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Results for “plasmid characterization”

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PubMed1986

Identification and characterization of recD, a gene affecting plasmid maintenance and recombination in Escherichia coli.

We isolated mutations that reduce plasmid stability in dividing cell populations and mapped these mutations to a previously undescribed gene, recD, that affects recombination frequency and consequently the formation of plasmid concatemers. Insertions of the transposable element Tn10 into recD resulted in increased concatemerization and loss of pSC101 and ColE1-like replicons during nonselective growth. Both concatemer formation and plasmid instability in recD mutants require a functional recA gene. Mutations in recD are recessive to recD+ and map to a small region of the Escherichia coli chromosome located between recB and argA. Although the recD locus is distinct from loci encoding the two previously identified subunits of the RecBC enzyme, mutations in recD appear to affect the exonuclease activity of this enzyme.

Chromosome Mapping↗

PubMed1991

Physical characterization of SCP1, a giant linear plasmid from Streptomyces coelicolor.

SCP1, coding for the methylenomycin biosynthesis genes in Streptomyces coelicolor, was shown to be a giant linear plasmid of 350 kb with a copy number of about four by analysis with pulsed-field gel electrophoresis. A detailed physical map of SCP1 was constructed by extensive digestion with six restriction endonucleases, by DNA hybridization experiments, and finally by cloning experiments. SCP1 has unusually long terminal inverted repeats of 80 kb on both ends and an insertion sequence at the end of the right terminal inverted repeat. Analysis by pulsed-field gel electrophoresis in agarose containing sodium dodecyl sulfate revealed that a protein is bound to the terminal 4.1-kb SpeI fragments derived from both ends of SCP1. Treatment with lambda exonuclease or exonuclease III and SpeI digestion also indicated that the 5' ends of SCP1 are attached to a protein.

Blotting, Southern↗

PubMed1996

Characterization and expression of the plasmid-borne bedD gene from Pseudomonas putida ML2, which codes for a NAD+-dependent cis-benzene dihydrodiol dehydrogenase.

The catabolic plasmid pHMT112 in Pseudomonas putida ML2 contains the bed gene cluster encoding benzene dioxygenase (bedC1C2BA) and a NAD+-dependent dehydrogenase (bedD) required to convert benzene into catechol. Analysis of the nucleotide sequence upstream of the benzene dioxygenase gene cluster (bedC1C2BA) revealed a 1,098-bp open reading frame (bedD) flanked by two 42-bp direct repeats, each containing a 14-bp sequence identical to the inverted repeat of IS26. In vitro translation analysis showed bedD to code for a polypeptide of ca. 39 kDa. Both the nucleotide and the deduced amino acid sequences show significant identity to sequences of glycerol dehydrogenases from Escherichia coli, Citrobacter freundii, and Bacillus stearothermophilus. A bedD mutant of P. putida ML2 in which the gene was disrupted by a kanamycin resistance cassette was unable to utilize benzene for growth. The bedD gene product was found to complement the todD mutation in P. putida 39/D, the latter defective in the analogous cis-toluene dihydrodiol dehydrogenase. The dehydrogenase encoded by bedD) was overexpressed in Escherichia coli and purified. It was found to utilize NAD+ as an electron acceptor and exhibited higher substrate specificity for cis-benzene dihydrodiol and 1,2-propanediol compared with glycerol. Such a medium-chain dehydrogenase is the first to be reported for a Pseudomonas species, and its association with an aromatic ring-hydroxylating dioxygenase is unique among bacterial species capable of metabolizing aromatic hydrocarbons.

Amino Acid Sequence↗

PubMed1998

Purification and characterization of thin pili of IncI1 plasmids ColIb-P9 and R64: formation of PilV-specific cell aggregates by type IV pili.

