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PubMed1973

Molecular and genetic studies of an R factor system consisting of independent transfer and drug resistance plasmids.

Certain genetic, structural, and biochemical properties of a class 2 R-factor system consisting of the conjugally proficient transfer plasmid I and the naturally occurring non-conjugative tetracycline (Tc) resistance plasmid 219 are reported. I and 219 exist as separate plasmid deoxyribonucleic acid (DNA) species in both Escherichia coli and Salmonella panama, having molecular weights of 42 x 10(6) and 5.8 x 10(6), respectively. The buoyant densities of I and 219 are 1.702 and 1.710 g/cm(3), respectively, in neutral cesium chloride. Although the Tc resistance plasmid is not transmissible in a normal conjugal mating, it is mobilized in a three-component mating by plasmid I and by certain other conjugative plasmids of the fi(+) or fi(-) phenotype. Mobilization does not appear to involve intermolecular recombination between plasmids, and no covalent linkage of resistance markers and fertility functions is observed. Transformation of CaCl(2)-treated E. coli by plasmid DNA is shown to be a useful procedure for studying the biological properties of different plasmid molecular species that have been fractionated in vitro, and for selectively inserting non-self-transmissible plasmids into specific bacterial strains. The effects of tetracycline on the rate of protein synthesis carried out by plasmid 219 were studied by using isolated E. coli minicells into which this plasmid had segregated. Consistent with the results of earlier investigations showing the inducibility of plasmid-mediated Tc resistance in E. coli, the antibiotic was observed to stimulate protein synthesis in minicells carrying the plasmid 219 and totally inhibit (3)H-leucine incorporation by minicells lacking the Tc resistance marker. Five discrete polypeptide species were synthesized by minicells carrying plasmid 219; exposure of minicells or parent bacteria to Tc resulted in specific and reproducible changes in polypeptide synthesis patterns.

Bacterial Proteins↗

PubMed1997

Multiplicity of the genes and plasmids conferring resistance to pristinamycin in staphylococci selected in an Algerian hospital.

In an Algerian hospital where pristinamycin (Pt) was extensively used for the treatment of chronic osteomyelitis and for prophylaxis in bone surgery, the prevalence of pristinamycin-resistant (PtR) staphylococci during a five-month period (20%) was higher than that among staphylococci isolated elsewhere in Algeria (4.5%). Analysis of 13 PtR staphylococci isolated in this hospital revealed a diversity of plasmids and genes conferring resistance to Pt and to related antibiotics. Most of the PtR staphylococci were unrelated: they belonged to either different taxa or types. Nevertheless, some of the unrelated staphylococci harboured structurally related plasmids carrying streptogramin resistance genes. Thus, these plasmids may have contributed to the dispersion of these genes.

Algeria↗

PubMed1979

Transposon A-generated mutations in the mercuric resistance genes of plasmid R100-1.

A series of 23 transposon 801(Tn801)-induced mutations of plasmid R100-1 from mercuric salts resistance to sensitivity was studied. Although Tn801 transposed frequently into the mer region of the plasmid, fine structural analysis showed that the site of insertion within mer varied. About one-half of the Tn801 insertion events also caused a deletion of greater than 1 megadalton. Genetic and restriction endonuclease EcoRI and BamHI analysis of the mutant plasmid deoxyribonucleic acid elucidated the organization of the mer operon and suggested the existence of a trans-acting regulatory factor governing resistance to mercuric salts. Tn801 insertions leading to mercuric sensitivity occurred in the restriction endonuclease fragments EcoRI-H and EcoRI-I. Regulatory mutations leading to a 50-fold-reduced synthesis of mercuric reductase enzyme occurred in two complementation classes thought to represent the gene for a trans-acting inducer molecule and a cis-acting operator-promoter sequence. Mutations leading to total loss of the enzyme mercuric reductase occurred on both the EcoRI-H and EcoRI-I fragments, showing that the structural gene for this enzyme (merA) bridges the EcoRI cleavage site separating the segments. Hypersensitivity to mercuric salts resulted when Tn801 insertion occurred in the reductase gene in the operatordistal portion of the operon. Hypersensitive cells inducibly bound three to five times more Hg2+ at low concentrations than did sensitive (plasmidless) cells. This finding led to the proposal that another gene (merT) controls uptake of Hg2+ by the cells. Transcription of the operon was deduced to start in the EcoRI-H fragment and to move into the EcoRI-I fragment of the plasmid genome.

DNA Transposable Elements↗

PubMed1992

Production of an immunoglobulin gene product by the plasmid expression vector L factor in mouse myeloma cells.

L factor, originally discovered in a subclone of mouse L cells, is a multicopy mammalian plasmid whose structure is related to that of polyoma. When a composite DNA consisting of L factor, pBR, bacterial neo, and an immunoglobulin (kappa) gene was introduced into mouse myeloma cells, the DNA was established as plasmids in the cells without rearrangement or integration into the chromosomes. The plasmid-bearing myeloma cells produced kappa mRNA and the gene product, kappa immunoglobulin, which were apparently derived from the gene located on plasmid L factor. These results suggest that L factor can be used as a plasmid expression vector for studies on gene expression and production of biologically active substances in mammalian cells.

Animals↗

PubMed1992

The enhanced mutagenic potential of the MucAB proteins correlates with the highly efficient processing of the MucA protein.

