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PubMed1996

Characterization and sequence analysis of a stable cryptic plasmid from Enterococcus faecium 226 and development of a stable cloning vector.

A small cryptic plasmid, pMBB1, isolated from Enterococcus faecium 226 was characterized. The plasmid contained an extremely stable replicon which has limited homology to the lactococcal plasmid pCI305. Sequence analysis of the replicon detected one open reading frame of 822 bp capable of encoding a 32-kDa protein. No detectable single-stranded intermediates were found for the replicon, suggesting that pMBB1 may be included in the same family as pCI305, although pCI305 exhibits a more narrow host range. A small stably maintained vector able to replicate in a variety of lactic acid bacteria, containing a large multiple cloning region, was constructed by using the pMBB1 replicon.

Amino Acid Sequence↗
PubMed2006

Genetic and biochemical characterization of the Streptococcus pneumoniae PcrA helicase and its role in plasmid rolling circle replication.

PcrA is a chromosomally encoded DNA helicase of gram-positive bacteria involved in replication of rolling circle replicating plasmids. Efficient interaction between PcrA and the plasmid-encoded replication initiator (Rep) protein is considered a requirement for the plasmid to replicate in a given host, and thus, the ability of a Rep protein to interact with heterologous PcrA helicases has been invoked as a determinant of plasmid promiscuity. We characterized transcription of the Streptococcus pneumoniae pcrA gene in its genetic context and studied the biochemical properties of its product, the PcrA(Spn) helicase. Transcription of the pneumococcal pcrA gene was directed by promoter Pa, consisting of an extended -10 box. Promoter Pa also accounted for expression of a second essential gene, radC, which was transcribed with much lower efficiency than pcrA, probably due to the presence of a terminator/attenuator sequence located between the two genes. PcrA(Spn) displayed single-stranded DNA-dependent ATPase activity. PcrA(Spn) showed 5'-->3' and 3'-->5' helicase activities and bound efficiently to partially duplex DNA containing a hairpin structure adjacent to a 6-nucleotide 5' or 3' single-stranded tail and one unpaired (flap) nucleotide in the complementary strand. PcrA(Spn) interacted specifically with RepC, the initiator of staphylococcal plasmid pT181. Although the pneumococcal helicase was able to initiate unwinding of the RepC-nicked pT181 DNA, it was much less processive in this activity than the cognate staphylococcal PcrA protein. Accordingly, PcrA(Spn) was inefficient in in vitro replication of pT181, and perhaps as a consequence, this plasmid could not be established in S. pneumoniae.

Adenosine Triphosphatases↗
PubMed1993

Characterization of Salmonella virchow phage types by plasmid profile and IS200 distribution.

The type strains of the 57 phage types of Salmonella virchow have been characterized by plasmid profile and by distribution of the insertion sequence IS200. Thirty-two strains carried plasmids and 21 profile types were identified; 17 strains were resistant to antimicrobial agents. In contrast only six of the type strains carried IS200 elements and three patterns were identified. Within Salm. virchow phage type 31, five of 10 wild-type isolates carried plasmids and two plasmid profiles were identified; in contrast, an IS200 element was identified in the genome of only one of these strains. It is concluded that for Salm. virchow, IS200 is unlikely to significantly extend the degree of discrimination achieved by phage typing which may be supplemented when appropriate by plasmid profile typing.

Animals↗
PubMedDate not supplied

Low-pH-sensitive PEG-stabilized plasmid-lipid nanoparticles: preparation and characterization.

The acid-labile poly(ethyleneglycol)-diorthoester-distearoylglycerol lipid (POD), was used with a cationic lipid-phosphatidylethanolamine mixture to prepare stabilized plasmid-lipid nanoparticles (POD SPLP) that could mediate gene transfer in vitro by a pH triggered escape from the endosome. Nanoparticles of 60 nm diameter were prepared at pH 8.5 using a detergent dialysis method. The DNA encapsulation efficiency in the nanoparticles was optimal between 10 and 13 mol % ratio of cationic lipid and at a POD content of 20 mol %. The apparent zeta potential of the nanoparticles at 1 mM salt and pH 7.5 was positive, indicating cationic lipid on the external surface. However, the external layer of the nanoparticles was depleted in the cationic component compared to the starting mole ratio. Low pH sensitivity of the POD SPLP was characterized by a lag phase followed by a rapid collapse; at pH 5.3 the nanoparticles collapsed in 100 min. Nanoparticles prepared from a pH-insensitive PEG-lipid, PEG-distearoylglycerol had similar physicochemical characteristics as the POD SPLP but did not collapse at low pH. The POD SPLP had up to 3 orders of magnitude greater gene transfer activity than did the pH-insensitive nanoparticles. Both the pH-sensitive and pH-insensitive nanoparticles were internalized to a qualitatively similar extent in a punctate pattern into cultured cells within 2 h of incubation with the cells; thus, increased gene transfer of the POD SPLP was due to a more rapid escape from the endosome rather than to greater cell association of these nanoparticles. These results suggest that the pH-sensitive stabilized plasmid-lipid nanoparticles may be a useful component of a synthetic vector for parenterally administered gene therapy.

