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PubMed1993

Characterization of a plasmid-encoded urease gene cluster found in members of the family Enterobacteriaceae.

Plasmid-encoded urease gene clusters found in uropathogenic isolates of Escherichia coli, Providencia stuartii, and Salmonella cubana demonstrated DNA homology, similar positions of restriction endonuclease cleavage sites, and manners of urease expression and therefore represent the same locus. DNA sequence analysis indicated that the plasmid-encoded urease genes are closely related to the Proteus mirabilis urease genes.

Amino Acid Sequence↗
PubMed1995

Physical mapping and characterization of a catabolic plasmid from the deep-subsurface bacterium Sphingomonas sp. strain F199.

A supercoiled 180-kb plasmid, pNL1, has been isolated from the deep-subsurface, chemoheterotrophic Sphingomonas sp. strain F199, and a physical map was generated. Analysis of a pNL1-derived cosmid library indicated that catechol 2,3-dioxygenase activity was linked to two distinct regions of the plasmid. Thus, the genes for aromatic catabolism in this Sphingomonas strain are, at least in part, plasmid encoded.

Catechol 2,3-Dioxygenase↗
PubMed1997

Characterization of large linear plasmids in mycobacteria.

Linear plasmids were found in Mycobacterium xenopi, M. branderi, and M. celatum. These elements represented a wide size range (from 20 to 320 kb), had Streptomyces-like terminal structures, and defined five hybridization groups. Cross-hybridization and common restriction fragment length polymorphism patterns of plasmids from the different species suggest either genetic exchange or a common ancestry of the species.

Blotting, Southern↗
PubMed1999

Isolation and characterization of two cryptic plasmids in the ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11.

Two plasmids were discovered in the ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11, which was isolated from activated sludge. The plasmids, designated pAYS and pAYL, were relatively small, being approximately 1.9 kb long. They were cryptic plasmids, having no detectable plasmid-linked antibiotic resistance or heavy metal resistance markers. The complete nucleotide sequences of pAYS and pAYL were determined, and their physical maps were constructed. There existed two major open reading frames, ORF1 in pAYS and ORF2 in pAYL, each of which was more than 500 bp long. The predicted product of ORF2 was 28% identical to part of the replication protein of a Bacillus plasmid, pBAA1. However, no significant similarity to any known protein sequences was detected with the predicted product of ORF1. pAYS and pAYL had a highly homologous region, designated HHR, of 262 bp. The overall identity was 98% between the two nucleotide sequences. Interestingly, HHR-homologous sequences were also detected in the genomes of ENI-11 and the plasmidless strain Nitrosomonas europaea IFO14298. Deletion analysis of pAYS and pAYL indicated that HHR, together with either ORF1 or ORF2, was essential for plasmid maintenance in ENI-11. To our knowledge, pAYS and pAYL are the first plasmids found in the ammonia-oxidizing autotrophic bacteria.

Amino Acid Sequence↗
PubMed2000

Identification and characterization of an active plasmid partition mechanism for the novel Lactococcus lactis plasmid pCI2000.

The replication region of the lactococcal plasmid pCI2000 was subcloned and analyzed. The nucleotide sequence of one 5.6-kb EcoRI fragment which was capable of supporting replication when cloned on a replication probe vector revealed the presence of seven putative open reading frames (ORFs). One ORF exhibited significant homology to several replication proteins from plasmids considered to replicate via a theta mode. Deletion analysis showed that this ORF, designated repA, is indeed required for replication. The results also suggest that the origin of replication is located outside repA. Upstream and divergently transcribed from repA, an ORF that showed significant (48 to 64%) homology to a number of proteins that are required for faithful segregation of chromosomal or plasmid DNA of gram-negative bacteria was identified. Gene interruption and transcomplementation experiments showed that this ORF, designated parA, is required for stable inheritance of pCI2000 and is active in trans. This is the first example of such a partitioning mechanism for plasmids in gram-positive bacteria.

Amino Acid Motifs↗
PubMed2000

Characterization of the endogenous plasmid from Pseudomonas alcaligenes NCIB 9867: DNA sequence and mechanism of transfer.

