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PubMedDate not supplied

[Characterization of antibiotic resistance plasmids in 2 strains of Salmonella typhi isolated in Belgium].

Two strains of Salmonella typhi resistant to streptomycin, tetracycline, chloramphenicol and sulfonamides were studied. Conjugation and analysis of plasmid DNA suggested that in the two strains, these resistance characters were mediated by a single R plasmid. The plasmids of both strains belonged to the FI-incompatibility group, whereas that isolated from the resistant Mexican S. typhi belonged to incomtibility group H. Therefore, the two resistant S. typhi isolated in Belgium do not represent an extension of the H plasmid which was responsible for chloramphenicol resistance in S. typhi during the 1972 Mexican epidemic.

Anti-Bacterial Agents↗
PubMed1993

Characterization of newly isolated plasmids from Actinobacillus pleuropneumoniae.

The genetic basis of drug-resistant strains of Actinobacillus pleuropneumoniae in Japan was studied. The A pleuropneumoniae strains AV277 and AV281 that belong to serotype 2 were resistant to streptomycin (SM) and sulfonamide (SA). Both strains had an 8.1-kilobase (kb) SM-SA plasmid that was previously classified in the H1 group. The AV177 (serotype 1) strain was resistant to SM, SA, ampicillin, and kanamycin (KM), but did not have any plasmids. The AV319 and AV324 (serotype 1) strains were resistant to SM, SA, tetracycline (TC), and chloramphenicol (CP). The AV318 (serotype 12) strain was resistant to SM, SA, TC, minocycline, and CP. These 3 strains (AV319, AV324, and AV318) had a 4.3-kb SM-SA plasmid and a 5.2-kb CP plasmid. The 4.3-kb plasmid was classified in the H2 group. The AV263 (serotype 1) strain was resistant to SM, SA, KM, TC, and CP. It had a 5.2-kb CP plasmid and a 6.6-kb SM-SA-KM plasmid. Both plasmids did not replicate stably in Escherichia coli strains. The former 5.2-kb plasmid was mobilized in E coli strains by plasmid RP4, which belonged to incompatibility P with broad host range, but the latter 6.6-kb plasmid was not so mobilized. Three 5.2-kb CP plasmids isolated from strains AV319, AV324, and AV318, had the same restriction endonuclease pattern after digestion with Ava I and EcoRI. They coexisted with H1 group plasmids in the incompatibility test, and coexisted also with H2 group plasmids of the original A pleuropneumoniae strains. Results indicated that the 5.2-kb CP plasmids could be classified in a new incompatibility group, H3. In this study, 4 types of plasmids were isolated, but no plasmids encoded TC and minocycline resistance.

Actinobacillus Infections↗
PubMed1995

Physicochemical and pharmacokinetic characteristics of plasmid DNA/cationic liposome complexes.

The objectives of this study were (i) to characterize the plasmid DNA encoding the chloramphenicol acetyltransferase reporter gene (pCAT) complexed with cationic liposomes (Lipofectin and LipofectACE) in terms of particle size and zeta potential, (ii) to compare pharmacokinetic characteristics, and (iii) to study the hepatic uptake mechanisms. DNA/LipofectACE complexes showed a negative zeta potential of -36 mV at 1:5 w/w ratio, but a positive zeta potential of (19 mV at 1:10 w/w ratio. Lipofectin samples showed a positive zeta potential) of 21-28 mV at these ratios. These preparations showed a wide particle size distribution ranging from 600 to 1200 nm. Following intravenous injection of 1:10 w/w ratio [32P]pCAT/liposome complexes at a dose of 0.1 mg DNA/kg into the tail vein of mice, radioactivity was rapidly eliminated from the plasma and almost 50-60% of the dose was taken up by the liver within 5 min after administration. Plasmid DNA/liposome complexes were predominantly taken up by the liver nonparenchymal cells. The hepatic uptake was inhibited by preceding administration of dextran sulfate (DS), but not by polycytidic acid (poly[C]) and polyinosinic acid (poly[I]), suggesting the involvement of a phagocytic process. We suggest that these complexes are preferentially taken up by the liver nonparenchymal cells mainly via Kupffer cell phagocytosis.