Animals↗
PubMed1996

Analysis of linear plasmid dimers in Borrelia burgdorferi sensu lato isolates: implications concerning the potential mechanism of linear plasmid replication.

The Borrelia genome is composed of a linear chromosome and a number of variable circular and linear plasmids. Atypically large linear plasmids of 92 to 105 kb have been identified in several Borrelia burgdorferi sensu lato isolates and characterized. These plasmids carry the p27 and ospAB genes, which in other isolates reside on a 50-kb plasmid. Here we demonstrate that these plasmids are dimers of the 50-kb ospAB plasmid (pAB50). The 94-kb plasmid from isolate VS116, pVS94, was an exception and did not hybridize with any plasmid gene probes. When this plasmid was used as a probe, homologous sequences in other isolates were not detected, suggesting that it is unique to isolate VS116. These analyses provide insight into the mechanism of linear plasmid replication and the mechanisms by which plasmid variability can arise.

Base Sequence↗
PubMed1985

[Plasmid resistance to antiseptics. Demonstration of methods].

Susceptibility to chlorhexidine, benzalkonium chloride and mercury chloride was studied for 70 Gram negative strains (56 Enterobacteriaceae and 14 Pseudomonas aeruginosa) recovered from humans or animals. Minimal inhibitory concentration (MIC) was determined on solid agar (replica plating) and in a liquid medium (laser nephelometry). For resistant strains, plasmidic mediation was looked for using conjugation. Isolation of lysates by the miniprep analytic method and physicochemical characterization of plasmids by agar electrophoresis were carried out using conjugative strains. MICs obtained using the two methods differed by one dilution at the most. Both methods yielded reproducible results. Regardless of the origin of strains, a resistant population was identified only for mercury chloride. This resistance was plasmid-mediated and transferred singly or associated with resistance to other antibiotics.

Anti-Infective Agents, Local↗
PubMed1982

Cloning, partial sequencing, and expression of glyceraldehyde-3-phosphate dehydrogenase gene in chick embryonic heart muscle cells.

Two recombinant plasmids containing structural gene sequences of chick embryonic heart glyceraldehyde-3-phosphate dehydrogenase (GAP dehydrogenase) were constructed and characterized. The plasmids pGAP 30 and pGAP 36 have inserts of 1200 and 950 base pairs, respectively. The identity of the clones was established by hybrid-arrested and hybrid-selection translation assays, and by immunoprecipitation of hybrid-selected translation product with GaP dehydrogenase antiserum. Hybridization of labeled pGAP 30 DNA to size-fractionated chick heart poly(A) RNA occurred at the region on the gel corresponding to the mobility of GAP dehydrogenase mRNA. Base sequence analysis of plasmid pGAP 30 and the comparison of the amino acid sequence derived from it with that of pig muscle GAP dehydrogenase revealed that the amino acid sequence of GAP dehydrogenase is strictly conserved between the chick and pig muscle tissues. Expression of GAP dehydrogenase mRNA in developing chick heart cells in cultures was monitored by in situ hybridization. The GAP dehydrogenase mRNA was present in 5-h-old dividing myoblasts, in contrast to mRNAs specific for contractile proteins, which appear late in myoblast development paralleling morphogenetic differentiation of myoblasts into myocytes (Jakowlew, S. B., Khandekar, P., Datta, K., Narula, S. K., Arnold, H. H., and Siddiqui, M. A. Q. (1982) J. Mol. Biol. 156, 673-682).

Amino Acid Sequence↗
PubMed1988

Cloning and preliminary characterization of the streptothricin resistance determinants of the transposons Tn1825 and Tn1826.

