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At least 685 records · Page 38Linked to original sources
PubMed2001

Characterization of In53, a class 1 plasmid- and composite transposon-located integron of Escherichia coli which carries an unusual array of gene cassettes.

Further characterization of the genetic environment of the gene encoding the Escherichia coli extended-spectrum beta-lactamase, bla(VEB-1), revealed the presence of a plasmid-located class 1 integron, In53, which carried eight functional resistance gene cassettes in addition to bla(VEB-1). While the aadB and the arr-2 gene cassettes were identical to those previously described, the remaining cassettes were novel: (i) a novel nonenzymatic chloramphenicol resistance gene of the cmlA family, (ii) a qac allele encoding a member of the small multidrug resistance family of proteins, (iii) a cassette, aacA1b/orfG, which encodes a novel 6'-N-acetyltransferase, and (iv) a fused gene cassette, oxa10/aadA1, which is made of two cassettes previously described as single cassettes. In addition, oxa10 and aadA1 genes were expressed from their own promoter sequence present upstream of the oxa10 cassette. arr-2 coded for a protein that shared 54% amino acid identity with the rifampin ADP-ribosylating transferase encoded by the arr-1 gene from Mycobacterium smegmatis DSM43756. While in M. smegmatis, the main inactivated compound was 23-ribosyl-rifampin, the inactivated antibiotic recovered from E. coli culture was 23-O-ADP-ribosyl-rifampin. The integrase gene of In53 was interrupted by an IS26 insertion sequence, which was also present in the 3' conserved segment. Thus, In53 is a truncated integron located on a composite transposon, named Tn2000, bounded by two IS26 elements in opposite orientations. Target site duplication at both ends of the transposon indicated that the integron likely was inserted into the plasmid through a transpositional process. This is the first description of an integron located on a composite transposon.

Amino Acid Sequence↗
PubMed2006

Characterization of pFMBL1, a small cryptic plasmid isolated from Leuconostoc mesenteroides SY2.

A 4661bp cryptic plasmid, pFMBL1, was isolated from Leuconostoc mesenteroides SY2, an isolate from Kimchi, and characterized. Nucleotide sequence analysis revealed two open reading frames, orf1 and orf2. orf2 was 453bp in size and its translation product had 58% identity with a putative protein possibly involved in the replication of pTXL1, a cryptic plasmid from L. mesenteroides ssp. mesenteroides Y110. RNA transcript from orf2 was detected but not from orf1 or intergenic region. Minimum 3.5kb fragment encompassing orf1 and orf2 was required for the replication of pFMBL1 and employed for the construction of Escherichia coli-Leuconostoc shuttle vector, pSJ33E. L. mesenteroides SY1 (another Kimchi isolate), Leuconostoc ssp., and Lactobacillus brevis were successfully transformed with pSJ33E, and the transformation efficiencies were ranged between 1.1x10(1) and 4x10(5)transformants/microg DNA. No single-stranded DNA intermediate was detected from L. mesenteroides SY1 cells harboring pSJ33E, indicating that pFMBL1 probably replicated via theta-type mechanism. pSJ33E was stably maintained in L. mesenteroides SY1 in the absence of erythromycin (Em, 5 microg/ml) and after 1 month of daily subculturing in MRS broth without selective pressure, three percent of cells still retained pSJ33E.

Base Pairing↗
PubMed1999

Isolation and partial characterization of Bac201: a plasmid-associated bacteriocin-like inhibitory substance from Staphylococcus aureus AB201.

Staphylococcus aureus AB201, a clinical isolate from wound pus, produced a bacteriocin-like inhibitory substance termed as Bac201 that exhibited a broad-spectrum activity against both gram-positive as well as gram-negative bacteria. Among gram-negative bacteria it was active against Neisseria meningitidis and Acinetobacter calcoaceticus both being gram-negative cocci. Inhibition due to the effect of organic acids, hydrogen peroxide, or bacteriophages was excluded. This inhibitory substance could not be induced or eluted. It was partially purified to 80% saturation by ammonium sulfate precipitation resulting in maximum specific activity of 829 AU/mg (25-fold increase). Proteolytic enzymes rapidly inactivated the antagonistic activity of the partially purified material, whereas glycolytic and lipolytic enzymes had no effect. It remained stable in the presence of mild organic solvents. It could be stored at -20 degrees C without loss of activity, stable at 60 degrees C and 80 degrees C for 30 min, 100 degrees C for 20 min and autoclaving temperature (121 degrees C for 15 min), and exhibited activity within a wide range of pH (2.5-10). Bac201 had an estimated M(r) of 41kDa, as indicated by activity detection after SDS-PAGE. Temperature-mediated (44 degrees C) plasmid curing studies suggested linkage of bacteriocin production to a 4.8 MDa plasmid. The Bac201 was bactericidal rather than bacteriolytic.

