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At least 199 records · Page 11Linked to original sources
PubMed1982

pIJ101, a multi-copy broad host-range Streptomyces plasmid: functional analysis and development of DNA cloning vectors.

Streptomyces lividans ISP 5434 contains four small high copy number plasmids: pIJ101 (8.9 kb), pIJ102 (4.0 kb), pIJ103 (3.9 kb) and pIJ104 (4.9 kb). The three smaller species appear to be naturally occurring deletion variants of pIJ101. pIJ101 and its in vivo and in vitro derivatives were studied after transformation into S. lividans 66. pIJ101 was found to be self-transmissible by conjugation, to elicit "lethal zygosis" and to promote chromosomal recombination at high frequency in both S. lividans 66 and S. coelicolor A3(2). A restriction endonuclease cleavage map of pIJ101 was constructed for 11 endonucleases; sites for five others were lacking. Many variants of pIJ101 were constructed in vitro by inserting DNA fragments determining resistance to neomycin, thiostrepton or viomycin, and having BamHI termini, into MboI or BclI sites on the plasmid, sometimes with deletion of segments of plasmid DNA. The physical maps of these plasmids were related to their phenotypes in respect of lethal zygosis and transfer properties. In vivo recombination tests between pairs of variant plasmids were also done. These physical and genetic studies indicated that determinants of conjugal transfer occupy less than 2.1 kb of the plasmid. A second segment is required for spread of the plasmid within a plasmid-free culture to produce the normal lethal zygosis phenotype: insertion of foreign DNA in this region caused a marked reduction in the diameter of lethal zygosis zones. The minimum replicon was deduced to be 2.1 kb or less in size; adjacent to this region is a 0.5 kb segment which may be required for stable inheritance of the plasmid. The copy number of several derivatives of pIJ101 in S. lividans 66 was between 40 and 300 per chromosome and appeared to vary with the age or physiological state of the culture. pIJ101 derivatives have a wide host range within the genus Streptomyces: 13 out of 18 strains, of diverse species, were successfully transformed. Knowledge of dispensable DNA segments and the availability of restriction sites for the insertion of DNA, deduced from the properties of plasmids carrying the E. coli plasmid pACYC184 introduced at various sites, was used in the construction of several derivatives of pIJ101 suitable as DNA cloning vectors. These were mostly designed to be non-conjugative and to carry pairs of resistance genes for selection. They include a bifunctional shuttle vector for E. coli and Streptomyces; a Streptomyces viomycin resistance gene of this plasmid is expressed in both hosts.

Chromosome Deletion↗
PubMed1984

A mathematical model for lambda dv plasmid replication: analysis of copy number mutants.

A mathematical model based on the molecular control mechanisms for lambda dv plasmid replication in a single Escherichia coli cell has been applied to simulate replication of mutant lambda dv plasmids. Model simulations of changes in repressor level and copy number resulting from mutations in the promoter-operator PROR region are consistent with experimental data. Calculated effects on lambda dv plasmid copy number of oligomer formation and of alternations in termination efficiency at tR1 also agree with experiment. The model has been employed to simulate the influence of cro mutants and of cro and tR1 double mutants on copy number and stable maintenance of lambda dv plasmid copy number. The genetic structure included in formulation of the replicon model provides a framework for relating changes in specific genetic loci on the plasmid with resulting alterations in host-plasmid system function.

Base Sequence↗
PubMed1990

Mercury resistance determined by a self-transmissible plasmid in Bacillus cereus 5.

