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PubMed1992

Identification of the CTAG-recognizing restriction-modification systems MthZI and MthFI from Methanobacterium thermoformicicum and characterization of the plasmid-encoded mthZIM gene.

Two CTAG-recognizing restriction and modification (R/M) systems, designated MthZI and MthFI, were identified in the thermophilic archaeon Methanobacterium thermoformicicum strains Z-245 and FTF, respectively. Further analysis revealed that the methyltransferase (MTase) genes are plasmid-located in both strains. The plasmid pFZ1-encoded mthZIM gene of strain Z-245 was further characterized by subcloning and expression studies in Escherichia coli followed by nucleotide sequence analysis. The mthZIM gene is 1065 bp in size and may code for a protein of 355 amino acids (M(r) 42,476 Da). The deduced amino acid sequence of the M.MthZI enzyme shares substantial similarity with four distinct regions from several m4C- and m6A-MTases, and contains the TSPPY motif that is so far only found in m4C-MTases. Partially overlapping with the mthZIM gene and in reverse orientation, an additional ORF was identified with a size of 606 bp potentially coding for a protein of 202 amino acids (M(r) 23.710 Da). This ORF is suggested to encode the corresponding endonuclease R.MthZI.

Amino Acid Sequence↗

PubMed1996

Construction of derivatives of the F plasmid pOX-tra715: characterization of traY and traD mutants that can be complemented in trans.

A derivative of the F plasmid, pOX38-tra715, expresses the entire F tra operon from a foreign promoter (PT7) derived from phage T7. A series of plasmids related to pOX38-tra715 were constructed which carry either deletion mutations or point mutations in traY. When the PT7 promoter was induced, these plasmids expressed the F pilus but were transfer deficient unless TraY was supplied In trans from compatible plasmids. Insertion of a kanamycin-resistance cassette in the traY gene of the pOX38 plasmid, which contains the wild-type PY promoter, resulted in loss of F piliation and transfer ability. Introduction of TraY in trans partially restored piliation and transfer suggesting that TraY has a role in positively regulating the PY promoter, pOX38-tra719-traD411, which contains a chloramphenicol-resistance cassette in place of the kanamycin-resistance cassette in pOX38-tra715 and a mutation in traD, was constructed to demonstrate the utility of this series of plasmids in studying the long (30 kb) F tra operon.

Bacterial Proteins↗

PubMed1975

Relaxation complexes of plasmid DNA and protein. II. Characterization of the proteins associated with the unrelaxed and relaxed complexes of plasmid ColE1.

The proteins of the DNA-protein relaxation complex of plasmid ColE1 were labeled with [3H]leucine by growth of ColE1 containing Escherichia coli cells in the presence of this radioactive labeled amino acid. Three major [3H]leucine-labeled proteins are found associated with the supercoiled DNA in the ColE1 relaxation complex. The molecular weights of these proteins, determined by sodium dodecyl sulfate-acrylamide gel electrophoresis, are 60,000, 16,000, and 11,000, respectively. Induction of relaxation of the supercoiled DNA by treatment of the complex with sodium dodecyl sulfate results in a dissociation of the two smaller proteins from the DNA. The 60,000 protein, however, remains associated specifically with the nicked strand of the open circular DNA. The strand-specific association of this protein with the relaxed DNA resists heat denaturation of the DNA, sedimentation through an alkaline (pH 12.5) sucrose gradient, and centrifugation to equilibrium in an alkaline (pH 12.5) CsCl gradient.

Bacterial Proteins↗

PubMed1985

An Escherichia coli recBCsbcBrecF host permits the deletion-resistant propagation of plasmid clones containing the 5'-terminal palindrome of minute virus of mice.

