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PubMed1985

Escherichia coli 0142.H6; a drug-resistant enteropathogenic clone?

For many years strains of Escherichia coli belonging to particular serotypes (EPEC) were a common cause of outbreaks of infantile enteritis in Europe and North America. E. coli 0142.H6 was first isolated from infants with diarrhoea in Indonesia in 1960 and a further outbreak occurred in Mexico in 1965. Between 1967 and 1972 outbreaks of infantile enteritis caused by E. coli 0142 were reported in hospitals in Scotland, England, Northern Ireland and Eire. Sporadic cases occurred in Canada in 1972 and a further outbreak occurred in Arizona, U.S.A. in 1975. Strains from all these incidents were examined by biochemical and serological methods. Their resistance to antimicrobial drugs was determined and their resistance plasmids characterized; their plasmid profiles were visualized by agarose gel electrophoresis. The cumulative evidence suggests that strains isolated in the British Isles all belonged to a single clone and were related to those isolated in Indonesia, the U.S.A. and Canada. However, the strains from Mexico appeared to be unrelated. This study demonstrates that single clones of enteropathogenic E. coli may spread throughout the world, causing outbreaks of diarrhoeal disease and acquiring resistance to antimicrobial drugs.

Anti-Bacterial Agents↗
PubMed1976

Simple agarose gel electrophoretic method for the identification and characterization of plasmid deoxyribonucleic acid.

Agarose gel electrophoresis may be employed effectively for the detection and preliminary characterization of plasmid deoxyribonucleic acid (DNA) present in clinical isolates and laboratory strains of gram-negative microorganisms. The method is sensitive and does not require radioisotopes or ultracentrifugation. The estimation of plasmid mass from the extent of DNA migration in gels compares favorably with results obtained by electron microscopy of plasmid DNA purified by equilibrium density centrifugation. The method has proved to be a useful tool for survey work and the epidemiological investigation of plasmid dissemination, as well as an important adjunct to the genetic analysis of plasmids.

Bacteriological Techniques↗
PubMed1982

Transformation of Bacillus stearothermophilus with plasmid DNA and characterization of shuttle vector plasmids between Bacillus stearothermophilus and Bacillus subtilis.

A thermophilic bacterium Bacillus stearothermophilus IFO 12550 (ATCC 12980) was transformed with each of the following plasmids, pUB110 (kanamycin resistance, Kmr), pTB19 (Kmr and tetracycline resistance [Tcr]), and its derivative pTB90 (Kmr Tcr), by the protoplast procedure in the presence of polyethylene glycol at 48 degrees C. The transformation frequencies per regenerant for pUB110, pTB19, and pTB90 were 5.9 x 10(-3), 5.5 x 10(-3), and 2.0 x 10(-1), respectively. Among these plasmids, pTB90 was newly derived, and the restriction endonuclease cleavage map was constructed. When tetracycline (5 micrograms/ml) was added into the culture medium, the copy number of pTB90 in B. stearothermophilus was about fourfold higher than that when kanamycin (5 micrograms/ml) was added instead of tetracycline. Bacillus subtilis could also be transformed with the plasmids extracted from B. stearothermophilus and vice versa. Accordingly, pUB110, pTB19, and pTB90 served as shuttle vectors between B. stearothermophilus and B. subtilis. The requirements for replication of pTB19 in B. subtilis and B. stearothermophilus appear to be different, because some deletion plasmids (pTB51, pTB52, and pTB53) derived from pTB19 could replicate only in B. subtilis, whereas another deletion plasmid pTB92 could replicate solely in B. stearothermophilus. Plasmids pTB19 and pTB90 could be maintained and expressed in B. stearothermophilus up to 65 degrees C, whereas the expression of pUB110 in the same strain was up to 55 degrees C.

Bacillus subtilis↗
PubMed1983

Plasmid transformation in Agmenellum quadruplicatum PR-6: construction of biphasic plasmids and characterization of their transformation properties.