Inducible mercuric reductase activity in Bacillus cereus 5 was plasmid-encoded. Plasmid analysis revealed three plasmids with molecular masses of 2.6, 5.2 and 130 MDa. A mating system permitted transfer of the resistance determinant among strains of B. cereus and B. thuringiensis. Transfer of mercury resistance from B. cereus 5 to B. cereus 569 and B. thuringiensis occurred during mixed culture incubation on agar surfaces. The 130-MDa plasmid (pGB130) was responsible for transfer; frequencies ranged from 10(-5) to 10(-4). B. cereus 569 transconjugants inheriting pGB130 were also effective donors. High transfer frequencies and the finding that cell-free filtrates of donor cultures were ineffective in mediating transfer suggested mercury-resistance transfer was not phage-mediated. Transfer was also insensitive to DNase activity. Further evidence that pGB130 DNA carried the mercury-resistance determinant was transformation of B. cereus 569 by electroporation with pGB130 DNA isolated from B. cereus 5 and a mercury-resistant B. cereus 569 transconjugant. Mercury-resistant transconjugants and transformants exhibited mercuric reductase activity. Plasmid pGB130 also conferred resistance to phenylmercuric acetate.

Bacillus cereus↗
PubMed1983

Colonization factor antigens I and II and type 1 somatic pili in enterotoxigenic Escherichia coli: relation to enterotoxin type.

Enterotoxigenic Escherichia coli (ETEC) isolates from 36 persons with acute traveler's diarrhea from whom no other pathogens were recovered were tested (after no more than three subcultures) for the presence of colonization factor antigens I and II (CFA/I and CFA/II) and type 1 somatic pili. CFA/I or CFA/II was identified in 7 of 10 strains with heat-labile and heat-stable enterotoxins (LT+/ST+), but in only 2 of 12 LT-/ST+ (P less than 0.05) and 0 of 14 LT+/ST- (P less than 0.02) strains. CFA pili were not found among 74 non-enterotoxigenic E. coli strains. Type 1 somatic pili were demonstrable in 42% of the 36 ETEC and in 49% of the 74 non-enterotoxigenic E. coli isolates. The nine ETEC isolates bearing a CFA were serially subcultured on 10 consecutive days and retested for CFA and toxin. After five subcultures only one strain had lost a CFA, but after 10 passages three strains were negative: two lost CFA/I and one lost CFA/II. The strain that lost CFA/II became negative for both LT and ST as well and was found to lack a 48- and a 60-megadalton plasmid. The two strains that lost CFA/I also became negative for ST, but plasmid analysis revealed no plasmid loss. Disappearance of the CFA/I phenotype without loss of a plasmid can be explained by phase variation, as exhibited by type 1 somatic pili, or by rearrangement of base sequences in the CFA/I plasmid genome. If purified pili vaccines are to provide broad-spectrum protection against ETEC diarrhea, the search must be intensified to identify the antigens responsible for adhesion to intestinal mucosa in the many ETEC strains that lack CFA/I and CFA/II.

Antigens, Bacterial↗
PubMed1985

Linkage of genes for heat-stable enterotoxin, drug resistance, K99 antigen, and colicin in bovine and porcine strains of enterotoxigenic Escherichia coli.

Linkages among genes for production of Escherichia coli heat-stable enterotoxin (ST), drug resistance, K99 antigen, and colicin were investigated in 2 bovine and 3 porcine strains of enterotoxigenic E coli. In conjugation experiments, all 5 isolates transferred enterotoxigenicity and the ability to produce K99; 4 of the 5 isolates also transferred antibiotic resistance markers, and 3 colicinogenic strains transmitted the ability to produce colicin. In 2 of the 3 colicin-producing strains, the genes for colicin were located along with those for K99 and ST on a single plasmid. One of these 2 strains transferred the genes for tetracycline resistance and production of both mouse-active ST (STa) and mouse-inactive ST, whereas the other transmitted the gene(s) for STa only. Transformation studies with the 3rd strain revealed that the K99 determinant resided on a 22-megadalton (Mdal)R plasmid, and that STa and colicin production were on a 65-Mdal plasmid. Analysis of the plasmids from the transformation experiments revealed that the larger plasmid was conjugative and the smaller plasmid was nonconjugative; stable cointegrate formation occurred between these 2 plasmids. Genetic information coding for the production of STa and K99 were also present on a single plasmid of approximately 80 Mdal in both noncolicinogenic strains.

