA extracted from SS1 using XSP buffer was digested using three restriction enzymes (DdeI, SpeI and MaeIII). The relative amount of DNA obtained using the protocol was also compared to the yield derived from four commercial kits commonly used in many laboratories. RESULTS: High and low molecular weight plasmids were extracted from H. pylori (n = 8) and H. felis (n = 3) isolates. The size range of these plasmids was from 3 kb to >16 kb. Attempts to isolate plasmids from H. hepaticus ATCC 51488, H. bilis ATCC 51630, H. rodentium MIT-95-2060, and H. mustelae NCTC 11574 were not successful, which was most likely due to the absence of endogenous plasmids from the strains examined. The relative amount of DNA obtained using the XSP buffer protocol was comparable to that obtained from commercial kits as assessed by direct examination of plasmid profiles on agarose gels. Plasmid DNA extracted from H. pylori SS1 using XSP buffer was successfully digested with restriction enzymes. CONCLUSION: This study reports the development of an efficient, inexpensive, and rapid method for extracting high and low molecular weight plasmids from Helicobacter species. Application of this novel method for the isolation and future characterization of plasmids from different Helicobacter species could promote a better understanding of the role of plasmids in the basic microbial physiology and ecology of this group of microorganisms.

Amino Acid Sequence↗
PubMed1997

Characterization of copy number mutants of plasmid pSC101.

We have characterized three copy number mutants of the plasmid pSC101. These mutations caused single amino acid substitutions at the 46th, 83rd and 115th codons in the rep gene and an increase in the copy number by 4- to 8-fold. Although the in vivo and in vitro repressor activities of these mutated Rep proteins were quite different from each other, the intracellular concentrations of the proteins were maintained at higher levels than the wild-type protein. It has been reported that excess amounts of Rep inhibit pSC101 replication (Ingmer and Cohen, 1993). This inhibitory activity of Rep was markedly decreased in all three mutants. When both the wild-type and one of the mutated rep genes were retained in the same plasmids, the copy number of these plasmids was decreased compared with plasmids retaining a single mutated rep gene. These results support the theory that the inhibitory activity of Rep for its own replication plays an important role in copy number regulation.

Journal Article↗