The deletion events that have plagued attempts to maintain molecular clones with long palindromic DNA sequences in Escherichia coli have been shown to be less frequent in recBCsbcB hosts [Collins et al., Gene 19 (1982) 139-146]. This study sought to determine if such hosts would permit the stable propagation of plasmid clones carrying the deletion-generating, 206 nucleotide (nt) long, imperfect palindrome derived from the 5' terminus of the genome of minute virus of mice (MVM), an autonomous parvovirus [Astell et al., Nucleic Acids Res. 11 (1983) 999-1018]. To this end these hybrid plasmids were used to transform several different mutant recBCsbcB hosts, followed by the isolation and restriction mapping of plasmid DNA from selected transformants. Characterization of plasmid DNA isolated from a recBCsbcBrecF host indicated deletion-resistant propagation of the intact species. Sequence analysis of unamplified and chloramphenicol (Cm)-amplified plasmid DNA obtained from these clones confirmed the integrity of the palindromic region of the viral DNA insert.

Base Sequence↗

PubMed1990

[Plasmid-encoded antibiotic resistance in Staphylococcus hyicus].

A total of 33 Staphylococcus hyicus-cultures from piglets with exudative epidermatitis were analyzed for the presence of antibiotic resistance plasmids. Four small plasmids encoding resistances to chloramphenicol, macrolide-lincosamide-antibiotics, streptomycin or tetracyclines could be identified in plasmid-curing and plasmid-transformation experiments. For further characterization these plasmids were digested with restriction endonucleases. This led to the construction of a specific restriction map for each of the 4 plasmids. On the basis of their restriction maps, these 4 antibiotic resistance plasmids from "porcine" S. hyicus-cultures were compared with the respective resistance plasmids of other staphylococcal species from infections of humans and animals.

Animals↗

PubMed1989

Characterization of mutant mitochondrial plasmids of Neurospora spp. that have incorporated tRNAs by reverse transcription.

The Mauriceville and Varkud mitochondrial plasmids of Neurospora spp. are closely related, closed-circular DNAs (3.6 and 3.7 kilobases, respectively) whose nucleotide sequences and genetic organization suggest relationships to mitochondrial introns and retroelements. We have characterized nine suppressive mutants of these plasmids that outcompete mitochondrial DNA and lead to impaired growth. All nine suppressive plasmids contain small insertions, corresponding to or including a mitochondrial tRNA (tRNATrp, tRNAGly, or tRNAVal) or a tRNA-like sequence. The insertions are located at the position corresponding to the 5' end of the major plasmid transcript or 24 nucleotides downstream near a cognate of the sequence at the major 5' RNA end. The structure of the suppressive plasmids suggests that the tRNAs were inserted via an RNA intermediate. The 3' end of the wild-type plasmid transcript can itself be folded into a secondary structure which has tRNA-like characteristics, similar to the tRNA-like structures at the 3' ends of plant viral RNAs. This structure may play a role in replication of the plasmids by reverse transcription. Major transcripts of the suppressive plasmids begin at the 5' end of the inserted mitochondrial tRNA sequence and are present in 25- to 100-fold-higher concentrations than are transcripts of wild-type plasmids. Mapping of 5' RNA ends within the inserted mtDNA sequences identifies a short consensus sequence (PuNPuAG) which is present at the 5' ends of a subset of mitochondrial tRNA genes. This sequence, together with sequences immediately upstream in the plasmids, forms a longer consensus sequence, which is similar to sequences at transcription initiation sites in Neurospora mitochondrial DNA. The suppressive behavior of the plasmids is likely to be directly related to the insertion of tRNAs leading to overproduction of plasmid transcripts.

DNA Mutational Analysis↗

PubMed1985

Isolation and characterization of antibiotic-resistance plasmids in thermophilic bacilli.

Four antibiotic-resistance plasmids isolated from thermophilic bacilli were characterized in detail. Three tetracycline-resistance (Tc1) plasmids were designated as pTHT9 (7.7 kilobases (kb], pTHT15 (4.5 kb) and pTHT22 (8.4 kb). From the results of restriction endonuclease analysis and the subsequent Southern hybridization, these were found to possess extensive genetic homology in the regions that include the replication origin and the Tcr gene. Detailed restriction maps of the smallest Tcr plasmid pTHT15 and a kanamycin-resistance (Kmr) plasmid pTHN1 (4.8 kb) were constructed. The positions of antibiotic-resistance loci and regions essential for plasmid replication were determined by cloning plasmid fragments in Bacillus subtilis. These four plasmids were found to replicate and express the resistance genes stably in both B. subtilis and B. stearothermophilus.