Animals↗
PubMed2004

Staphylococcus aureus multiresistance plasmid pSK41: analysis of the replication region, initiator protein binding and antisense RNA regulation.

The vast majority of large staphylococcal plasmids characterized to date appear to possess an evolutionarily common replication system, which has clearly had a major impact on the evolution of antimicrobial resistant staphylococci worldwide. Related systems have also been found in plasmids from other Gram-positive genera, including enterococci, streptococci and bacilli. The 46.4 kb plasmid pSK41 is the prototype of a family of conjugative staphylococcal multiresistance plasmids. The replication region of pSK41 encodes a protein product, Rep, which was shown to be essential for replication; mutations that truncated Rep could be complemented in trans. Rep was found to bind in vitro to four tandem repeat sequences located centrally within the rep coding region. An A + T-rich inverted repeat sequence upstream of rep was required for efficient replication, whereas no sequences downstream of rep were necessary. An antisense countertranscript, RNAI, encoded upstream of rep was identified and transcriptional start points for both RNAI and the rep-mRNA were defined.

Amino Acid Sequence↗
PubMed1986

Characterization of the ColE9-J plasmid and analysis of its genetic organization.

We have determined the restriction and functional map of the ColE9-J plasmid. By sub-cloning and transposon mutagenesis we have shown that the ColE9imm gene and the ColE5imm gene present on the ColE9-J plasmid are located on separate EcoRI fragments. Using an expression vector we have demonstrated the presence of two lys genes on the ColE9-J plasmid, both of which are dependent upon the colicin E9 structural gene promoter. Promoter mapping studies imply that the colicin E9 structural gene and the ColE5imm gene are transcribed in the same direction, but that the ColE9imm gene is transcribed in the opposite orientation.

Bacteriocin Plasmids↗
PubMed1985

Analysis of plasmid deletional instability in Bacillus subtilis.

Using a model system, we have studied deletion formation in Bacillus subtilis. When the staphylococcal plasmids pSA2100 (7.1 kilobases) and pUB110 (4.5 kilobases) were ligated to one another at their unique XbaI sites and transformed into either rec+ or recE4 strains of B. subtilis, an intramolecular recombination event usually occurred. Two plasmids, one of 2.6 kilobases and the other of 9.0 kilobases, were consistently isolated and shown by restriction enzyme analysis to be derived by recombination occurring in the pSA2100-pUB110 cointegrate. Analysis of the sequence of the junctions of the recombinant plasmids and of the crossover regions of the parental plasmids suggested that a reciprocal, conservative, intramolecular recombination event had occurred between short 18-base-pair homologous sequences that were oriented as direct repeats and bounded by regions of dyad symmetry. Evidence is presented that the above illegitimate recombination event is biased to occur intramolecularly and that randomly chosen direct repeats of either 22 or 29 base pairs are not sufficient to support recombination. The recombination event occurs in recA1, recB2, recD3, recE5, recL16, recM13, polA59, polA13, uvr-22, uvr-13, and stb mutants of B. subtilis and does not require that the competent state be established.

Bacillus subtilis↗
PubMed1988

Plasmid profile analysis of a salmonellosis outbreak and identification of a restriction and modification system.

After an outbreak of salmonellosis in humans caused by Salmonella typhimurium bacteriophage type 135, 62 isolates from human, animal, and water sources were retained for further analysis. Most of the isolates (92%) could be placed in one of five plasmid pattern groups, with a majority containing a common 60-kilobase plasmid and a smaller 3.8-kilobase-pair plasmid. This small plasmid, pIMVS1, was labeled with [32P]phosphate and used as a probe in subsequent colony and Southern hybridization studies. We concluded that pIMVS1 from isolates obtained from humans was genetically different from plasmids of a similar size found in isolates from chickens. Studies to characterize pIMVS1 were undertaken to determine if it codes for known virulence factors. It did not appear to be associated with the formation of attachment pili or major outer membrane proteins. By using transposon mutagenesis techniques, Tn3(Apr) was inserted into pIMVS1, and the existence of a restriction and modification system was deduced.