The endogenous plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 was determined to have 32,743 bp with a G+C content of 59.8%. Sequence analysis predicted a total of 29 open reading frames, with approximately half of them contributing towards the functions of plasmid replication, mobilization, and stability. The Pac25I restriction-modification system and two mobile elements, Tn5563 and IS1633, were physically localized. An additional eight open reading frames with unknown functions were also detected. pRA2 was genetically tagged with the OmegaStr(r)/Spc(r) gene cassette by homologous recombination. Intrastrain transfer of pRA2-encoded genetic markers between isogenic mutants of P. alcaligenes NCIB 9867 were observed at high frequencies (2.4 x 10(-4) per donor). This transfer was determined to be mediated by a natural transformation process that required cell-cell contact and was completely sensitive to DNase I (1 mg/ml). Efficient transformation was also observed when pRA2 DNA was applied directly onto the cells, while transformation with foreign plasmid DNAs was not observed. pRA2 could be conjugally transferred into Pseudomonas putida RA713 and KT2440 recipients only when plasmid RK2/RP4 transfer functions were provided in trans. Plasmid stability analysis demonstrated that pRA2 could be stably maintained in its original host, P. alcaligenes NCIB 9867, as well as in P. putida RA713 after 100 generations of nonselective growth. Disruption of the pRA2 pac25I restriction endonuclease gene did not alter plasmid stability, while the pRA2 minireplicon exhibited only partial stability. This indicates that other pRA2-encoded determinants could have significant roles in influencing plasmid stability.

Bacterial Proteins↗
PubMed2003

Identification and characterization of a pSLA2 plasmid locus required for linear DNA replication and circular plasmid stable inheritance in Streptomyces lividans.

Streptomyces linear plasmids and linear chromosomes can replicate also in a circular form when their telomeres are deleted. The 17-kb linear plasmid pSLA2 has been a useful model in studies of such replicons. Here we report that the minimal origin initiating replication of pSLA2-derived plasmids as circular molecules cannot propagate these plasmids in a linear mode unless they also contain a novel plasmid-encoded locus, here named rlrA (required for linear replication). In contrast with the need for rlrA to accomplish replication of telomere-containing linear plasmids, expression of rlrA, which encodes two LuxR family regulatory domains, interferes with the establishment of pSLA2 in circular form in Streptomyces lividans transformants. The additional presence of an adjacent divergently transcribed locus, rorA (rlrA override), which strongly resembles the kor (kil override) transcription control genes identified previously on Streptomyces plasmids, reversed the detrimental effects of rlrA on plasmid establishment and additionally stabilized circular plasmid inheritance by spores during the S. lividans life cycle. While the effects of the rlrA/rorA locus of pSLA2 were seen also on linear plasmids derived from the unrelated SLP2 replicon, they did not extend to plasmids whose replication was initiated at a cloned chromosomal origin. Our results establish the existence of, and provide the initial description of, a novel plasmid-borne regulatory system that differentially affects the propagation of linear and circular plasmids in Streptomyces.

Amino Acid Sequence↗
PubMed2004

Detection and characterization of conjugative degradative plasmids in xenobiotic-degrading Sphingomonas strains.