Bacteriocins↗
PubMed1993

Characterization of the replicon from plasmid pAC1 from Acetobacter pasteurianus.

A panel of recombinant plasmids pACK5 and pACT7 was prepared by introducing kanamycin and tetracycline resistance into the partially split plasmid pAC1 which contained replicon isolated from Acetobacter pasteurianus. The replicon in plasmid pAC1 is compatible with the ColE1 replicon. Compared to pBR322, the plasmid had more than 30 copies per chromosome in Escherichia coli cells. Plasmids were transformed into E. coli DH1, Acetobacter pasteurianus 3614, Acetobacter aceti 3620, Shigella, Citrobacter, and Brevibacterium flavum cells, and the stability of plasmid DNA was tested after cultivation in nonselective conditions.

Acetobacter↗
PubMed1995

Characterization of a 3.5-kbp plasmid from Helicobacter pylori.

A 3.5-kbp plasmid (pHPM180) was isolated from Helicobacter pylori (HPM180) and the DNA sequence was determined. Two open reading frames (ORF1 and ORF2) were identified which could encode polypeptides of 54,517 and 27,629 Da, respectively. Ribosome binding and promoter consensus sequences were identified, as well as two 232-bp direct repeats and four 22-bp direct repeats. DNA sequence homology was found between pHPM180 ORF1 and a 684-base pair HindIII fragment from a 5.6-kbp H. pylori plasmid. ORF1 showed amino acid homology with six replication proteins from bacterial plasmids with theta-type replicons. Additional sequence identity was found between pHPM180 noncoding DNA and a segment of H. pylori pHPK255, a plasmid that replicates via a rolling circle type mechanism. A ribonuclease protection assay determined that ORF1 was transcribed in H. pylori HPM180.

Amino Acid Sequence↗
PubMed2000

Characterization of the IncW cryptic plasmid pXV2 from Xanthomonas campestris pv. vesicatoria.

The gram-negative plant pathogen Xanthomonas campestris pv. vesicatoria strain Xv2 harbors an indigenous, cryptic plasmid pXV2 of 14.6 kb. This plasmid can only be maintained in Xanthomonas and is incapable of self-transmission. However, incompatibility testing classified it in IncW, a group containing the smallest number of naturally occurring, broad-host-range, conjugative plasmids. A pXV2 derivative containing only a 5.5-kb PstI fragment is stably maintained. Deletion of a 3.0-kb region from the PstI fragment causes a loss of plasmid stability. Nucleotide sequencing of the 2. 1-kb region essential for autonomous replication revealed a repA gene and a downstream noncoding region containing four iterons, two 17- and two 19-nt direct repeats, and an AT-rich region lying between the two sets of iterons. The sequence of the deduced RepA and the iterons shows homology to the RepA (39% identity) and the iterons, respectively, of the IncW plasmid pSa. Maxicell expression of the repA gene produced a protein of 35 kDa, a size similar to that deduced from the nucleotide sequence. Trans-complementation test confirmed that the repA gene and the iterons are indeed the essential elements for pXV2 replication.

Amino Acid Sequence↗
PubMed2000

Characterization of a novel plasmid-encoded HsdS subunit, S.LlaW12I, from Lactococcus lactis W12.

A novel type I restriction-modification specificity subunit, S. LlaW12I, has been identified on the naturally occurring 8.0-kb plasmid pAW122 in the lactic acid bacterium Lactococcus lactis subsp. cremoris W12. Presence of the HsdS protein together with a complete type I restriction-modification system conferred increased phage restriction to the host, indicating exchange of specificity subunits. Sequence analysis showed that the S.LlaW12I subunit is most probably of type IC. Presumably, the hsdS gene is organized together with the repB gene on one transcriptional unit.

Amino Acid Sequence↗
PubMed2001

Molecular characterization of the klebicin B plasmid of Klebsiella pneumoniae.

The nucleotide sequence of a bacteriocin-encoding plasmid isolated from Klebsiella pneumoniae (pKlebB-K17/80) has been determined. The encoded klebicin B protein is similar in sequence to the DNase pyocins and colicins, suggesting that klebicin B functions as a nonspecific endonuclease. The klebicin gene cluster, as well as the plasmid backbone, is a chimera, with regions similar to those of pore-former colicins, nuclease pyocins and colicins as well as noncolicinogenic plasmids. Similarities between pKlebB plasmid maintenance functions and those of the colicin E1 plasmid suggest that pKlebB is a member of the ColE1 plasmid replication family.