Anti-Bacterial Agents↗

PubMed1991

Rhodococcus equi plasmids: isolation and partial characterization.

Fifty-four strains of Rhodococcus equi from different clinical sources (mainly horses and pigs) were examined for their plasmid content by two screening methods. Plasmids were detected in 49 of 54 strains. A plasmid of approximately 80 kb was isolated from 21 of 22 isolates from horses and 20 of 28 isolates from pigs, and a 105-kb plasmid was isolated from 7 of 28 isolates from pigs. The 80-kb plasmid was significantly associated with strains of equine rather than porcine origin, and the 105-kb plasmid was significantly associated with strains of porcine origin. The type strain, ATCC 6939, consistently failed to yield a plasmid. Restriction enzyme analysis of purified plasmid DNA confirmed the relatedness of the 80-kb plasmids isolated from strains of equine and porcine origin. More differences between the restriction patterns of plasmids from strains isolated from horses and from pigs than among strains from either species were observed. Restriction enzyme analysis also showed relatedness of the 105-kb plasmid to the 80-kb plasmid. Three strains shown by others to be virulent in horses or mice possessed the 80-kb plasmid, whereas three other strains not virulent for horses or mice lacked the plasmid, although one had the 105-kb plasmid. There was a significant but not perfect association between the presence of the 80-kb plasmid and production of a diffuse 17.5-kDa thermoregulated, virulence-associated protein. Further study is needed to determine whether this plasmid is associated with virulence in R. equi.

Animals↗

PubMedDate not supplied

Characterization of two cryptic plasmids from Spiroplasma citri and occurrence of their DNA sequences among various spiroplasmas.

Two plasmids, pMH1 with 7 kilobase pairs (Kbp) and pM41 with 8 Kbp, were purified from Spiroplasma citri strains MH and M4, respectively, and characterized by restriction mapping. Upon in vitro DNA recombination with plasmid pBR328 as a vector, pMH1 was cloned in Escherichia coli. Radioactive probes specific of the plasmids were used to investigate the occurrence of pMH1 and pM41 DNA sequences among various spiroplasmas. pM41 or a closely related plasmid was found in three other S. citri strains and also seems to be present as an 8-Kbp plasmid in three spiroplasmas not belonging to the S. citri species. Up to now, pMH1 had been found as a free 7-Kbp plasmid only in the S. citri strain MH. However, DNA sequences corresponding to the entire pMH1 DNA have been found to be integrated in the high-molecular-weight-DNA molecules and perhaps the chromosomal DNA itself of two other S. citri strains. DNA sequences hybridizable with pMH1 DNA have also been found to be integrated into high-molecular-weight-DNA molecules from several spiroplasma strains not belonging to the S. citri species.

Base Sequence↗

PubMed1992

The Mauriceville plasmid of Neurospora crassa: characterization of a novel reverse transcriptase that begins cDNA synthesis at the 3' end of template RNA.

The Mauriceville and Varkud plasmids are retroid elements that propagate in the mitochondria of some Neurospora spp. strains. Previous studies of endogenous reactions in ribonucleoprotein particle preparations suggested that the plasmids use a novel mechanism of reverse transcription that involves synthesis of a full-length minus-strand DNA beginning at the 3' end of the plasmid transcript, which has a 3' tRNA-like structure (M. T. R. Kuiper and A. M. Lambowitz, Cell 55:693-704, 1988). In this study, we developed procedures for releasing the Mauriceville plasmid reverse transcriptase from mitochondrial ribonucleoprotein particles and partially purifying it by heparin-Sepharose chromatography. By using these soluble preparations, we show directly that the Mauriceville plasmid reverse transcriptase synthesizes full-length cDNA copies of in vitro transcripts beginning at the 3' end and has a preference for transcripts having the 3' tRNA-like structure. Further, unlike retroviral reverse transcriptases, the Mauriceville plasmid reverse transcriptase begins cDNA synthesis directly opposite the 3'-terminal nucleotide of the template RNA. The ability to initiate cDNA synthesis directly at the 3' end of template RNAs may also be relevant to the mechanisms of reverse transcription used by LINEs, group II introns, and other non-long terminal repeat retroid elements.