A systematic survey for the presence of plasmids in 17 different xenobiotic-degrading Sphingomonas strains was performed. In almost all analyzed strains, two to five plasmids with sizes of about 50 to 500 kb were detected by using pulsed-field gel electrophoresis. A comparison of plasmid preparations untreated or treated with S1 nuclease suggested that, in general, Sphingomonas plasmids are circular. Hybridization experiments with labeled gene probes suggested that large plasmids are involved in the degradation of dibenzo-p-dioxin, dibenzofuran, and naphthalenesulfonates in S. wittichii RW1, Sphingomonas sp. HH69, and S. xenophaga BN6, respectively. The plasmids which are responsible for the degradation of naphthalene, biphenyl, and toluene by S. aromaticivorans F199 (pNL1) and of naphthalenesulfonates by S. xenophaga BN6 (pBN6) were site-specifically labeled with a kanamycin resistance cassette. The conjugative transfer of these labeled plasmids was attempted with various bacterial strains as putative recipient strains. Thus, a conjugative transfer of plasmid pBN6 from S. xenophaga BN6 to a cured mutant of strain BN6 and to Sphingomonas sp. SS3 was observed. The conjugation experiments with plasmid pNL1 suggested a broader host range of this plasmid, because it was transferred without any obvious structural changes to S. yanoikuyae B1, Sphingomonas sp. SS3, and S. herbicidovorans. In contrast, major plasmid rearrangements were observed in the transconjugants after the transfer of plasmid pNL1 to Sphingomonas sp. HH69 and of pBN6 to Sphingomonas sp. SS3. No indications for the transfer of a Sphingomonas plasmid to bacteria outside of the Sphingomonadaceae were obtained.

Biodegradation, Environmental↗
PubMed1979

Plasmid-determined 2-hydroxypyridine utilization by Arthrobacter crystallopoietes.

Arthrobacter crystallopoietes has the ability to utilize 2-hydroxypyridine (2-HP) as a source of carbon and nitrogen and forms a blue extracellular pigment when grown in the presence of 2-HP. Ultracentrifugal analyses of pigment producing (Pig+) and pigment nonproducing (Pig-) strains of A. crystallopoietes revealed the presence of plasmid material in both strains. Recovery of plasmid DNA from Pig+ strains is two or three times greater than from Pig- strains. The molecular weight of plasmid DNA recovered from Pig+ strains (62 Mdaltons) is slightly higher than the molecular weight of plasmid DNA from Pig- strains. Consistent with the characterization of plasmid DNA from the two strains is that Pig+ strains contain a 63-Mdalton plasmid encoding 2-HP utilization as well as a cryptic plasmid of very nearly equal molecular weight. Pig- strains contain only the cryptic plasmid.

Arthrobacter↗
PubMed2000

Cloning and characterization of a retroviral plasmid, pCC1, for combination suicide gene therapy.

Introduction of the herpes simplex virus thymidine kinase (HSV-TK) gene into mammalian cells confers specific sensitivity to killing by the anti-herpes drug ganciclovir (GCV). This gene has therefore been used in a number of cancer gene therapy protocols as a therapeutic gene. However, the therapeutic efficacy of HSV-TK/GCV in cancer gene therapy experiments can be augmented by additional therapeutic genes. We have cloned a retroviral plasmid, pCC1, containing a fusion gene of HSV-TK and Sh-ble driven by an internal simian virus 40 early promoter. This gene encodes a fusion protein that confers GCV sensitivity and Zeocin resistance when introduced into mammalian cells. A multiple cloning site (MCS) allows the introduction of a second therapeutic gene under the transcriptional control of the Moloney murine leukemia virus 5' long terminal repeat. We have generated packaging cell lines electroporated with pCC1 or pCC1 rtIL-2 S (rat interleukin-2 gene cloned in the sense direction in the MCS), the supernatants of which transfer GCV sensitivity only, or both GCV sensitivity and rtIL-2 production, respectively to rat ovarian cancer cells. This plasmid may be useful for the study of combination suicide gene therapy strategies.

Animals↗
PubMed1998

Construction and characterization of F plasmid-based expression vectors.

A low-copy expression vector has been constructed from a 9 Kbp region of the Escherichia coli F plasmid containing the oriV and oriS origins of replication. This plasmid carries the beta-lactamase gene (Apr) and the araBAD promoter/araC regulator for arabinose-inducible gene expression. A derivative which carries a lacZ reporter gene was found to be stably maintained for at least 150 generations. A related multi-copy plasmid was stably maintained in arabinose-free medium, but no plasmid-bearing segregants remained after 60 generations when lacZ expression was induced. Induced expression resulted in 27% (multi-copy) and 12% (low-copy) decreases in growth rate. The uninduced levels of beta-galactosidase were 200 units (multi-copy) and 15 units (low-copy).