Biphasic, chimeric plasmids for the transformation of Agmenellum quadruplicatum PR-6 (Synechococcus sp. strain 7002) were constructed by splicing the 3.0-megadalton cryptic plasmid from strain PR-6 into plasmids pBR322 and pBR325 from Escherichia coli. Transformants of either E. coli or strain PR-6 by these plasmids could be detected on the basis of the drug resistance marker(s) carried by the chimeric plasmids. Plasmid DNA isolated from a PR-6 transformant transformed PR-6 much more efficiently than plasmid DNA prepared from E. coli. Plasmids from which the AvaI recognition site was deleted (AvaI is an isoschizomer of the AquI restriction endonuclease of strain PR-6) also transformed strain PR-6 much more efficiently than did plasmids containing the AvaI recognition site. These and other results suggest that AquI strongly effects plasmid transformation when the donor plasmid contains an unmodified AquI recognition site. Multimeric forms of the chimeric plasmids are also much more efficient at transforming strain PR-6 than are the analogous monomeric forms.

Chimera↗
PubMed1998

Characterization of plasmid regions of foodborne Yersinia enterocolitica biogroup 1A strains hybridizing to the Yersinia enterocolitica virulence plasmid.

The aim of our study was to find out if plasmids of foodborne Yersinia enterocolitica biogroup 1A strains harbour genes related to the virulence genes located on the virulence plasmid pYV of Yersinia enterocolitica. The foodborne strains were isolated from pork, as pigs are considered as an important reservoir for enteropathogenic Y. enterocolitica 0:3 and 0:9 strains. The plasmids of the foodborne strains were characterized by restriction enzyme analysis and hybridized to the virulence plasmid pYV of pathogenic Y. enterocolitica strains (0:3 biogroup 4; 0:9 biogroup 2). In several cases the plasmids of the foodborne strains showed homologies to parts of the pYV plasmid. Analysis of the hybridizing regions revealed that genes involved in replication, sequences of transposable elements and an endonuclease gene caused the observed hybridization to the virulence plasmid. In cause of the study also a remnant of a Tn3-like transposon was shown to be present adjacent to the yadA gene on the pYV plasmid. Although there is evidence that at least some strains of Y. enterocolitica biogroup 1A might possess pathogenic properties none of the well known plasmid encoded virulence genes were present on the plasmids of the investigated foodborne biogroup 1A strains.

Animals↗
PubMed1995

Transcription-induced deletions in Escherichia coli plasmids.

Characterization of functions that render DNA susceptible to rearrangement is important for a better understanding of genome instability. In a previous work, we showed that sequences located downstream of a strong promoter are particularly prone to deletion. In this paper, the parameters that influence transcription-induced deletions were studied. pBR322 derivatives carrying the M13 (+) replication origin and a PTac-dependent transcription region were used. Deletion formation was analysed in the presence of the replication protein of M13, which introduces a nick at the phage replication origin, and in a rep- strain to avoid M13-driven replication. Our study showed that: (i) 4 h after induction of transcription, a few per cent of the plasmids have experienced a deletion; (ii) these deletions result in joining of the M13 replication origin to a nucleotide located in or downstream of the transcribed region; (iii) deletion formation strongly depends on the orientation of transcription, on promoter strength and transcript length, but is independent of translation; (iv) formation of transcription-induced supercoiling domains does not induce deletion formation. We propose that deletions in the transcribed region result from collisions between converging replication and transcription machineries.

Base Sequence↗
PubMed2002

Characterization of two cryptic Helicobacter pylori plasmids: a putative source for horizontal gene transfer and gene shuffling.