Base Sequence↗

PubMed1985

Characterization of the plasmid-mediated beta-lactamase in Branhamella catarrhalis, with special reference to substrate affinity.

The plasmid-mediated Branhamella catarrhalis beta-lactamase BRO-1, also found in Moraxella nonliquefaciens, was characterized as regards substrate profile, isoelectric point and relative substrate affinity index (RSAI) to various substrates and compared in these aspects with the TEM-1 enzyme of Haemophilus influenzae. As measured by a biological assay and with high performance liquid chromatography (HPLC), BRO-1 was found to hydrolyse carbenicillin, mecillinam, methicillin and cefaclor with a higher rate than TEM-1. The only substrates having a relative rate of hydrolysis higher for TEM-1 than for BRO-1 were ampicillin and cephaloridine. The rates of hydrolysis registered with these two methods were comparable for all but 2 of 13 tested substrates. Isoelectric focusing yielded a main band at pH 5.6 and several satellite bands consistent with those reported by other authors for Branhamella enzymes having a substrate profile similar to that of BRO-1. A tenfold or higher difference in RSAI between BRO-1 and TEM-1 was recorded for five of the 15 compounds tested. BRO-1 seems to be the most common beta-lactamase in Bran. catarrhalis, irrespective of geographic origin. Its substrate profile, isoelectric pattern and RSAI differ from those of other known plasmid-mediated beta-lactamases described, thus justifying a specific designation.

Anti-Bacterial Agents↗

PubMed1998

Construction of improved plasmid vectors for promoter characterization in Pseudomonas aeruginosa and other gram-negative bacteria.

We report the construction of two broad host range promoter-probe plasmid vectors for rapid analysis of promoters in Gram-negative bacteria. The new vectors, pME4507 and pME4510, carry carbenicillin and gentamycin resistance genes, respectively, and are small sized (4 kb) with a flexible multiple cloning site to facilitate directional cloning of putative promoter elements. The vectors allow rapid plate-based screening for promoter activities, using beta-galactosidase as the reporter enzyme. In the absence of an inserted promoter fragment, they display very low background activity, making them a useful tool for analysis of low expression level promoters.

Carbenicillin↗

PubMed2001

Isolation and characterization of a Streptococcus thermophilus plasmid closely related to the pMV158 family.

Twenty-two Streptococcus thermophilus strains used for milk fermentations were analyzed for their plasmid content and 13 of them (59%) were found to contain one or two plasmids. Fifteen S. thermophilus plasmids were divided into four groups using DNA homology. Ten plasmids were classified within group A and they shared homologies with all the previously sequenced S. thermophilus plasmids. Three plasmids (group B) hybridized with each other and two plasmids only hybridized with themselves (groups C and D). Single-stranded DNA was detected within strains containing plasmids of groups A, C, and D, indicating that they replicate via a rolling-circle mode. The only plasmid of group C, named pSMQ172, was further characterized. This 4230-bp plasmid replicates in Escherichia coli, Lactococcus lactis, and Streptococcus salivarius and does not confer phage resistance. Comparisons with databases showed that pSMQ172 was related to pMV158 of Streptococcus agalactiae and to pSSU1 of Streptococcus suis. These results suggest that genetic exchanges may have occurred between pathogenic and nonpathogenic streptococci.

Base Sequence↗

PubMed2003

A small cryptic plasmid from Rhodococcus erythropolis: characterization and utility for gene expression.