Animals↗
PubMed1978

Characteristics of PR5, a lipid-containing plasmid-dependent phage.

An extensive characterization of plasmid-dependent phage PR5 isolated from sewage has been carried out. The phage has a head diameter of 65--68 nm, is isometric with a double-layered capsid, and a minority possess tails. It adsorbs to many but not all types of bacteria possessing P, N, or W plasmids. The phage contains 20% lipid, 15.1% DNA, and 64.9% protein by weight and has a buoyant density of 1.265 g/ml in CsCl. The DNA is double-stranded with a G + C content of 49% and a molecular weight of 7.4 +/- 0.6 x 10 (6) as shown by electron microscopy. Phospholipid content is 66% of lipid and consists of cardiolipin (13%), phosphatidylethanolamine (43%), and phosphatidylglycerol (44%) and differ quantitatively from that of host bacteria. Anti-PR5 serum inactivates other similar phages, PR3 and PR4. Phage adsorption is impaired in deep rough mutants of Salmonella minnesota.

Bacteriophages↗
PubMed2000

Characterization of a phage resistance plasmid, pLKS, of silage-making Lactobacillus plantarum NGRI0101.

Phage contamination has resulted in abnormal fermentation in silage. We isolated a phage-resistant strain, Lactobacillus plantarum NGRI0101 from silage. The strain carried two plasmids, pLKL (6.8 kb) and pLKS (2.0 kb). By curing and retransformation of the plasmids, we clarified that pLKS has phage resistant activity, characterized as no adsorption inhibition. pLKS has 2,025 bp and three orfs, orfl23, orf132, and orf918. The predicted amino acid sequence of the orf918 product showed high similarity to those of Rep proteins of Pediococcus halophilus plasmid pUCL287 and Lactobacillus acidophilus plasmid pLA103. The replication origin (ori) was upstream from orf918. There was no gene similar to typical phage resistant genes encoded by known plasmids. The phage resistance of L. plantarum NGRI0101 may possibly be due to a plasmid-encoded abortive infection.

Amino Acid Sequence↗
PubMed1998

Molecular characterization of the erythromycin resistance plasmid pPV142 from Staphylococcus simulans.

The 2.5-kb erythromycin resistance (EmR) plasmid pPV142 of Staphylococcus simulans 13044 was isolated and characterized. Sequence analysis identified ORF1 and ORF2 encoding a 158-residue replication protein (Rep142) and a 244-residue erythromycin resistance protein (Erm, rRNA adenine N-6-methyltransferase), respectively. Structural analysis and Southern hybridization showed that the rep and ermM genes in pPV142 shared homology with the EmR plasmid pPV141 (2.4 kb) of S. chromogenes 3688 and other EmR plasmids known to exist in staphylococci and bacilli. Based on the presence of a 61-bp repeat upstream of the ermM gene, pPV142 is apparently a unique member of the pSN2 family of EmR plasmid able to express erythromycin resistance constitutively.

Amino Acid Sequence↗
PubMed1996

Characterization of a chromosomally encoded 2,4-dichlorophenoxyacetic acid/alpha-ketoglutarate dioxygenase from Burkholderia sp. strain RASC.

The findings of previous studies indicate that the genes required for metabolism of the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) are typically encoded on broad-host-range plasmids. However, characterization of plasmid-cured strains of Burkholderia sp. strain RASC, as well as mutants obtained by transposon mutagenesis, suggested that the 2,4-D catabolic genes were located on the chromosome of this strain. Mutants of Burkholderia strain RASC unable to degrade 2,4-D (2,4-D- strains) were obtained by insertional inactivation with Tn5. One such mutant (d1) was shown to have Tn5 inserted in tfdARASC, which encodes 2,4-D/alpha-ketoglutarate dioxygenase. This is the first reported example of a chromosomally encoded tfdA. The tfdARASC gene was cloned from a library of wild-type Burkholderia strain RASC DNA and shown to express 2,4-D/alpha-ketoglutarate dioxygenase activity in Escherichia coli. The DNA sequence of the gene was determined and shown to be similar, although not identical, to those of isofunctional genes from other bacteria. Moreover, the gene product (TfdARASC) was purified and shown to be similar in molecular weight, amino-terminal sequence, and reaction mechanism to the canonical TfdA of Alcaligenes eutrophus JMP134. The data presented here indicate that tfdA genes can be found on the chromosome of some bacterial species and suggest that these catabolic genes are rather mobile and may be transferred by means other than conjugation.