Many Helicobacter pylori isolates carry cryptic plasmids of extremely variable size. In this study we analyzed two H. pylori plasmids, pHel4 and pHel5, from H. pylori strains P8 and P29, respectively. Plasmid pHel4 consists of 10,970 bp, constituting 15 putative open reading frames (ORFs), whereas pHel5 consists of 18,291 bp, constituting 17 ORFs. The findings that both plasmids encode a conserved RepA protein and that both have an origin of replication containing an iteron place them in the group of theta plasmids. In pHel4, the products of the overlapping orf4C, orf4D, orf4E, and orf4F sequences are homologous to MobA, MobB, MobC, and MobD, encoded by colicinogenic plasmids, suggesting that pHel4 might be mobilizable. A further putative operon consists of orf4B and orf4A, the products of which are homologous to microcin C7 (MccC7) biosynthesis and secretion proteins MccB and MccC, respectively. Plasmid pHel5 carries putative genes encoding proteins with homology to an endonuclease and gene products of an H. pylori chromosomal plasticity zone. Both plasmids contain repeat sequences, such as the previously identified R2 repeat, which are considered preferred recombination sites. In pHel4, a new repeat sequence (R4 repeat), which seems to act as a hot spot for site-specific recombination, was identified. All H. pylori plasmids characterized so far have a modular structure. We suggest a model that explains the existing plasmids by insertions and deletions of genetic elements at the repeat sequences. A genetic exchange between plasmids and the bacterial chromosome, combined with plasmid mobilization, might add a novel mechanism to explain the high genetic macrodiversity within the H. pylori population.

Amino Acid Sequence↗
PubMedDate not supplied

[Characterization of plasmids in Escherichia coli strains].

BACKGROUND: Strains of Escherichia coli are frequently plasmid carriers. In this species, resistance to beta-lactam antibiotics is almost always conditioned by the production of enzymes coded by plasmidic genes. The present is a study of the plasmids of 44 ampicillin-sensitive strains and 134 ampicillin-resistant (ampS and ampR). The possibility that the number and size of the plasmids are different and that this data may be added to the information to be considered in these two groups of strains is suggested. METHODS: The 178 strains selected had been isolated from human products. Sensitivity to ampicillin was studied by diffusion and was confirmed with the study of MIC (Mueller-Hinton agar, innoculum: 5 x 10 CFU). The plasmid type beta-lactamases were identified by analytical isoelectrofocus. Characterization of the plasmids was performed according to a variant of the Birnboim and Doly alkaline lysis technique. RESULTS: Among the ampR and ampS strains no plasmid were observed in 9 (6.72%) and 11 (25%) respectively. The mean number of plasmids was 2.53 and 1.57, ranging between 0-10 and 0-5. The number of strains with plasmids larger than, or equal to, 38 Kb was 113 and 27 respectively. The largest plasmids observed in the ampS strains were of 99 Kb and in the ampR of 109 Kb. A total of 3.73% of the ampR strains presented plasmids larger than 99 Kb and 8.20% more than 5 plasmids. CONCLUSIONS: No plasmids, presence of up to five and sizes smaller than or equal to 99 Kb were observed in strains of ampS and ampR. The presence of more than five and/or plasmids larger than or equal to 100 Kb was observed in 11.94% of the ampR.

Ampicillin Resistance↗
PubMed1979

Physical mapping of Staphylococcus aureus penicillinase plasmid pI524: characterization of an invertible region.

The staphylococcal penicillinase plasmid pI524 and a series of derivatives have been extensively mapped by restriction endonuclease digestion and by heteroduplex analysis. We report here the identification of a 2.2 kb region that undergoes a reversible, rec-independent inversion. This sequence is bounded by a pair of inverted repeats 650 base pairs in length, and has asymmetrically located recognition sites for at least three restriction endonucleases. A series of deleted derivatives and one naturally occurring, closely related plasmid, were studied. Two of these retain the inversion; the remainder are incapable of inverting and were all found to be locked in the same orientation of the inversion. The invertible sequence is adjacent to the region of the plasmid encoding beta-lactamase (bla); this entire region appears to be transposable and the inversion may be involved in the regulation of beta-lactamase expression or in translocation.

DNA Restriction Enzymes↗
PubMed1991

deoP1 promoter and operator mutants in Escherichia coli: isolation and characterization.