Thin pili of the closely related IncI1 plasmids ColIb-P9 and R64 are required only for liquid mating and belong to the type IV family of pili. They were sedimented by ultracentrifugation from culture medium in which Escherichia coli cells harboring ColIb-P9- or R64-derived plasmids had been grown, and then the pili were purified by CsCl density gradient centrifugation. In negatively stained thin pilus samples, long rods with a diameter of 6 nm, characteristic of type IV pili, were observed under an electron microscope. Gel electrophoretic analysis of purified ColIb-P9 thin pili indicated that thin pili consist of two kinds of proteins, pilin and the PilV protein. Pilin was demonstrated to be the product of the pilS gene. Pilin was first synthesized as a 22-kDa prepilin from the pilS gene and subsequently processed to a 19-kDa protein by the function of the pilU product. The N-terminal amino group of the processed protein was shown to be modified. The C-terminal segments of the pilV products vary among six or seven different types, as a result of shufflon DNA rearrangements of the pilV gene. These PilV proteins were revealed to comprise a minor component of thin pili. Formation of PilV-specific cell aggregates by ColIb-P9 and R64 thin pili was demonstrated and may play an important role in liquid mating.

Amino Acid Sequence↗

PubMed2003

Conservation of plasmid maintenance functions between linear and circular plasmids in Borrelia burgdorferi.

The Lyme disease agent Borrelia burgdorferi maintains both linear and circular plasmids that appear to be essential for mammalian infection. Recent studies have characterized the circular plasmid regions that confer autonomous replication, but the genetic elements necessary for linear plasmid maintenance have not been experimentally identified. Two vectors derived from linear plasmids lp25 and lp28-1 were constructed and shown to replicate autonomously in B. burgdorferi. These vectors identify internal regions of linear plasmids necessary for autonomous replication in B. burgdorferi. Although derived from linear plasmids, the vectors are maintained in circular form in B. burgdorferi, indicating that plasmid maintenance functions are conserved, regardless of DNA form. Finally, derivatives of these vectors indicate that paralogous gene family 49 is apparently not required for either circular or linear plasmid replication.

Borrelia burgdorferi↗

PubMed2005

Characterization of the replication region of plasmid pLS32 from the Natto strain of Bacillus subtilis.

Plasmid pL32 from the Natto strain of Bacillus subtilis belongs to a group of low-copy-number plasmids in gram-positive bacteria that replicate via a theta mechanism of replication. We studied the DNA region encoding the replication protein, RepN, of pLS32, and obtained the following results. Transcription of the repN gene starts 167 nucleotides upstream from the translational start site of repN. The copy number of repN-coding plasmid pHDCS2, in which the repN gene was placed downstream of the IPTG (isopropyl-1-thio-beta-D-galactopyranoside)-inducible Pspac promoter, was increased 100 fold by the addition of IPTG. Histidine-tagged RepN bound to a specific region in the repN gene containing five 22-bp tandem repeats (iterons) with partial mismatches, as shown by gel retardation and foot printing analyses. Sequence alterations in the first three iterons resulted in an increase in plasmid copy number, whereas those in either the forth or fifth iteron resulted in the failure of plasmid replication. The iterons expressed various degrees of incompatibility with an incoming repN-driven replicon pSEQ243, with the first three showing the strongest incompatibility. Finally, by using a plasmid, pHDMAEC21, carrying the sequence alterations in all the five iterons in repN and thus unable to replicate but encoding intact RepN, the region necessary for replication was confined to a 96-bp sequence spanning the 3'-terminal half of the fourth iteron to an A+T-rich region located downstream of the fifth iteron. From these results, we conclude that the iterons in repN are involved in both the control of plasmid copy number and incompatibility, and we suggest that the binding of RepN to the last two iterons triggers replication by melting the A+T-rich DNA sequence.

Alanine↗

PubMed1983

Identification and physical characterization of a Col E1 hybrid plasmid containing a catalase gene of Escherichia coli.

A hybrid Escherichia coli: Col E1 plasmid, pLC36-19, containing a catalase gene has been identified in the Clarke and Carbon colony bank. Catalase activity was amplified two- to three-fold in the pLC36-19-containing strain relative to other hybrid-plasmid-containing strains and this activity could be induced three- or four-fold by hydrogen peroxide or ascorbic acid. The plasmid was transferred to a strain chromosomally deficient in catalase synthesis, resulting in a strain with high and inducible levels of catalase. The plasmid was also transferred to a minicell-producing strain and minicells harbouring the plasmid were found to synthesize a labelled protein with a molecular weight of 84 000 characteristic of catalase from E. coli. A catalase activity was also synthesized by the plasmid-containing minicells. Two catalase activities with associated peroxidase activities coded for by the plasmid were separable by polyacrylamide gel electrophoresis and migrated coincident with chromosomally encoded catalase-peroxidase activities. A third catalase activity which did not have an associated peroxidase activity was not coded for by the plasmid. A physical map of the 25.5-kilobase pair plasmid was constructed by restriction nuclease analysis and the relative positions of 38 restriction sites were defined.