2,4-Dichlorophenoxyacetic Acid↗
PubMed2005

TraA and its N-terminal relaxase domain of the Gram-positive plasmid pIP501 show specific oriT binding and behave as dimers in solution.

TraA is the DNA relaxase encoded by the broad-host-range Grampositive plasmid pIP501. It is the second relaxase to be characterized from plasmids originating from Gram-positive organisms. Full-length TraA (654 amino acids) and the N-terminal domain (246 amino acids), termed TraAN246, were expressed as 6xHis-tagged fusions and purified. Small-angle X-ray scattering and chemical cross-linking proved that TraAN246 and TraA form dimers in solution. Both proteins revealed oriTpIP501 (origin of transfer of pIP501) cleavage activity on supercoiled plasmid DNA in vitro. oriT binding was demonstrated by electrophoretic mobility shift assays. Radiolabelled oligonucleotides covering different parts of oriTpIP501 were subjected to binding with TraA and TraAN246. The KD of the protein-DNA complex encompassing the inverted repeat, the nick site and an additional 7 bases was found to be 55 nM for TraA and 26 nM for TraAN246. The unfolding of both protein constructs was monitored by measuring the change in the CD signal at 220 nm upon temperature change. The unfolding transition of both proteins occurred at approx. 42 degrees C. CD spectra measured at 20 degrees C showed 30% a-helix and 13% b-sheet for TraA, and 27% alpha-helix and 18% beta-sheet content for the truncated protein. Upon DNA binding, an enhanced secondary structure content and increased thermal stability were observed for the TraAN246 protein, suggesting an induced-fit mechanism for the formation of the specific relaxase-oriT complex.

Base Sequence↗
PubMed2005

Plasmid replicons of Rhizobium.

Rhizobium spp. are found in soil. They are both free-living and found symbiotically associated with the nodules of leguminous plants. Traditionally, studies have focused on the association of these organisms with plants in nitrogen-fixing nodules, since this is regarded as the most important role of these bacteria in the environment. Rhizobium sp. are known to possess several replicons. Some, like the Rhizobium etli symbiotic plasmid p42d and the plasmid pNGR234b of Rhizobium NGR234, have been sequenced and characterized. The plasmids from these organisms are the focus of this short review.

Plasmids↗
PubMed1994

Homology among Treponema denticola plasmids.

Three of 16 isolates of Treponema denticola were found to contain small (2.0-2.7 kb) cryptic plasmids. These were pTD1 from T. denticola ATCC 33520, pTD2 from strain T32A, and pTD3 from strain D3A1. These plasmids were characterized by restriction mapping and cloned into E. coli plasmid pUC19. Extensive homology between these plasmids was revealed by Southern blot, and single-stranded DNA regions were found by neutral Southern blots and S1 nuclease mapping. These plasmids were not found in serovars usually associated with human periodontal disease nor are they universal in all T. denticola strains and serovars.

Blotting, Southern↗
PubMed1978

R-factor inheritance and plasmid content in mucoid Pseudomonas aeruginosa.