Amino Acid Sequence↗
PubMed2001

Nucleotide sequence, structural organization, and functional characterization of the small recombinant plasmid pOM1 that is specific for Francisella tularensis.

pOM1 is a recombinant 4442-bp plasmid that includes the replicon of the Francisella novicida-like strain F6168 cryptic plasmid pFNL10 and the tetracycline resistance gene (tetC) of plasmid pBR328. pOM1 can stably replicate and is maintained in Francisella tularensis biovars tularensis, palaearctica, and palaearctica var. japonica. The replicon of pOM1 includes the ori region and the repA gene. The ori region, located upstream of the repA gene includes two sets of 31- and 13-bp direct repeats (DR), with AT-rich regions preceding each of the DRs. Two putative promoters of the repA gene were found connected with the DR regions. A 40-kDa protein was encoded by the repA gene and found essential for replication. Expression of the tetC gene is regulated by an Escherichia coli sigma(70)-like promoter and is dependent on the F. tularensis strain and its environment.

Bacterial Proteins↗
PubMed2001

Complete nucleotide sequence and characterization of pUA140, a cryptic plasmid from Streptococcus mutans.

Approximately 5% of strains of Streptococcus mutans contain plasmid DNA. Strain UA140 harbors a 5.6-kb cryptic plasmid, pUA140, with an overall G+C content of 32.7%. Five open reading frames (ORF), encoding peptides of larger than 100 amino acid residues, were initially designated as ORF1 to ORF5. These five ORFs were located on the same strand of pUA140. ORF1 (258 amino acids) resembled a replication protein, Rep. Upstream of the putative Rep gene, a double-stranded origin for plasmid replication that showed strong similarity to those of a number of plasmids in the pT181 family was identified. Further upstream was a region constituting the single-stranded origin of replication. A single-stranded DNA intermediate was detected during plasmid replication. Taken together, these results suggest that pUA140 replicated by the rolling circle replication mechanism but exhibited several characteristics that differ from those of other members of the pT181 plasmid family.

Amino Acid Motifs↗
PubMed1995

Site-specific integration of the phage phi CTX genome into the Pseudomonas aeruginosa chromosome: characterization of the functional integrase gene located close to and upstream of attP.

The site-specific integration of the phage phi CTX genome, which carries the gene for a pore-forming cytotoxin, into the Pseudomonas aeruginosa chromosome was analysed. The 1,167 bp integrase gene, int, located immediately upstream of the attachment site, attP, was characterized using plasmid constructs, harbouring the integration functions, and serving as an integration probe in both P. aeruginosa and Escherichia coli. The attP plasmids p1000/p400 in the presence of the int plasmid pIBH and attP-int plasmids pINT/pINTS can be stably integrated into the P. aeruginosa chromosome. Successful recombination between the attP plasmid p1000 and the attB plasmid p5.1, in the presence of the int plasmid pIBH in E. coli HB101 showed that the int gene is active in trans in E. coli. The int gene product was detected as a 43 kDa protein in E. coli maxicells harbouring pINT. Proposed integration arm regions downstream of attP are not necessary for the integration process. pINT and phage phi CTX could be integrated together into P. aeruginosa chromosomal DNA, yielding double integrates.

Attachment Sites, Microbiological↗
PubMed1988

Isolation and characterization of a linear DNA plasmid from Streptomyces clavuligerus.

A linear DNA plasmid (pSCL) has been isolated from Streptomyces clavuligerus by a method employing high concentrations of protease. Rate-zonal sedimentation on sucrose gradients was used to purify the plasmid. The plasmid is 12 kb in length and appears to be linked to protein at its 5' termini. A restriction endonuclease map of the plasmid for ten enzymes has been determined. Evidence for terminally repeated sequences is provided by cross-hybridization analysis.

DNA Restriction Enzymes↗
PubMed1976

Characterization of the antibiotic resistance plasmid ERL1 from Streptococcus pyogenes.

The streptococcal plasmid ERL1 determining inducible resistance to erythromycin, lincomycin, and staphylomycin S was isolated by dye-buoyant density centrifugation and shown to have a molecular weight of about 17.5 Mdal, as revealed by sedimentation through neutral sucrose gradients. In SM60 cells entering the stationary phase its covalently closed circular form was present to the extent of 5 copies per chromosomal genome equivalent, ERL1 was subject to the DNA restriction and modification mechanism discovered in strain 56188. It did not appear to exercise restriction of phage DNA but mediated a partial release of the restricted growth of A25.