Animals↗
PubMed2000

Nucleotide sequence and analysis of plasmid pMD136 from Pediococcus pentosaceus FBB61 (ATCC43200) involved in pediocin A production.

The complete sequence of the 19515-bp plasmid pMD136 from Pediococcus pentosaceus FBB61 (ATCC43200) has been determined. This plasmid is involved in Pediocin A production, a bacteriocin active against a wide range of gram-positive bacteria. It appears to replicate via a theta mechanism, with structures closely related to those of many lactococcal plasmids. Genes homologous to mobilization functions are also present, which are similar in sequence and arrangement to mobA, mobB, and mobC of some staphylococcal plasmids, although the last one contains a deletion in its central part. The region involved in bacteriocin activity has been limited to a 9.4-kb fragment, containing 10 open reading frames organized in a single operon. Since Pediocin A has a molecular weight of about 80 kDa (Piva and Headon, Microbiology, 140, 697-702, 1994), and a gene long enough to encode it is not present in pMD136, it is proposed that genes residing on the plasmid are responsible for the regulation of bacteriocinogenic activity. Gene arrangement and sequence homologies suggest the presence of a two-component-like regulatory mechanism.

Amino Acid Sequence↗
PubMed1978

Restriction enzyme analysis of plasmids from Haemophilus influenzae.

Examination of Haemophilus influenzae strains isolated in New Orleans revealed ampicillin-resistant strains with plasmids of size classes not previously detected in North America. The molecular weight of plasmids in five ampicillin-resistant strains ranged from 0.8 x 10(6) daltons (0.8 Mdal) to 36 Mdal. The molecular weights of the plasmids were determined by sucrose gradient centrifugation, electron microscopy, and agarose gel electrophoresis. Plasmids of the previously detected 30-Mdal size class were found in three of the five ampicillin-resistant strains. Restriction enzyme analysis is consistent with a close relationship between these three 30-Mdal plasmids. Of the two remaining ampicillin-resistant strains, one contained a single plasmid of 36 Mdal and the other contained two plasmids of 0.8 and 2.3 Mdal.

Centrifugation, Density Gradient↗
PubMed1996

The FinOP repressor system of plasmid R1: analysis of the antisense RNA control of traJ expression and conjugative DNA transfer.

A key determinant of the frequency of IncF plasmid-mediated DNA transfer between enterobacterial cells is the FinOP system. traJ, a positive regulator of the transfer (tra) genes is controlled at the post-transcriptional level by two negative elements, finP and finO. FinP is a plasmid-specific antisense RNA, whereas finO encodes a proteic co-repressor which is not plasmid specific but exchangeable among F-like plasmids. We designed a traJ-lacZ test system that allowed us to monitor the effects of FinP and various FinP mutants on traJ expression. Furthermore, the introduction of finO into the test system enabled us to assess the function of FinO in the interaction of FinP with its target, the traJ mRNA. In this test system, FinP, expressed from a single-copy plasmid, in the absence of FinO, repressed traJ expression six-fold. When expressed from a pBR322-derived multicopy plasmid FinP repressed traJ expression approx. 2000-fold. This result unambiguously demonstrated that FinP is sufficient to repress traJ expression in a gene dosage-dependent manner. Mutations of finP creating base exchanges either in loop I or loop II of the two stem-loop structures of the antisense RNA led to a dramatic decrease in the repressor activity. In a combined loop I-loop II mutation the repressor activity was almost completely lost, supporting the model that the first critical interaction between the two RNA molecules occurs via 'kissing' of both loops of the RNAs. Addition of finO to the test system enhanced the repression of traJ expression by FinP by up to two orders of magnitude. This effect of FinO on FinP activity in vivo might indicate that FinO, in addition to its function as an RNA stabilizer, promotes complex formation between the target mRNA and the antisense RNA. Such a function of FinO has recently been shown to exist in vitro (van Biesen and Frost (1994) Mol Microbiol 14: 427-436).

