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The 2.7-kbZymomonas mobilisATCC10988 plasmid pZMO3 contains a coding region (ORF1) indispensable for mobilization. Acis-acting 409-bp sequence between ORF2 (C-terminal) and ORF1 (N-terminal) conferred mobilization activity to pUC19, when the product of ORF1 was providedin trans.In this area, two segments showed homology with previously characterizedoriTregions.
Tn4371is a 55-kb catabolic transposon originally isolated fromRalstonia eutrophaA5 that encodes enzymes catalyzing the complete degradation of biphenyl. Unlike previously described transposons encoding similar genes for aromatic compound degradation. Tn4371carries a phage-like degradation, Tn4371integrase gene and RP4/Ti-like transfer genes. Tn4371transposition involves an excision/integration process...
TheKlebsiella pneumoniae ozenaeKIIIA strain was isolated from the River Rhine soon after a serious mercury pollution episode and was selected for mercury resistance as well as for intergeneric DNA mobilization helper potential. This transfer helper capacity was shown to be related to the presence of a Tn3-like transposable element,Tn5403.Because transposon-mediated fusion was found to be involved...
Bacterial chromosome replication is tightly regulated at the initiation stage to coordinate with mass increase. Together with chromosome partition at cell division, this regulation mechanism ensures the proper number of chromosomes in daughter cells at any growth rate. Therefore, elucidation of this regulation mechanism is important for understanding the bacterial cell cycle. Despite much effort inEscherichia...
Cryptic plasmids pDP1 and pSMB1 from clinical strains ofStreptococcus pneumoniaeisolated 74 years apart were found to be essentially identical in their nucleotide sequence. pDP1, 3161 bp, contains five codirectional ORFs and presents all the general features of plasmids replicating by the rolling circle mechanism. Therepgene, 963 bp, is highly homologous to therepgene of other streptococcal plasmids...
A series of cloning vectors with conditional, temperature-sensitive replication that are selectable with ampicillin, chloramphenicol, and kanamycin has been constructed. These vectors are derivatives of a pSC101 mutant that can replicate only at low temperatures. The cloning vectors carry a number of unique restriction sites and provide for screening of recombinant plasmids by α complementation. These...
We have isolated spontaneous rifampicin-resistant mutants fromEscherichia colithat showed allele-specific suppression of the copy-number phenotype of ColE1 high-copy-number mutantsin vivo.The key step in the regulatory circuitry of the initiation of ColE1 DNA replication is the formation of the persistent hybrid between the primer RNA and the DNA template around the replication origin. Three host-encoded...
The higher copy number of pUC19, compared to its parent plasmid pBR322, is known to be due to deletion ofrop,also known asrom,and to anorimutation that impedes RNAI:RNAII interaction. pUC19, unlike pBR322, fails to transformE. coli rhomutantrho026cells. Here we identify two features of pUC19 that contribute to this transformation defect. (1) The pUCorimutation is involved because replacing the pUCoriwith...
Amino acid starvation of bacterial cells leads to expression of the stringent (in wild-type strains) or relaxed (inrelAmutants) response (also called the stringent or relaxed control, respectively). The stringent control is a pleiotropic response which changes drastically almost the entire cell physiology. Although starvation is a rule rather than an exception in natural environments of bacteria,...
TheEnterococcus faecalisconjugative cytolysin plasmid pAD1 encodes a specific aggregation (clumping) response to the peptide sex pheromone cAD1 secreted by plasmid-free strains. Here it is shown that, in the absence of cAD1, exposure ofE. faecaliscells harboring pAD1 to subinhibitory concentrations of chloramphenicol, erythromycin, or tetracycline also results in an aggregation response that appears...
Theimldeterminant of IncM plasmid R446 was described initially as a short fragment of DNA causing (when cloned in multicopy plasmids) insensitivity to pilus-dependent bacteriophage lysis in bacteria harboring coresident IncM plasmids. We have performed a computational analysis of theimldeterminant of IncM plasmid R446 and found that a potential polypeptide (Orf4) shows similarity to the H-NS family...
Dictyosteliumplasmids Dgp1 and Dfp1, two members of the Ddp2 plasmid family, are 86% identical in nucleotide sequence. These small (4481 and 5015 bp), high copy number, nuclear plasmids carry both a gene homologous to the Ddp2repgene and a long 0.47- to 0.48-kb inverted repeat region. Their Rep proteins are 82.8% identical in amino acid sequence and carry all 10 of the conserved peptide sequence motifs...
A new cryptic plasmid, pRS1, from anOenococcus oenistrain isolated from Spanish wines is reported. Nucleotide sequence analysis (2523 bp) revealed the presence of three major open reading frames (ORFs) whose nucleotide sequence and encoded proteins exhibit high homology with those of pOg32, a previously described plasmid ofO. oeni.Common features in other plasmids fromO. oeni(i.e., pLo13 and pOg32)...
TheStaphylococcus aureustransposon Tn4001and derivatives thereof have been transformed successfully in several mycoplasma species. In order to expand the versatility of Tn4001for other genetic manipulations and for use in mycoplasma species resistant to gentamicin (Gm), chloramphenicol acetyltransferase (Cat) fromS. aureuswas evaluated as a selectable marker. Thecatgene was cloned in both orientations...
Escherichia coli–mycobacteria shuttle vectors, derived from pAL5000 (a mycobacterial plasmid) and pUC19, were frequently found to undergo structural alterations due to transposition of IS1096,aMycobacterium smegmatistransposable element, at a cluster of sites located within a small region of 60 bp, immediately upstream of a kanamycin resistance gene present in these vectors. The structural alterations...
The copy number per cell mass of plasmid pBR322 and arom − derivative was measured as a function of generation time. In fast growing cells the copy number per cell mass was virtually identical forrom + androm − derivatives. However, the copy number of pBR322 only increased 3- to 4-fold from a 20- to 80-min generation time, whereas the copy number of therom − derivative...
The 5846-bp circular plasmid pHPS1 ofHelicobacter pyloriSydney strain, SS1, was cloned, sequenced, and structurally characterized. The SS1 strain is widely used in animal studies ofH. pyloriinfection. The sequence of pHPS1 revealed three open reading frames (ORFs), all of which are transcribed. Two ORFs encode putative plasmid replication proteins, RepA and RepB, similar to replicases resident on...
Ectopic expression of genes from recombinant plasmids is commonly used to study gene function. In Dictyostelium, three drug resistance cassettes are commonly used as selectable markers in vectors. We report here a comparative study of the expression of green fluorescent protein (GFP) gene from vectors containing each of the drug-resistant cassettes. The expression was highest in cells transformed...
The two-hybrid system was used to show that the Rep proteins from three members of the Dictyostelium discoideum Ddp2 plasmid family, Ddp2, Ddp5, and Ddp6, form homomultimers but not heteromultimers when expressed in yeast cells. The results with deletion mutations suggest that multiple regions of the Rep proteins are involved in the multimerization. Electrophoretic mobility shift assays with heterologously...
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