Bacteriocin Plasmids↗

PubMed1990

[Identification and characterization of the two promoters of plasmid pKYM].

Plasmid pKYM is a multicopy plasmid isolated from Shigella sonnei and multiples stably in Escherichia coli. The plasmid encodes Rep protein which is essential for its multiplication and synthesizes cop ribonucleic acid (RNA) which is a short RNA complementary to the 5' region of rep m-RNA. This RNA controls the copy number and the incompatibility of the plasmid. The previous analysis located the promoters of rep m-RNA RNA (PR) and cop RNA (PL) in the inc region. This report confirmed the presence of these promoters by analyzing the RNAs isolated from the cells carrying pKYM and those synthesized in vitro. The initiation sites of these transcriptions were also determined. Analysis of in vivo RNA suggested that the quantity of cop RNA whose size was about 90 nucleotides was larger than that of rep m-RNA and these RNAs easily formed RNA-RNA hybrid. The analysis also suggested that the synthesis of rep m-RNA was repressed by cop RNA and Rep protein itself.

Base Sequence↗

PubMed1981

Characterization of chromosomal and membrane associated plasmid in Bacillus brevis ATCC 9999.

A covalently closed circular (ccc) DNA, with a weight of 44.7 x 10(6) daltons, has been isolated from Bacillus brevis ATCC 999 (a gramicidin S producer) and from the gramicidin S-negative mutant EB16. The ccc DNA in the case of the parent strain, is mainly (99%) attached to the chromosome and membrane fraction. A restriction enzyme map of the plasmid DNA was constructed for the enzymes SalI, SmaI and BamHI, which cleaved the plasmid DNA into two, two and six fragments respectively. Further digestion with the endonucleases EcoRI and HindIII cleaved the plasmid into 17 and 22 fragments.

Anti-Bacterial Agents↗

PubMed1984

Isolation and characterization of a pock-forming plasmid pTA4001 from Streptomyces lavendulae.

A covalently closed circular (ccc) DNA with a size of 5.9 kb was isolated from a strain producing streptothricins , Streptomyces lavendulae No. 1080, and its restriction map was determined. Colonies of the plasmid-carrying strain formed pocks on the lawn of the plasmid-free derivatives of the same organism. The pock-forming ability of pTA4001 was confirmed by transformation of S. lividans with the plasmid DNA. Insertion of the chromosomal streptothricin resistance gene of S. lavendulae No. 1080 into pTA4001 gave various composite plasmids with marked deletion in S. lividans as a host. By analyzing these derivatives, a 2.2 kb region essential for replication and a possible region for pock formation were determined in the map of pTA4001 . A cloning vector, pKST2 (4.3 kb) containing resistance genes to streptothricin and thiostrepton with several unique cloning sites was constructed from pTA4001 .

Base Sequence↗

PubMed1989

Characterization of cytotoxicity and R-plasmid in Salmonella krefeld.

From 1981 to 1984, 254 isolates of Salmonella krefeld were isolated from newborns and infants with acute gastroenteritis in southern Taiwan. All the crude enterotoxin preparations of S. krefeld caused the cytotoxic elongation reaction in Chinese hamster ovary K1 (CHO-K1) cells. Cytotoxic enterotoxin was also produced by S. krefeld inducing Vero cells to round up and appear partially detached from the culture plate. It was noted that S. krefeld showed internalization and multiplication in CHO-K1 cells. S. krefeld exhibited 12 different resistant patterns. And the predominant patterns were found to be resistant to kanamycin and ampicillin (Ka-Amr), and resistant to kanamycin, ampicillin, chloramphenicol and tetracycline (Ka-Am-Cm-Ter). It was found that two distinct plasmids of 34 megadalton (Md) and 120 Md were commonly present in these strains. S. krefeld haboured 34 Md and 120 Md R-plasmid, which conferred resistance to Ka-Amr and Ka-Am-Cm-Ter, respectively. From the resistance transferred patterns, Ka-Amr was the most common resistance among transconjugants. The frequency of transfer of the 34 Md R-plasmid (2.71 x 10(-3) transconjugants/donor cell) from S. krefeld to E. coli K-12 14R525 was 20 times higher than that of the 120 Md R-plasmid (1.48 x 10(-4) transconjugants/donor cell). In analysis of the restriction endonuclease digest of the 34 Md plasmid obtained from different bacterial sources, their specific identical DNA fragment pattern suggested that the outbreak infection due to S. krefeld had a common origin.