Bacterial Outer Membrane Proteins↗
PubMed2000

Molecular identification of Acetobacter isolates from submerged vinegar production, sequence analysis of plasmid pJK2-1 and application in the development of a cloning vector.

Three new Acetobacter strains were isolated from vinegar. By plasmid profiling they were recognized as genotypically different from each other. Sequencing of the genes for 16S and 23S rRNA and DNA-DNA hybridization of total DNA against DNA of all type strains of Acetobacter identified Acetobacter strains JK2 and V3 as A. europaeus, and Acetobacter strain JK3 as A. intermedius. In contrast to the type strain of A. europaeus (DSM 6160), A. europaeus JK2 and V3 do not require acetic acid for growth and can be successfully transferred between media with and without acetic acid. This phenotypic characteristic enables convenient handling of both strains in genetic studies. Plasmid pJK2-1 from A. europaeus JK2 was used as the basis for shuttle plasmid construction with the aim of developing an efficient vector system for these strains. The entire nucleotide sequence of pJK2-1 was determined. High amino acid identities were found for three open reading frames: Rep (replication protein); Dinjl (DNA damage inducible enzyme); and Dinj2 proteins. A recombinant plasmid pUCJK2-1 (5.6 kb) consisting of the entire plasmid pJK2-1 and the entire plasmid pUC18 was successfully used in transformation experiments. Plasmid pJT2 (5.8 kb) was constructed from pUCJK2-1 with the aim of reactivating the lacZ' gene.

Acetic Acid↗
PubMed1979

Restriction map of the region surrounding the EcoRI site in the pCR1 plasmid and analysis of an inserted ovalbumin gene.

We have determined a restriction map of a 1650 base pair region surrounding the EcoRI site of the bacterial plasmid, pCR1. We have used pCR1 as a vector in cloning synthetic ovalbumin double-stranded cDNA. Using the pCR1 restriction map, we have characterized the ovalbumin sequences inserted in one recombinant plasmid, pOvE12. POvE12 appears to contain all, or nearly all, of the sequences found in full length, double-stranded cDNA synthesized in vitro.

Base Sequence↗
PubMed1986

Plasmid profile analysis of bovine isolates of Pasteurella haemolytica.

Pasteurella haemolytica isolates, obtained from cattle with respiratory tract disease, were characterized as to serotype, antimicrobial susceptibility, and plasmid content. Strains isolated from 2 groups of cattle were compared. Remarkable similarity was seen in the plasmid profiles of isolates of the same serotype. In contrast, isolates of 2 different serotypes had totally different plasmid profiles.

Animals↗
PubMed2006

Topological analysis of plasmid chromatin from yeast and mammalian cells.

Yeast has proven to be a powerful system for investigation of chromatin structure. However, the extent to which yeast chromatin can serve as a model for mammalian chromatin is limited by the significant number of differences that have been reported. To further investigate the structural relationship between the two chromatins, we have performed a DNA topological analysis of pRSSVO, a 5889 base-pair plasmid that can replicate in either yeast or mammalian cells. When grown in mammalian cells, pRSSVO contains an average of 33 negative supercoils, consistent with one nucleosome per 181 bp. This is close to the measured nucleosome repeat length of 190 bp. However, when grown in yeast cells, pRSSVO contains an average of only 23 negative supercoils, which is indicative of only one nucleosome per 256 bp. This is dramatically different from the measured nucleosome repeat length of 165 bp. To account for these observations, we suggest that yeast chromatin is composed of relatively short ordered arrays of nucleosomes with a repeat of 165 bp, separated by substantial gaps, possibly corresponding to regulatory regions.

Animals↗