Streptothricin resistance determinants have been cloned from the transposons Tn1825 and Tn1826 to the vector plasmid pUC8. The recombinant plasmids were characterized with respect to their physical structure. The resistance properties of their hosts were characterized with respect to the minimal inhibitory concentration of streptothricin and the activity of the streptothricin acetyltransferase. The proteins encoded by the cloned resistance determinants were analysed in a maxicell system. The results of our investigations suggest that the resistance to streptothricin is mediated by the activity of a streptothricin acetyltransferase with both, Tn1825 and Tn1826. However, the resistance determinant of Tn1825 is more complex than Tn1826 and additional functions involved in realizing the resistance phenotype must be recognized.

Acetyltransferases↗
PubMed1995

Enterotoxin production and plasmid profiles in Salmonella typhimurium isolated from man.

One hundred twenty-six isolates of Salmonella typhimurium from various clinical sources were tested for enterotoxin production and characterization of plasmid profile. Cell-free culture supernates and polymyxin B extracts of all the strains were assayed by rabbit ileal loop and skin permeability tests. Enterotoxic activity was detected in culture supernates of 32 strains. Twenty-one strains by both rabbit ileal loop and skin permeability tests, nine strains by skin permeability test, and two strains by rabbit ileal loop test were positive. Live culture of three enterotoxic strains, positive in culture supernates produced ileal secretion. Polymyxin B extracts from 6 hours and 18 hours broth cultures of all the strains were devoid of enterotoxicity. Ileal mucosa exposed to culture supernate of enterotoxigenic strains showed swollen and blunted villi with submucosal oedema while those exposed to polymyxin B extracts showed shortening of villi and sloughing of epithelial lining. Plasmid profiles of enterotoxigenic strains were heterogenous and grouped into 20 different profiles. No correlation could be established between plasmid profile, R-pattern, and enterotoxin production.

Animals↗
PubMed1976

Phage group II staphylococcal strains with chromosomal and extrachromosomal genes for exfoliative toxin production.

Staphylococcal phage group 2 strain UT0007 was previously shown to contain a high-molecular-weight plasmid containing genes for exfoliative toxin (ET) and bacteriocin production. Phage group 2 strains UT0002 and UT0003 (Tox+Bac-) underwent a twofold and ninefold loss of ET activity, respectively, after growth at 44 C for 18 h. Strain UT0002 also lost total bacteriocin activity. Both strains contained (i) a 56S plasmid that was lost from those substrains showing reduced ET activity and (ii) a 21S plasmid with a gene for cadmium resistance that could be transduced into two recipient strains. Since the ET plasmid-negative substrains still made ET, it was postulated that this residual toxin was made from chromosomal genes. In characterizing the plasmid species from strains UT0002 and UT0003, the 21S but little or no 56S plasmid deoxyribonucleic acid could be isolated after centrifugation of cleared lysates from these strains on dye-buoyant density gradients. Treatment of cleared lysates from strain UT0002 with ethidium bromide, Pronase, or sodium dodecyl sulfate, but not heat at 60 C, induced conversion of the 56S closed circular ET plasmid to a 38S open circular form as determined after centrifugation on 5 to 20% neutral sucrose gradients.

Cadmium↗
PubMed1995

Plasmid content in different serovars of Salmonella isolated in Sicily: an ecology study.

This study determined plasmid relative abundance and distribution among a representative collection of Sicilian strains of Salmonella, with the purpose of gaining more insight into bacterial and plasmid ecology. 161 Salmonella strains of different serovars, isolated in Sicily from human and environmental sources between 1981 and 1985 were characterized by plasmid profile typing. Two main sets of Salmonella serovars were identified, one composed by plasmid-free serovars, rarely associated with clinical salmonellosis but typical of environmental niches; the other comprising the serovars usually encountered as major agents of human and animal salmonellosis. The biological relevance of the baseline information so provided on the genetic profiles of different serovars in Sicily is discussed.

Animals↗
PubMed1997

pPV: a novel IRES-containing vector to facilitate plasmid immunization and antibody response characterization.