Cell Line↗

PubMed1989

Characterization of a wide host range plasmid pANV-6 from Citrobacter diversus.

pANV-6 is a 5.85 kb, streptomycin resistant, high copy number plasmid isolated from a multi drug resistant clinical isolate of C. diversus by transformation in Escherichia coli C-600. The plasmid was very stable, noncurable and could not be amplified with chloramphenicol. It was non-conjugative among Enterobacteriaceae hosts. Plasmid was transferred by transformation to several Gram negative and Gram positive hosts. These included Esch. coli, Serratia marcescens, Salmonella typhimurium, Klebsiella pneumoniae, Staphylococcus aureus, Corynebacterium glutamicum. From Staph. aureus it was transferred by conjugation to other Staph. aureus, Staph. epidermidis and Bacillus subtilis. Plasmid was incompatible with standard plasmids belonging to incompatibility groups H1, H2 = S,J,P and T of coliform bacteria. Plasmid has one site for restriction enzyme EcoRI and Pst I, three for Bgl 1 and non for Bgl 1, Bam HI, Hind III and X ba I.

Citrobacter↗

PubMed1988

Isolation, characterization, and molecular cloning of cryptic plasmids isolated from Edwardsiella ictaluri.

Fifty-five isolates of Edwardsiella ictaluri were examined for the presence of plasmid DNA by a rapid alkaline extraction procedure. All 49 isolates from channel catfish and a single isolate from Bengal danio carried 2 plasmids with molecular masses of approximately 3.2 and 3.7 megadaltons (Mdal). Five E ictaluri isolates from other fish contained 1 to 3 plasmids, which had molecular masses ranging from 2.5 to 45 Mdal. The 2 plasmids (3.2 and 3.7 Mdal) from the type strain of E ictaluri (ATCC 33202) were ligated into pUC19 cloning vectors, and restriction endonuclease maps of each insert were prepared.

Animals↗

PubMed1985

Biochemical characterization of E. coli transconjugants with plasmids derived from Klebsiella pneumoniae.

Sixty-three strains of E. coli transconjugants derived from E. coli K12 J62-1 containing plasmids derived from Klebsiella pneumoniae, were examined for the presence of phenotypic markers other than antibiotic resistance. This investigation was carried out using API 50CHE and API ZYM tests. Beta-glucosidase was found in 63/63 J62-1 transconjugants. Dulcitol dehydrogenase was present in 92.1% while beta-galactosidase was present in 70% of transconjugants. None of the three enzymes were present in the recipient. Dulcitol dehydrogenase was present only in the transconjugants and is absent from the donors and recipient. The transconjugants, cured of their antibiotic resistant plasmids retained dulcitol dehydrogenase activity. The Klebsiella donors were not cured of antibiotic resistance by the curing process.

Anti-Bacterial Agents↗

PubMed1979

Characterization of a naturally occurring R plasmid in Salmonella isangi conferring unusually high resistance to ampicillin.

A strain of Salmonella isangi was isolated from HUMANA milk, used a baby food in Bulgarian hospitals. It harbours two stable plasmids, distinguishable by their molecular weight of 21 Mdal for the transfer factor (rRB 1) and 9,3 Mdal for R-factor rRB 2, confering resistance to ampicillin, cephalothin, streptomycin and sulfonamides. Both plasmids are maintained in different copy numbers per chromosome genome equivalent, 8 copies for rRB 1 and 158 copies for rRB 2, associated with an unusual high level of resistance to ampicillin (MIC 65 mg/ml).

Ampicillin↗

PubMed1982

Characterization of the in vitro constructed plasmid composed of the nif gene cluster of Lignobacter and RP4.