Plasmid DNA containing deoP1, one of the two major promoters of the deo operon, has been mutagenized using hydroxylamine, and promoter down-mutations and operator mutations were selected. The isolated mutants are all located within a 16 bp palindromic sequence containing the -10 region of deoP1. The results show that RNA polymerase and DeoR repressor compete for the same DNA target. The deoP1 promotor activity is dependent on a TG motif one base pair upstream of the -10 consensus sequence. The sequence of the deo operator site was further verified by use of a synthetic linker.

Base Sequence↗
PubMed1978

Improved lysis of group N streptococci for isolation and rapid characterization of plasmid deoxyribonucleic acid.

Procedures for effective cellular lysis and plasmid deoxyribonucleic acid (DNA) isolation from group N streptococci were developed. Cells were grown at 32 degrees C for 4 h in a modified Elliker broth containing 20 mM DL-threonine. After cellular digestion with 2 mg of lysozyme per ml for 7 min at 37 degrees C, 1% sodium dodecyl sulfate exposure resulted in complete and immediate lysis. Lactose (Lac) plasmid species in Streptococcus lactis C2 and S. cremoris B1 (30 and 37 megadaltons, respectively) were demonstrated upon examination of DNA from the cleared lysates by agarose gel electrophoresis. Increasing the lysozyme treatment to 20 min or more resulted in loss of the Lac plasmid, whereas other resident plasmids were unaffected and demonstrable in agarose gels. Diethylpyrocarbonate added before lysis prevented Lac plasmid loss in 20-min lysozyme-treated cells, but was not effective after 40 min of lysozyme treatment. The results suggested that endogenous nuclease activity during the lysozyme treatment period initiated Lac plasmid DNA loss. The development of an efficient lysis procedure for the group N streptococci allowed rapid identification and characterization of plasmid DNA by agarose gel electrophoresis. The plasmid composition of S. lactis C2 and S. cremoris B1, as determined by agarose gel electrophoresis, compared favorably to previous electron microscopic observations.

Bacteriolysis↗
PubMed1987

Characterization of plasmids encoding the adherence factor of enteropathogenic Escherichia coli.

Volunteer studies have shown that a 60-megadalton plasmid is required for full virulence of the human enteropathogenic Escherichia coli (EPEC) strain E2348/69 (O127:H6). The plasmid, designated pMAR2, encodes localized adherence to HEp-2 cells in tissue culture via the adhesin known as the EPEC adherence factor (EAF). Using a DNA probe for the EAF, we have previously shown that these genes are specific for EPEC and are usually encoded on plasmids ranging from 55 to 65 megadaltons. In this study, Southern blot analysis and S1 nuclease homology determination reveal a high degree of sequence conservation among these plasmids, despite some variation in restriction maps. Phenotypic characterization of the prototype EAF plasmid pMAR2 reveals that the plasmid belongs to the group IncFII and is negative for alpha-hemolysin, colicin, and aerobactin synthesis, as well as biochemical markers and antibiotic resistance. Regions encoding adherence to HEp-2 cells were localized by Tn801 insertion mutagenesis. Adherence genes were then cloned as two distinct plasmid regions which confer the adherence phenotype only when complementing each other in trans.

Adhesins, Escherichia coli↗
PubMed1981

Characterization of plasmids in Erwinia stewartii.

Plasmids in 39 strains of Erwinia stewartii were examined by agarose gel electrophoresis. Most virulent strains had from 11 to 13 plasmids ranging in molecular mass from 2.8 to 210 megadaltons and contained plasmids of 210, 70, 49, 43, 29.5, 16.8, 8.8, and 2.8 megadaltons. Plasmids in strains SW2 and SS104 were characterized by both electron microscopy and agarose gel electrophoresis and may be useful as convenient references for sizing plasmids by electrophoresis. Specific size classes of plasmids could not be associated with antibiotic and heavy metal resistance, carbohydrate utilization, bacteriocin production, or pathogenicity to corn. However, avirulent strains tended to have fewer plasmids than virulent strains.

DNA↗
PubMed1983

Construction and characterization of plasmid vectors for cloning in Staphylococcus aureus and Staphylococcus carnosus.