BACKGROUND: The ability to derive immunological reagents for basic and applied research in a timely fashion is a basic requirement of many research projects and is becoming increasingly important as the number of novel gene products of potential interest continues to evolve rapidly. DNA immunization provides a means of facilitating the production of antibody reagents by circumventing the need to derive either purified protein or define peptides before initiating an in vivo immunization protocol. OBJECTIVES: The DNA construct pPV, for plasmid vaccination, has been designed to facilitate the generation and characterization of antibody reagents against either random or defined molecular targets. STUDY DESIGN: pPV incorporates mammalian regulatory and structural features that promote expression of a bifunctional messenger RNA (mRNA) from a single promoter within mammalian cells both in vitro and in vivo. The bifunctional mRNA encodes a control epitope (human IL5), and the 'test' epitope expressed as a tagged recombinant polypeptide in either a random 'shot-gun' mode or a predetermined fashion. In addition, to aid subsequent characterization of antibody responses elicited in vivo, a T7 promoter is included to enable in vitro expression of tagged recombinant polypeptides. RESULTS: The utility and functionality of pPV for the in vitro expression of recombinant protein and the in vivo elicitation of antibody responses is illustrated using a defined 'test' epitope, human proIL1 beta. CONCLUSION: It is anticipated pPV will find particular utility in the future rapid generation and characterization of antibody reagents against the plethora of novel genes emerging from ongoing genomics activity in a directed or genome wide fashion.

Amino Acid Sequence↗
PubMed1977

Construction and restriction endonuclease mapping of hybrid plasmids containing Saccharomyces cerevisiae ribosomal DNA.

Fragments produced by partial digestion of Saccharomyces cerevisiae ribosomal DNA (rDNA) with the restriction endonuclease EcoRI were ligated in vitro to the bacterial plasmid RSF2124. The resulting hybrid plasmids were cloned in Escherichia coli. Three hybrid plasmids which contain at least one intact repetitive unit of the multiple, tandem sequences of the yeast rDNA genes have been further characterized. These plasmids have been used to construct a map of the EcoRI, SmaI, HindII and HindIII restriction sites in the individual repetitive units of yeast rDNA.

DNA↗
PubMed1989

Genetic relatedness of antibiotic resistance determinants in multiply resistant Hemophilus influenzae.

Antimicrobial resistance determinants and plasmids present in 10 multiply antibiotic-resistant strains of Hemophilus influenzae isolated from patients in different geographic regions of Spain were characterized. Conjugative plasmids with molecular sizes of 38-50 MDa encoded resistance to ampicillin, chloramphenicol, kanamycin, streptomycin, sulfamethoxazole, and tetracycline. Trimethoprim resistance was not linked to the other antibiotic resistance determinants and trimethoprim-resistant transconjugants and transformants lacked detectable plasmid DNA, suggesting that this determinant is chromosomal. Restriction endonuclease analysis revealed similarities among the plasmids, but several restriction patterns could be distinguished. Three hybridization patterns were found with DNA probes coding for H. parainfluenzae beta-lactamase and chloramphenicol-acetyltransferase. Resistance to kanamycin was due to drug modification by aminoglycoside-phosphotransferase (3')I. In Spain, it appears that multiple antibiotic resistance phenotypes in H. influenzae did not arise from acquisition of a single R plasmid; rather, both plasmid and chromosomal resistance evolved independently from several sources.

Blotting, Southern↗
PubMed1974

Isolation of a lambda dv plasmid carrying the bacterial gal operon.

A lambdadvgal plasmid carrying genes for controlled plasmid replication from phage lambda and the bacterial gal operon was isolated as a deletion mutant of phage lambdagalq4, which carries the gal operon between lambda genes P and Q. The plasmid DNA was found in cell extracts as covalently closed circular molecules. The plasmid was characterized by using genetic crosses, digestion with the specific endonuclease EcoRI, sucrose gradient centrifugation, and electron microscopy. In one clone analyzed, the plasmid was a complete dimer (O(lambda)P(lambda)galO(lambda)P(lambda)gal); in a subclone derived from it, the plasmid was a partial dimer with only one copy of gal (O(lambda)P(lambda)O(lambda)P(lambda)gal). The partial dimer may be a recombination product of the complete dimer, since test crosses show that the gal and lambda sequences in the plasmid can be separated by recombination. Analyses of the EcoRI digests of plasmid DNAs indicated one cleavage site per lambda gene sequence and none in the gal operon. A lambdadvgal monomer was approximately 6.7 x 10(6) daltons and the lambda gene and gal components were 3.9 x 10(6) and 2.8 x 10(6) daltons, respectively. The lambdadvgal plasmid can be introduced into a new bacterial host by transfection at an efficiency of 10(-6) per DNA molecule.

Centrifugation, Density Gradient↗
PubMed1982

Effects of neighboring DNA homopolymers on the biochemical and physical properties of the Escherichia coli lactose promoter. I. Cloning and characterization studies.

