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At least 217 records · Page 12Linked to original sources
PubMed1980

Characterization of promoter-cloning plasmids: analysis of operon structure in the rif region of Escherichia coli and isolation of an enhanced internal promoter mutant.

Using the promotor-cloning vehicle described by An and Friesen (J. Bacteriol. 140:400-410, 1979), Escherichia coli chromosomal deoxyribonucleic acid fragments derived from the lambda drifd18 transducing phage were cloned in one of several unique restriction endonuclease sites adjacent to tetracycline(tet) genes that lack their own promotor. One of these plasmids has been used to isolate nine variants having mutations that lie in a putative internal promoter which is located between rplL and rpoB. Deoxyribonucleic acid sequence analysis revealed that, in all nine mutants, a single base change, C to T, in the ribonucleic acid polymerase recognition site led to a large increase in promoter activity. Analysis of a variety of plasmids in which tet is fused to various promoters yielded the following results: (i) rplK and rplA, genes for ribosomal protein L11 and L1, respectively, were cotranscribed from a common promoter located upstream from rplK; (ii) there was a strong promoter in the region between the rplKA operon and rplJ, the gene for ribosomal protein L10; (iii) an attenuator region was located between rplL, the gene for ribosomal protein L12, and rpoB, the gene for ribonucleic acid polymerase subunit beta; (iv) transcription terminated immediately after rpoC, the gene for ribonucleic acid polymerase subunit beta'; (v) a gene coding for unknown protein U, which is located between tufB and the rplKA operon, had its own promoter; (vi) the tufB gene was separated from all of the genes described above and had its own promoter.

Bacterial Proteins↗
PubMed1980

Recombinant plasmids containing avian vitellogenin structural gene sequences derived from complementary DNA.

Purified mRNA coding for chicken vitellogenin, a precursor of egg yolk proteins, was transcribed to complementary DNA (cDNAvit) with avian myeloblastosis virus (AMV) reverse transcriptase. Double-stranded cDNA was synthesized with Escherichia coli DNA polymerase I (fragment A) using the self priming ability of the cDNA. Following S1 nuclease digestion the double-stranded cDNA was inserted into the Hind III site of plasmid pBR322 using the poly(dA) . poly(dT) tailing method, and the hybrid molecules were used to transform Escherichia coli chi 1776. Ampicillin-resistant colonies were screened by colony hybridization with 125I-labeled vitellogenen mRNA. Further screening of positive clones was done by agarose gel electrophoresis and in situ hybridization with 125I-labeled vitellogenin mRNA. In addition, plasmid DNA covalently bound to diazotized paper was used to select complementary mRNA sequences. The cloned vitellogenin sequences were shown to hybridize to a mRNA which directs the synthesis of immunoprecipitable vitellogenin when translated in a reticulocyte lysate cell-free system. The length of the inserted cDNA was determined by agarose gel electrophoresis and heteroduplex mapping. The largest insertion was about 2500 base pairs. Restriction mapping indicates that at least three plasmids out of four have different sequences.

Animals↗
PubMedDate not supplied

[Effect of naphthalene biodegradation plasmids on physiological characteristics of rhizospheric bacteria of the genus Pseudomonas].

Specific growth rate, duration of the lag phase, stability of plasmids, and activities of the key enzymes involved in naphthalene biodegradation were studied in rhizospheric pseudomonades carrying structurally similar plasmids pOV17 and pBS216. It was demonstrated that these plasmids determined various levels of catechol 2,3-dioxygenase activities. The structural rearrangements in the plasmid pBS216 could "switch off" the genes of catechol oxidation meta-pathway. It was shown that certain combinations of biodegradation plasmids and bacterial hosts, such as Pseudomonas chlororaphis PCL1391(pBS216), P. chlororaphis PCL1391(pOV17), and P. putida 53a(pOV17), were considerably more efficient than natural variants in their growth characteristics and stability of the biodegradation activity, having a potential for bioremediation of soils polluted with polycyclic aromatic hydrocarbons (PAHs).

Biodegradation, Environmental↗
PubMed2005

Functional analysis and annotation of the virulence plasmid pMUM001 from Mycobacterium ulcerans.