Eighteen strains of alginate-producing mucoid Pseudomonas aeruginosa were evaluated with respect to plasmid content and the ability to maintain well-characterized R plasmids. The spontaneous loss of alginate production in these strains varied from 0.01 to 0.7% and was not significantly increased by plasmid curing regimens. Examination of cleared lysates of these strains and their isogenic nonmucoid derivatives by agarose gel electrophoresis failed to reveal plasmid DNA. R-plasmid (P-incompatibility-group) transfer to mucoid P. aeruginosa was unaffected by the presence of the alginate capsule. Maintenance and expression of such plasmids in the mucoid strains were confirmed by agarose gel electrophoresis and by verification of plasmid-linked drug resistance and pilus-specific bacteriophage sensitivity. These studies demonstrate that alginate production does not appear to be plasmid linked and that mucoid P. aeruginosa are capable of receiving and donating certain drug resistance plasmids. Since some of the plasmids used here have been shown to mobilize chromosomal DNA, strains constructed in this study should afford the means for exploring the genetic basis of the mucoid phenotype.

Alginates↗
PubMed1989

[A new plasmid pBS1001 with broad range of hosts].

A new broad host-range plasmid capable of conjugative transfer has been isolated and characterized. The plasmid has the high frequency of conjugation transfer, is capable of conjugative transfer mobilization of nonconjugative plasmids, carries no known phenotypic markers. The plasmid demonstrates the specific interaction with the plasmids of P incompatibility group. The comparatively small size of the plasmid permits one to use it efficiently for comparative study of organization of the broad host range plasmids.

Caprolactam↗
PubMed1986

Plasmid vectors useful in the study of translation initiation signals.

The construction and characterization of plasmid vectors useful in the analysis of translation initiation signals in Escherichia coli and in the construction of lacZ gene hybrids are described. Transcription on the vectors proceeds from a cAMP-independent lac promoter through several restriction sites into a truncated lacZ structural gene lacking its first eight codons. Because this gene has no translation initiation signal, its level of expression is extremely low. A DNA fragment containing a translation start signal can be inserted between the promoter and truncated lacZ gene to produce a hybrid protein with functional beta-galactosidase activity. The vectors described here differ in sequence between the EcoRI cloning site and the lacZ gene to allow easy, in-frame joining of DNA containing a translation initiation signal to the lacZ gene. Cells containing plasmids can be screened directly for in-frame inserts by colony color on indicator plates.

Genetic Vectors↗
PubMed1993

Complete nucleotide sequence of a linear plasmid from Streptomyces clavuligerus and characterization of its RNA transcripts.

The complete nucleotide sequence of a small linear plasmid (pSCL1) from Streptomyces clavuligerus has been determined. This plasmid is 11,696 bp in length, has a 72% G+C content, and has approximately 900-bp inverted terminal repeat sequences. A comparison of the inverted terminal repeats of pSCL1 with those of a linear plasmid from S. rochei shows that the two terminal sequences have a high degree of similarity (approximately 70%). Several small inverted repeats found in the long terminal sequences of both plasmids are also conserved. An analysis of the sequence and codon preferences indicates that pSCL1 has seven or eight highly probable protein-coding open reading frames (ORFs). However, only two RNA species encoded by pSCL1 were detected in S. clavuligerus grown in liquid culture. The larger of these transcripts (900 nucleotides) corresponds to an ORF and is likely to be an mRNA for a protein similar to the KorA protein of pIJ101. The smaller transcript (460 nucleotides) does not correspond to any ORF; however, its 5' end is complementary to the 5' end of a predicted mRNA, suggesting that it may function as an antisense RNA. The larger of the two RNA species was present at a high level during the early stage of growth in liquid medium, and then its apparent rate of transcription decreased and remained at a lower level through the later stages; the level of the smaller RNA species remained relatively constant through all stages of growth.

Amino Acid Sequence↗
PubMed1992

Characterization of the virulence regions in the plasmids of three live Salmonella vaccines.