DNA, Bacterial↗
PubMed1988

Construction of full-length cDNA copies of viral double-stranded RNA.

A method is described for the construction of full-length cDNA clones of dsRNAs. All dsRNA viruses have a capsid-associated transcriptase that is responsible for synthesis of the plus strand that is then extruded from viral particles. We have used in vitro transcripts synthesized by the segmented Saccharomyces cerevisiae virus (ScV) as templates for first-strand cDNA synthesis. Synthesis was primed by a 33-base synthetic oligonucleotide. This contained 27 nucleotides complementary to the 3' end of the plus strand from one ScV viral dsRNA segment (S14), and 6 additional nucleotides encoding an XbaI restriction site at the 5' end. The second cDNA strand was synthesized using a similar XbaI linker-synthetic oligonucleotide and the ds cDNA was cloned by standard ligation techniques. All four cDNA plasmid isolates characterized by sequence analysis contained the complete 5' end sequence of S14. Two of these were complete at the 3' end, and one lacked a single base here. Of these four clones, one also retained the XbaI sites at either end. Preparing full-length cDNA clones with unique restriction-site linkers by the use of synthetic oligonucleotides allows for easier screening for complete cDNA clones if neither the vector nor the cDNA has the chosen restriction site. It also provides for easier sequence analysis and manipulation of the genome for later studies, such as cloning into expression vectors. This method is more efficient than any previously described for production of full-sized cDNA clones.

Cloning, Molecular↗
PubMed1996

Characterization of the replicon of plasmid pSW500 of Erwinia stewartii.

A 1.6-kb DNA region required for the replication of pSW500 from Erwinia stewartii SW2 has been identified. DNA sequencing analysis revealed that this DNA fragment consists of a DnaA box, seven 16-bp direct repeats, and a 1005-bp open reading frame. The seven direct repeats have been demonstrated to mediate the incompatibility function of the plasmid. Primer extension analysis showed that the 1005-bp ORF is transcribed in vivo and the +1 site of the transcript is located 113 bp upstream from the translation initiation codon of the ORF. Complementation studies showed that this ORF is required for the replication of the plasmid and may encode a replication protein, RepA. Gene fusion studies revealed that the expression of repA is autoregulated by RepA. We also found that the pSW500 replicon has a copy number of approximately two and that the plasmid is stably maintained in Escherichia coli, thus demonstrating that the replicon contains all the elements required for copy number control and plasmid stability in E. coli. Curing of pSW500 from E. stewartii SW2 revealed that loss of pSW500 did not have any obvious effect on morphology or physiology of the cells, suggesting that pSW500 does not encode a function that is indispensable for the survival of the organism.

Amino Acid Sequence↗
PubMed2006

Identification and characterization of IS1 transposition in plasmid amplification mutants of E. coli clones producing DNA vaccines.

Merck Research Laboratories has developed a highly productive Escherichia coli fermentation process to produce plasmid DNA for use as vaccines. The process consists of a fed-batch fermentation in a chemically defined medium. Initiation of the feed stream precedes a growth-limited phase in which plasmid DNA is amplified. The fermentation is only maximally productive for a small fraction of E. coli transformants designated as high-producers, while the predominant low-producer population does not amplify plasmid DNA. In experiments undertaken to probe this phenomenon, transposition of the 768-bp E. coli insertion sequence IS1 into an HIV DNA vaccine vector was observed in several low-producer clones. IS1 was found to insert in or near the neomycin resistance gene in nearly a dozen unique sites from within a single population of plasmid molecules. The fraction of IS1-containing plasmids within several clones was determined by quantitative polymerase chain reaction and was found to increase with increasing cultivation time in the chemically defined medium. Because transposition into an antibiotic-resistance gene is unlikely to affect plasmid amplification, the genomes of high- and low-producers of three different HIV DNA vaccine vectors were subsequently profiled by restriction fragment length polymorphism analysis. In all three cases, IS1 insertional mutations were found in the genomes of the predominant low-producers, while the genomes of the high-producers were indistinguishable from untransformed cells. The insertions reside on similarly sized fragments for two of the low-producer clones, and the fragment size is smaller for the third clone. The third clone also produces much less plasmid DNA than a typical low-producer. The results suggest the presence of an IS1 insertional mutation that affects plasmid replication and amplification, possibly in a position-dependent manner.

DNA Fingerprinting↗