The presence of a 174 kb plasmid called pMUM001 in Mycobacterium ulcerans, the first example of a mycobacterial plasmid encoding a virulence determinant, was recently reported. Over half of pMUM001 is devoted to six genes, three of which encode giant polyketide synthases (PKS) that produce mycolactone, an unusual cytotoxic lipid produced by M. ulcerans. In this present study the remaining 75 non-PKS-associated protein-coding sequences (CDS) are analysed and it is shown that pMUM001 is a low-copy-number element with a functional ori that supports replication in Mycobacterium marinum but not in the fast-growing mycobacteria Mycobacterium smegmatis and Mycobacterium fortuitum. Sequence analyses revealed a highly mosaic plasmid gene structure that is reminiscent of other large plasmids. Insertion sequences (IS) and fragments of IS, some previously unreported, are interspersed among functional gene clusters, such as those genes involved in plasmid replication, the synthesis of mycolactone, and a potential phosphorelay signal transduction system. Among the IS present on pMUM001 were multiple copies of the high-copy-number M. ulcerans elements IS2404 and IS2606. No plasmid transfer systems were identified, suggesting that trans-acting factors are required for mobilization. The results presented here provide important insights into this unusual virulence plasmid from an emerging but neglected human pathogen.

Bacterial Proteins↗
PubMed1993

Antimicrobial susceptibility of Pediococcus spp. and genetic basis of macrolide resistance in Pediococcus acidilactici HM3020.

We determined the MICs of 28 antimicrobial agents against 36 clinical strains of Pediococcus spp. (25 P. acidilactici, 9 P. pentosaceus, and 2 P. urinaeequi strains). Penicillin G, imipenem, gentamicin, netilmicin, erythromycin, clindamycin, rifampin, chloramphenicol, daptomycin, and ramoplanin were the most active. All strains of P. acidilactici were susceptible to novobiocin, whereas all isolates of P. pentosaceus were resistant. Novobiocin could therefore be helpful for differentiation of these two closely related species. P. acidilactici HM3020 was inducibly resistant to macrolide, lincosamide, and streptogramin B-type (MLS) antibiotics. Resistance was due to a determinant homologous to ermAM and carried by a nontransferable 46-kb plasmid, pVM20. This plasmid was structurally distinct from two enterococcal MLS resistance plasmids, pIP819 and pAM beta 1. The 34 strains of P. acidilactici and P. pentosaceus were resistant to tetracycline, and total DNA of these strains did not hybridize to probes specific for tetK, tetL, tetM, and tetO.

Anti-Bacterial Agents↗
PubMed1983

R plasmid dihydrofolate reductase with a dimeric subunit structure.

Dihydrofolate reductase specified by plasmid R483 from a trimethoprim-resistant strain of Escherichia coli has been purified 2,000-fold to homogeneity using dye-ligand chromatography, gel filtration, and polyacrylamide gel electrophoresis. The protein migrated as a single band on nondenaturing polyacrylamide gel electrophoresis and had a specific activity of 250 mumol/mg min(-1). The molecular weight was estimated to be 32,000 by gel filtration and 39,000 by Ferguson analysis of polyacrylamide gel electrophoresis. When subjected to electrophoresis in the presence of sodium dodecyl sulfate, the protein migrated as a single 19,000-molecular weight species, a fact that suggests that the native enzyme is a dimer of similar or identical subunits. Antibody specific for R483-encoded dihydrofolate reductase did not cross-react with dihydrofolate reductase encoded by plasmid R67, T4 phage, E. coli RT500, or mouse L1210 leukemia cells. The amino acid sequence of the first 34 NH2-terminal residues suggests that the R483 plasmid dihydrofolate reductase is more closely related to the chromosomal dihydrofolate reductase than is the enzyme coded by plasmid R67.

Amino Acid Sequence↗
PubMed2003

A fluorescence study of the structure and accessibility of plasmid DNA condensed with cationic gene delivery vehicles.

The cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane and dimethyldioctadecylammonium bromide, with or without the helper lipids 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine or cholesterol, and the cationic polymer polyethyleneimine, were compared for their ability to displace fluorescent dyes from DNA. Differences in displacement of the intercalating dyes ethidium bromide and ethidium homodimer correlate with their relative affinities with DNA, with the extent of ethidium homodimer displacement significantly less. Differences in ethidium homodimer and ethidium bromide displacement as a function of the ratio of polycation to DNA and the charge density of the polycation suggest a greater sensitivity of the former to topological changes in condensed DNA. Marked differences in the ability of these cationic delivery systems to displace the minor groove binding dyes 4',6-diamidino-2-phenylindole and Hoechst 33258 upon interaction with DNA are also apparent, with the majority of Hoechst 33258 remaining bound to DNA. Changes in the spectral properties of Hoechst 33258 were further used to characterize polycation-induced changes in solvent accessibility of the DNA minor groove. Taken together, these studies demonstrate differences in the interaction of various cationic lipids and polyethyleneimine in terms of regional displacement of dyes, polycation-induced structural changes in DNA, as well as polycation-mediated changes in solvent accessibility of the minor groove. The relevance of these studies to current models of the structure and assembly of polycation/DNA complexes are discussed.