The nitrogen fixation of Lignobacter K17 is plasmid mediated. Nif plasmid was transferred from Lignobacter to other bacterial species and the transposon Tn9 was inserted into it. The molecular weight of this plasmid designated pUCS101, is of 19.8 Mdal. In this study we constructed in vitro a hybrid plasmid (pUCS110) by ligating HindIII digests of pUCS101 nif:: Tn9 and of RP4. Next it was proved that pUCS110 is able to complement the total deletion of the nif region in Klebsiella pneumoniae. The 50 Mdal plasmid pUCS110 was not maintained stably in Escherichia coli recA+ as in E. coli recA-. After being transferred to K. pneumoniae, pUCS110 showed a tendency to generate plasmids of various size from 2.8 to 78 Mdal. Bacteria harbouring plasmids of various size classes were more resistant to chloramphenicol than K. pneumoniae (pUCS110). Altered cleavage patterns were found in derivatives of pUCS110. The obtained results suggest that translocation of the transposon Tn9 can be responsible for the instability of pUCS110.

Anti-Bacterial Agents↗

PubMed1981

Klebsiella and Enterobacter strains derived from hospital infections. II. Occurrence and characterization of R-, Lac- and Col- plasmids and their clinical-epidemiological significance.

A total of 269 hospital Klebsiella strains and 103 hospital Enterobacter strains showed 34 and 10 different antibiotic resistance patterns, respectively. Among multiple resistant Klebsiella and Enterobacter strains the Ap Sm Cm Tc resistance pattern was the most frequent (K. aerogenes). Antibiotic resistant strains carried R-plasmids in 27.5%. The presence of R-plasmids was demonstrable in 2.9% of single antibiotic resistant, in 12.8% of double antibiotic resistant, and in 71.4% of multiple antibiotic resistant Klebsiella strains. R-plasmid carriage was most frequent in strains of the species K. pneumoniae and K. atlantae. Transfer frequency of R-plasmids of multiple resistant strains was generally higher into Escherichia coli K12 recipient (10-1 to 10-2) than into K. pneumoniae recipient (10-3 to 10-5). Regarding the individual resistance determinants, transfer frequency of Km-Nm resistance was the highest (65.1%). The lac-plasmid was demonstrable in 18 out of the 77 R-plasmid carrying Klebsiella strains. R- and Lac-plasmids of single and multiple resistant Klebsiella strains could be transferred into E. coli and Klebsiella recipients in mice in vivo. As many as 67% showed fi- and 33% showed fi+ character among the demonstrated 112 R-plasmids. Regarding their incompatibility, the studied five plasmids belonged to groups FI, FII and I alpha. Altogether 14 different groups could be distinguished among R-plasmids on the basis of their phage restriction capability on phage lambda and coliphages T, and another group of R-plasmids showing no restriction at all. Fi+ character was demonstrated in 54.5% of R-plasmids showing phage restriction capability and in 10.9% of R-plasmids showing no phage restriction. Nearly twice as many R-plasmids carrier strains were found among non-typable ones as among those typable by phages. Three R-plasmids showed phage-modification among 51 R-plasmids restrictive for phages. In addition to the phage-type of Klebsiella strains, R-plasmids can also be used as an epidemiologic marker.

Bacteriocin Plasmids↗

PubMed1989

Transcription events in the origin of replication of plasmid pSC101.

Insertion mutations were isolated in the origin fragment of the plasmid pSC101 after random cleavage with DNase I. The replication properties of the resulting plasmids confirmed previous findings and extended the characterization of the essential regions. Using these plasmids, we analyzed by various methods the transcription events in the pSC101 origin. In addition to the mRNA of repA, a gene coding for the self-regulated RepA protein which is essential for replication of the plasmid, we characterized a transcript, which we called RNA Y, that runs in the opposite direction and that starts in the middle of the second repeated sequence in the origin region. Like the self-regulated repA mRNA, RNA Y is weakly expressed. It does not code for a complete protein within the origin fragment but may do so in the wild-type plasmid. We also found indications for one or, possibly, two small RNA species, called RNA X, which run in the same direction as RepA and which are partially complementary to RNA Y. We postulate that RNA Y and, possibly, RNA X are implicated in the initiation of replication of pSC101.

Base Sequence↗