Three live Salmonella vaccines, Zoosaloral "Dessau", Bovisaloral "Dessau", and Suisaloral "Dessau" successfully used in veterinary medicine in eastern Germany were analysed for their plasmid DNA content. Plasmids of 60 MDa (S. typhimurium), 55 MDa and 26 MDa (S. dublin) and 34 MDa (S. choleraesuis) were found. The three large plasmids contained a virulence region as shown by restriction enzyme analysis and hybridization studies with virulence-region-specific DNA probes. Restriction enzyme pattern analysis of the whole plasmids revealed differences between vaccine strains and parental strains. We wanted to know if there were alterations within the virulence region of the vaccine strain plasmids compared with parental strains caused by the attenuation procedure. Therefore, the 6 kb-Cla I fragments of several plasmids were cloned and characterized by restriction enzyme fingerprinting. The expression of the Cla I fragment-encoded proteins was analysed in the minicell-producing strain DS 410. The experiments revealed that the restriction pattern of the 6 kb-Cla I fragments of Zoosaloral and Bovisaloral as well as the PAGE pattern of virulence-region-encoded proteins were in complete accordance with one another, between vaccine strains and the parental strain of Zoosaloral, and with a wild-type strain of S. typhimurium.

Nucleic Acid Hybridization↗
PubMed1987

Characterization of self-transmissible plasmids determining lactose fermentation and multiple antibiotic resistance in clinical strains of Klebsiella pneumoniae.

The lactose fermentation (Lac+) and antibiotic resistance (R+) phenotypes were conjugally transferred from Klebsiella pneumoniae strains (K166, K182, K186, K218, and K220) to Salmonella typhi, S. typhimurium, Shigella flexneri, and Vibrio cholerae. The genes for lactose fermentation and antibiotic resistance were located on the plasmids. Further analysis of plasmid DNA from these isolates indicated the presence of multiple plasmids (Mr ranged less than 2.7 to 70 X 10(6)). The Lac+R+ plasmids p166 and p182 were members of the FII incompatibility group. The fertility inhibition property of plasmids, p182, p218, and p220 was fi+ type. Furthermore, phage typing experiments showed that plasmids p166 and p218 (Lac+R+) conferred the ability to inhibit the multiplication of bacteriophages 12 and 13 in S. typhimurium. However, the plasmids p182, p186, and p220 (Lac+R+) could inhibit the visible lysis of all the 30 phages in S. typhimurium. This study describes the characterization of Lac+R+ plasmids and the medical significance of an intergeneric transfer of lactose fermentation to non-lactose-fermenting pathogens.

Escherichia coli↗
PubMed2002

The application of fluorescence correlation spectroscopy in detecting DNA condensation.

We report the application of fluorescence correlation spectroscopy (FCS) in characterizing conformational changes (condensation) of chemically well-defined DNA plasmids. The plasmids: pHbetaAPr-1-neo (10 kbp, contour length 3.4 microm) and pBluescript SKt (2.96 kbp, contour length 1.02 microm) were imaged by a confocal fluorescence microscope using two fluorescent probes: ethidium bromide (EtBr) and propidium iodide (PrIo). It became clear that the DNA molecule exhibits discrete conformational change between the coil and globule states with the addition of a small amount (the order of magnitude being 10(-5) M) of cationic surfactant, spermine and hexadecyltrimethyl ammonium bromide (HTAB). When the concentrations of both condensing agents are smaller than 6.0x10(-6) M and 2.0 x 10(-6) M for the 10 and 2.96 kbp, both plasmids are in the extended coil state with diffusion constants D(10 kbp)=9.6 x 0(-13) m(2) s(-1) and D(2.96 kbp)=2.5x10(-12) m(2) s(-1), respectively. When the condensing agent in a concentration higher than 1.10 x 10(-5) M is added to pHbetaAPr-1-neo (10 kbp), plasmids are in the condensed globular state and their diffusion constants are D(10 kbp)=8.0 x 10(-12) m(2) s(-1) (spermine) and D(10 kbp)=5.5x10(-12) m(2) s(-1) (HTAB). The globular state of the pBluescript SKt (2.96 kbp) plasmids is characterized by diffusion constants equal to D(2.96 kbp)=9.2x10(-12) m(2) s(-1) (spermine) and D(2.96 kbp)=8.2x10(-12) m(2) s(-1) (HTAB).

Algorithms↗