To assess the role of neighboring DNA sequences in gene regulation, poly(dA).poly(dT) and poly(dG).poly(dC) were cloned adjacent to promoters of the lactose control region. Recombinant plasmids were constructed which were suitable for large scale purification of restriction fragments containing these promoters, 95-base pair (bp) AluI fragments containing the lack operator and promoter for the lac wild type and for the catabolite gene activating the protein-independent mutant, lac UV5, were cloned into pBR322. Homopolymers of varying lengths were inserted into the -60 region of these promoters using recombinant DNA techniques. Six of the recombinant plasmids were chosen for detailed analysis: wild type (wt); wt-AT, containing 70 bp of poly(dA).poly(dT); wt-GC, containing 23 bp of poly(dG).poly(dC); UV5; UV5-AT, containing 70 bp of poly(dA).poly(dT) and finally UV5-GC, containing 43 bp of poly(dG).poly(dC). These plasmids were characterized by restriction mapping and DNA sequencing. The effects of the DNA homopolymers on the interaction of the Escherichia coli RNA polymerase with the promoters were studied using nitrocellulose filter binding. The results show that poly(dA).poly(dT) increases the level of RNA polymerase binding, whereas poly(dG).poly(dC) has no detectable effect.

Base Sequence↗
PubMed1984

Characterization of an R-plasmid dihydrofolate reductase with a monomeric structure.

A plasmid-encoded dihydrofolate reductase that originated in a clinical isolate of Salmonella typhimurium (phage type 179) moderately resistant to trimethoprim has been isolated and characterized. The dihydrofolate reductase (called type III) was purified to homogeneity using a combination of gel filtration, hydrophobic chromatography, and methotrexate affinity chromatography. Polyacrylamide gel electrophoresis under denaturing and nondenaturing conditions indicated that the enzyme is a 16,900 molecular weight monomeric protein. Kinetic analyses showed that trimethoprim is a relatively tight binding inhibitor (Ki = 19 nM) competitive with dihydrofolate. The enzyme is also extremely sensitive to methotrexate inhibition (Ki = 9 pM) and has a high affinity for dihydrofolate (Km = 0.4 microM). The sequence of the first 20 NH2-terminal residues of the protein shows 50% homology with the trimethoprim-sensitive chromosomal Escherichia coli dihydrofolate reductase and suggests that the two enzymes may be closely related. This is the first example of a plasmid encoding for a monomeric dihydrofolate reductase only moderately resistant to trimethoprim, and a resistance mechanism, dependent in part on the high dihydrofolate affinity of the type III enzyme, is proposed.

Amino Acid Sequence↗
PubMed2000

Molecular and evolutionary characterization of the cp32/18 family of supercoiled plasmids in Borrelia burgdorferi 297.

In this study, we characterized seven members of the cp32/18 family of supercoiled plasmids in Borrelia burgdorferi 297. Complete sequence analysis of a 21-kb plasmid (cp18-2) confirmed that the strain 297 plasmids are similar in overall content and organization to their B31 counterparts. Of the 31 open reading frames (ORFs) in cp18-2, only three showed sequence relatedness to proteins with known functions, and only one, a ParA/SopA ortholog, was related to nonborrelial polypeptides. Besides the lipoproteins, none of the ORFs appeared likely to encode a surface-exposed protein. Comparison with the B31 genomic sequence indicated that paralogs for most of the ORFs in cp18-2 can be identified on other genetic elements. cp18-2 was found to lack a 9- to 10-kb fragment present in the 32-kb homologs which, by extrapolation from the B31 cp32 sequences, contains at least 15 genes presumed to be unnecessary for plasmid maintenance. Sequence analysis of the lipoprotein-encoding variable loci provided evidence that recombinatorial processes within these regions may result in the acquisition of exogenous DNA. Pairwise analysis with random shuffling revealed that the multiple lipoproteins (Mlp; formerly designated 2.9 LPs) fall into two distinct homology groups which appear to have arisen by gene fusion events similar to those recently proposed to have generated the three OspE, OspF, and Elp lipoprotein families (D. R. Akins, M. J. Caimano, X. Yang, F. Cerna, M. V. Norgard, and J. D. Radolf, Infect. Immun. 67:1526-1532, 1999). Comparative analysis of the variable regions also indicated that recombination within the loci of each plasmid may occur independently. Last, comparison of variable loci revealed that the cp32/18 plasmid complements of the B31 and 297 isolates differ substantially, indicating that the two strains have been subject to divergent adaptive pressures. In addition to providing evidence for two different types of recombinatorial events involving cp32/18 plasmids, these findings underscore the need for genetic analysis of diverse borrelial isolates in order to elucidate the Lyme disease spirochete's complex parasitic strategies.

Amino Acid Sequence↗