Arabinose↗
PubMed1992

Molecular characterization of R-plasmids pST1 and pST2 from Salmonella typhimurium S24.

Salmonella typhimurium S24 was isolated in September 1986 at Kaohsiung Medical College Hospital, Kaohsiung, Taiwan, from a patient suffering from gastroenteritis during an outbreak of salmonellosis. Two conjugative R-plasmids have been isolated from Escherichia coli K-12 14R525, which was mated with S. typhimurium S24. The two R-plasmids found in S. typhimurium S24 belong to two different incompatibility (Inc) groups: the 130-kilobase IncFI plasmid pST1 and the 56-kb IncN plasmid pST2. These two R-plasmids of pST1 and pST2 together mediate resistance to multiple antibiotics in S. typhimurium S24. By DNA probes hybridization, plasmid pST1 was shown to carry an enteric type II chloramphenicol acetyltransferase (CAT) gene, a class C tetracycline resistance (TetR) gene and a type III dihydrofolate reductase (DHFR) gene, all of which confer resistance to chloramphenicol, tetracycline, and trimethoprim respectively. A Richmond's type III beta-lactamase gene was located on each plasmid of pST1 and pST2. beta-lactamases specified by both plasmids pST1 and pST2 conferred high level resistance to amoxicillin, carbenicillin, piperacillin, sulbenicillin, ticarcillin in addition to ampicillin. A novel aminoglycoside 6'-N-acetyltransferase [AAC(6')] was demonstrated on plasmid pST2. This AAC(6') enzyme modified kanamycin, amikacin, dibekacin, tobramycin, gentamicin, netilmicin, sisomicin, butirosin and ribostamycin.

Aminoglycosides↗
PubMedDate not supplied

Isolation and characterization of Bordetella avium plasmids.

Experiments were conducted to study the plasmids of Bordetella avium, B. avium-like, and B. bronchiseptica isolates from turkeys and the plasmids of the Art-Vax commercial vaccine strain. Plasmids were observed in 6 of 20 B. avium isolates, in 6 of 20 B. avium-like isolates, in all 5 B. bronchiseptica isolates, and in the Art-Vax strain. Plasmids of B. avium correlated with resistance to antibiotics but not with pathogenicity, hemagglutination of guinea pig erythrocytes, or expression of pili.

Animals↗
PubMed1986

Characterization of the F plasmid TraJ protein synthesized in F' and Hfr strains of Escherichia coli K-12.

Using purified F plasmid TraJ protein (Cuozzo, M., Silverman, P., and Minkley, E. (1984) J. Biol. Chem. 259, 6659-6666), we prepared rabbit anti-TraJ protein antibodies to analyze for the first time the TraJ protein as it is synthesized in normal F' and Hfr conjugal donor strains. Using affinity-purified antibody, we identified the protein on immuno-overlay blots of whole cell proteins separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. In contrast to the TraJ protein synthesized in large quantity by heat-induced lambda (traJ) lysogens, the TraJ protein synthesized in normal donor cells was soluble, even after sedimentation at 100,000 X g. The soluble protein was found with the cytoplasmic fraction after separation of cytoplasmic and periplasmic proteins. Velocity sedimentation analysis indicated an S20,w of 3.5 for the single molecular species composed of or including all the TraJ polypeptide in crude extracts. Quantitative analyses showed that conjugal donor strains normally contain 2000-4000 TraJ monomers/cell. However, that level depended on other plasmid and chromosomal genes.

Bacterial Outer Membrane Proteins↗
PubMed1983

Isolation and characterization of Streptomyces erythreus plasmids.

Streptomyces erythreus strains were found to carry several plasmids of molecular weights ranging from about 2 X 10(6) Mr to 40 X 10(6) Mr. Restriction enzyme maps for the streptomycete plasmids pPC7 and pPC8 were constructed for the enzymes Bg/II, EcoRI, XbaI, HindIII, BamHI and SalI. The smaller, pPC8, plasmid appears to be a naturally occurring deletion variant of pPC7. These plasmids belong to the group of conjugative streptomycete plasmids.

Culture Media↗