Several plasmid vectors for cloning in Staphylococcus aureus and S. carnosus have been constructed and characterized. The chimeric plasmids are composed of parts of the following parental plasmids: The chloramphenicol-resistance plasmid, pC194, the tetracycline-resistance plasmid, pMK148, and the erythromycin-resistance plasmid, pE12. All the chimeric plasmids confer two selectable antibiotic-resistance markers on host cells. Insertional inactivation of the various antibiotic-resistance markers occurred at the BclI site of pE12, and the Sau96- or AvaII-site of pMK148; only a slight inactivation of the chloramphenicol-resistance marker occurred at the HaeIII-site of pC194. The chimeric plasmids pCT20 and pCE10 are both stable in S. aureus and S. carnosus. In addition, the hybrid plasmids of pCT20 and pCE10, containing lambda-DNA fragments in various restriction sites between 0.4 and 1.2 kb, are stably maintained. The inserted lambda-DNA fragments appear unchanged.

Chloramphenicol↗
PubMed1979

Use of recombinant plasmids to characterize collagen RNAs in normal and transformed chick embryo fibroblasts.

Two recombinant plasmids containing chick collagen DNA sequences have been used to characterize messenger RNAs for pro-alpha1 (type I) and pro-alpha2 collagen. Poly(A)-containing RNA from chick embryo calvaria and long bones, tissues which are very active in collagen synthesis, were electrophoresed on agarose gels containing methylmercuric hydroxide and transferred to diazobenzyloxymethyl paper; these covalently bound RNAs were hybridized to 32P-labeled pro-alpha1 or pro-alpha2 collagen DNA sequences derived from the recombinant plasmids. The pro-alpha1 collagen probe identified two RNAs, a major species of 5000 bases and a minor species of 7100 bases; the pro-alpha2 collagen probe hybridized to a major species very similar in size to the pro-alpha1 mRNA, about 5200 bases, and a minor species of 5700 bases. It is possible that the 7100 and 5700 base RNAs represent precursors of pro-alpha1 and pro-alpha2 collagen mRNA, respectively. When similar hybridization experiments were performed with RNA from chick embryo fibroblasts, both the pro-alpha1 and pro-alpha2 collagen mRNAs were observed, as well as their corresponding larger species. With RNAs from fibroblasts transformed by Rous sarcoma virus, however, the levels of all RNA species which hybridized with the pro-alpha1 and pro-alpha2 collagen DNA probes were significantly reduced.

Animals↗
PubMed1983

Physical characterization of plasmids isolated from Streptosporangium.

Plasmids were isolated from two species of Streptosporangium by CsCl-ethidium bromide equilibrium density gradient centrifugation. A plasmid isolated from S. brasiliense, designated pSgB-1, was characterized by electron microscopy and agarose gel electrophoresis. The pSgB-1 plasmid is a closed circular DNA molecule of 9.4 X 10(6) Da. A restriction endonuclease map was generated and unique cleavage sites were found for EcoRI, ClaI, XbaI, and MstII. Another plasmid, pSgV-1, isolated from S. viridognriseum, has an estimated Mr of 54 X 10(6). The pSgB-1 plasmid is phenotypically cryptic but an unusual phenotypic trait, resembling phage plaques, may be associated with the S. viridogriseum plasmid pSgV-1.

Actinomycetales↗
PubMed1990

A unique plasmid profile characterizing Salmonella enteritidis isolates from patients and employees in a hospital.

Plasmid profiling was used as an epidemiological tool during a period of frequent Salmonella enteritidis infection in a hospital. S. enteritidis was isolated from 22 patients and employees. Isolates from 18 persons harbored one 29 and one 36 megadalton (MDa) plasmid. The 29 MDa plasmid has not been previously described in this species and was not found in 54 control strains of S. enteritidis from other sources. The respective restriction endonuclease digest fragments of the 36 and the 29 MDa plasmids were always identical. This plasmid pattern thus served as a marker for the isolates from the outbreak.

Humans↗