Exploration of metabolically diverse rhodococci is generally hampered by the lack of genetic tools. A small cryptic plasmid (pAN12) isolated from Rhodococcus erythropolis strain AN12 was sequenced. Plasmid pAN12 encodes proteins that share homology to replication proteins and putative cell division proteins. Based on in vitro transposon mutagenesis, we determined that the Rep protein of pAN12 is essential for plasmid replication in Rhodococcus spp., and the putative cell division protein Div is important for plasmid stability. The pAN12 replicon is able to replicate in R. erythropolis strains AN12 and CW23 (ATCC 47072) and is compatible with the nocardiophage Q4 replicon present on a Rhodococcus shuttle plasmid pDA71. pAN12 appears to belong to the pIJ101/pJV1 family of rolling circle replication plasmids. Expression of an isoprenoid pathway gene ( dxs) on the pAN12-derived multicopy shuttle vector increased production of carotenoid pigments in R. erythropolis ATCC 47072.

Adenosine Triphosphatases↗

PubMed1979

Characterization of R-plasmids coding for ampicillin resistance from Salmonella species.

A sudden increase in the incidence of ampicillin resistance was observed among Salmonella species isolated within New Zealand in 1973--4. This increase was due mainly to the apperance and proliferation of Salmonella newington and Salmonella anatum serotypes resistant to ampicillin. The plasmid complements of 14 ampicillin-resistant S. newington and S. anatum isolates obtained from widely separated geographical areas within New Zealand between 1973 and 1974 were characterized by agarose gel electrophoresis. Each contained one or more plasmids ranging in molecular weight from 1.4 to 42 Mdal. Eleven isolates contained a self-transmissible plasmid of 33 Mdal which encoded resistance to ampicillin. After transfer to Escherichia coli, the 33 Mdal R-plasmids from each of these isolates were shown to be identical by restriction endonuclease analysis. The remaining three strains contained ampicillin R-plasmids having molecular weights of 35, 37.5 and 42 Mdal. These plasmids were shown by restriction endonuclease analysis to be related to the 33 Mdal R-plasmid. We conclude that the 33 Mdal plasmid and its derivatives were responsible for the increase in the incidence of ampicillin-resistant S. newington and S. anatum serotypes among the total Salmonella population.

Ampicillin↗

PubMed1981

Characterization of conjugative R plasmids belonging to the new incompatibility group IncU.

Five conjugative plasmids governing different antibiotic resistance patterns were identified in wild strains of enteric bacteria isolated in Czechoslovakia and the G.D.R. between 1976 and 1979. They have been characterized as members of the new incompatibility group IncU (reference plasmid RA3 from Japan). The molecular sizes of the IncU plasmids ranged between 18 and 37 megadaltons; their restriction fragment patterns indicated them to be distinct types.

Citrobacter↗

PubMed1988

Characterization of a circular plasmid from Borrelia burgdorferi, etiologic agent of Lyme disease.

Borrelia burgdorferi, the etiologic agent of Lyme disease, was recently shown to contain plasmid DNA. Two plasmid species have been described in strain CT1, a Wisconsin tick isolate: a 9.2-kilobase entity; and a larger, 70-kilobase entity. Characterization of the 9.2-kilobase entity by using DNase I and restriction endonucleases demonstrated that the plasmid is supercoiled and exists as a stable dimer in this strain. The role played by the plasmid in B. burgdorferi is unknown.

Borrelia↗

PubMed1998

[Characterization of the Streptomyces plasmid pSGL1].

The plasmid pSGL1(7.4 kb) which was isolated from antitumor antibiotic C-1027 producing strain Streptomyces globisporus C-1027 was characterized. It could be introduced into Streptomyces lividans by transformation. Following a series of deletion experiments, the minimal replication region was located within a 2.0 kb Sau 3AI fragment. pSGL1 is compatible with pIJ101. pSGL1 and its derivatives have a copy number of 70-250. Some of the derived plasmids such as pSGLN and pSGLS3 have suitable selectable antibiotic resistance marker and unique restriction endonuclease sites and may be potentially useful as cloning vectors. These plasmids may be used to design and construct efficient secretory expression vectors.

Aminoglycosides↗