Bisbenzimidazole↗
PubMed1983

DNA restriction--modification enzymes of phage P1 and plasmid p15B. Subunit functions and structural homologies.

We have purified the type III restriction enzymes EcoP1 and EcoP15 to homogeneity from bacteria that contain the structural genes for the enzymes cloned on small, multicopy plasmids and which overproduce the enzymes. Both of the enzymes contain two different subunits. The molecular weights of the subunits are the same for both enzymes and antibodies prepared against one enzyme cross-react with both subunits of the other. Bacteria containing a plasmid derivative in which a large part of one of the structural genes has been deleted have a restriction- modification+ phenotype and contain only the smaller of the two subunits. This subunit therefore must be the one that both recognizes the specific DNA sequence and methylates it in the modification reaction (the restriction enzyme itself also acts as a modification methylase). We have purified the P1 and P15 modification subunits from these deletion derivatives and have shown that in vitro they have the expected properties: they are sequence-specific modification methylases. In addition, we have demonstrated that strains carrying the full restriction/modification system also contain a pool of free modification subunits that might be responsible for in vivo modification.

Adenosine Triphosphate↗
PubMed2003

The effect of protective agents on the stability of plasmid DNA by the process of spray-drying.

The effect of several protective agents was assessed on the stability of spray-dried plasmid DNA. The spray-drying process had adverse effects on the tertiary structure of plasmid DNA with the protective agents of sucrose, glycine and agarose. With the protection of these noncondensing agents, a band corresponding to the linear form of plasmid DNA was observed in the gel electrophoresis between the supercoiled circular (SC) form and the open circular (OC) form. On the contrary, spray-dried plasmid DNA maintained some degree of structural integrity under the protection of condensing agents. For the protection by neutral condensing polymers, such as polyethylene glycol 1000 and 4000, no linear form between the SC form and the OC form of plasmid DNA was revealed in the gel electrophoresis. Also, excess cationic condensing polymer, polyethyleneimine, had the ability to provide the plasmid DNA with protection from degradation as indicated by the preservation in SC and OC forms of plasmid DNA on the agarose gel electrophoresis. Moreover, DNA topology was unchanged after six-month storage at 4 degrees C by the protection of these neutral and cationic condensing agents. Accordingly, DNA condensation induced by condensing agents may provide a way to minimize damage to plasmid DNA by the process of spray drying.

DNA↗
PubMed1991

Unidirectional theta replication of the structurally stable Enterococcus faecalis plasmid pAM beta 1.

Numerous bacterial replicons remain poorly characterized due to difficulties in localization of the replication origin. We have circumvented this problem in the characterization and fine mapping of the origin of plasmid pAM beta 1 by exploiting the Bacillus subtilis termination signal, terC. In terC-containing derivatives, theta-form molecules with two invariant endpoints accumulate. The endpoints, which correspond to plasmid origin and terC, were mapped with single-nucleotide precision. Analysis of the replication intermediates of wild-type molecules by two-dimensional gel electrophoresis confirmed the location of the plasmid origin. Our results demonstrate that pAM beta 1 replication proceeds unidirectionally by a theta mechanism. This work confirms the use of termination signals to localize origins, suggests that termination in B. subtilis occurs by a mechanism similar to that of Escherichia coli and establishes that in addition to rolling circle replicating plasmids, Gram positive bacteria harbour plasmids which replicate by a theta mechanism.

Base Sequence↗
PubMed2006

Structural analysis of the plasmid pTA1 isolated from the thermoacidophilic archaeon Thermoplasma acidophilum.

Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at pH1.8 and 56 degrees C and has no cell wall. Plasmid pTA1 was found in some strains of the species. We sequenced plasmid pTA1 and analyzed the open reading frames (ORFs). pTA1 was found to be a circular DNA molecule of 15,723 bp. Eighteen ORFs were found; none of the gene products except ORF1 had sequence similarity to known proteins. ORF1 showed similarity to Cdc6, which is involved in genome-replication initiation in Eukarya and Archaea. T. acidophilum has two Cdc6 homologues in the genome. The homologue found in pTA1 is most similar to Tvo3, one of the three Cdc6 homologues found in the genome of Thermoplasma volcanium, among all of the Cdc6 family proteins. The phylogenetic analysis suggested that plasmid pTA1 is possibly originated from the chromosomal DNA of Thermoplasma.

Amino Acid Sequence↗