Inducible mutagenesis in Escherichia coli requires the direct action of the chromosomally encoded UmuDC proteins or functional homologs found on certain naturally occurring plasmids. Although structurally similar, the five umu-like operons that have been characterized at the molecular level vary in their ability to enhance cellular and phage mutagenesis; of these operons, the mucAB genes from the N-group plasmid pKM101 are the most efficient at promoting mutagenesis. During the mutagenic process, UmuD is posttranslationally processed to an active form, UmuD'. To explain the more potent mutagenic efficiency of mucAB compared with that of umuDC it has been suggested that unlike UmuD, intact MucA is functional for mutagenesis. To examine this possibility, we have overproduced and purified the MucA protein. Although functionally similar to UmuD, MucA was cleaved much more rapidly both in vitro and in vivo than UmuD. In vivo, restoration of mutagenesis functions to normally nonmutable recA430, recA433, recA435, or recA730 delta(umuDC)595::cat strains by either MucA+ or mutant MucA protein correlated with the appearance of the cleavage product, MucA'. These results suggest that most of the differences in mutagenic phenotype exhibited by MucAB and UmuDC correlate with the efficiency of posttranslational processing of MucA and UmuD rather than an inherent activity of the unprocessed proteins.

Bacterial Proteins↗

PubMed1991

The rate of processing and degradation of antisense RNAI regulates the replication of ColE1-type plasmids in vivo.

We show that the rate of degradation of RNAI, an anti-sense repressor of the replication primer RNAII, is a key element of control in the replication of ColE1-type plasmids in vivo. Cleavage of RNAI by RNAase E, a ribosomal RNA-processing enzyme encoded or controlled by the rne (also known as ams) locus, relieves repression by endonucleolytically converting RNAI to a very rapidly decaying product, pRNAI-5. A 5' triphosphate-terminated homolog of pRNAI-5 is degraded slowly and consequently inhibits replication. Nucleotide substitutions within the RNAase E cleavage sequence alter RNAI half-life and plasmid copy number, changing also the incompatibility phenotype. RNAI variants lacking the sequence cleaved by RNAase E are eliminated by growth rate-dependent degradation, resulting in growth-responsive control of plasmid replication and copy number.

Bacteriocin Plasmids↗

PubMed1994

Genetic structure of the Enterococcus faecalis plasmid pAD1-encoded cytolytic toxin system and its relationship to lantibiotic determinants.

Pheromone-responsive conjugative plasmids are unique to the species Enterococcus faecalis. Many pheromone-responsive plasmids, including those frequently isolated from sites of infection, express a novel cytolysin that possesses both hemolytic and bacteriocin activities. Further, this cytolysin has been shown to be a toxin in several disease models. In the present study, nucleotide sequence determination, mutagenesis, and complementation analysis were used to determine the organization of the E. faecalis plasmid pAD1 cytolysin determinant. Four open reading frames are required for expression of the cytolysin precursor (cylLL, cylLS, cylM, and cylB). The inferred products of two of these open reading frames, CyILL and CyILS, constitute the cytolysin precursor and bear structural resemblance to posttranslationally modified bacteriocins termed lantibiotics. Similarities between the organization of the E. faecalis cytolysin determinant and expression units for lantibiotics exist, indicating that the E. faecalis cytolysin represents a new branch of this class and is the first known to possess toxin activity.

Adenosine Triphosphate↗

PubMed1997

DNA inoculation with a plasmid vector carrying the hepatitis E virus structural protein gene induces immune response in mice.

The plasmid construct, pJHEV, containing the full-length open reading frame 2 (ORF-2) of hepatitis E virus (HEV) gene, expresses the HEV structural protein ORF-2 in Cos-7 cells under the control of a hCMV promoter. No ORF-2 protein could be detected in Cos-7 cells transfected with either vector alone or with a vector containing the ORF-2 of HEV in the incorrect orientation. The successful construct was further tested in BALB/c (H-2d) mice for the induction of an ORF-2 specific immune response. Intramuscular (i.m.) immunization of mice pretreated 24 h earlier with bupivacaine with the naked DNA construct elicited a humoral immune response in 80% and 100% of two separate groups of mice, respectively. No anti-ORF-2 responses were observed in mice immunized with the vector only. Sera from mice injected with pJHEV specifically recognized HEV ORF-2 structural protein expressed in recombinant baculovirus in an enzyme-linked-immunosorbent assay (ELISA) and Western blot. Anti-ORF-2 serum titers peaked at ca 1:5000 in the ELISA and 1:1000 in the Western blot. These titers have remained constant for over 12 months after the last boost of pJHEV. To our knowledge, this is the first report of the use of DNA-based immunization for the generation of an immune response to a HEV structural protein.

Animals↗

PubMed2001

Heterologous over-expression of alpha-1,6-fucosyltransferase from Rhizobium sp.: application to the synthesis of the trisaccharide beta-D-GlcNAc(1-->4)-[alpha-L-Fuc-(1-->6)]-D-GLcNAc, study of the acceptor specificity and evaluation of polyhydroxylated indolizidines as inhibitors.

An efficient heterologous expression system for overproduction of the enzyme alpha-1,6-Fucosyltransferase (alpha-1,6-FucT) from Rhizobium sp. has been developed. The gene codifying for the alpha-1,6-FucT was amplified by PCR using specific primers. After purification, the gene was cloned in the plasmid pKK223-3. The resulting plasmid, pKK1,6FucT, was transformed into the E. coli strain XL1-Blue MRF'. The protein was expressed both as inclusion bodies and in soluble form. Changing the induction time a five-fold increase of enzyme expressed in soluble form was obtained. In this way five units of enzyme alpha-1,6-FucT can be obtained per liter of culture. A crude preparation of the recombinant enzyme was used for the synthesis of the branched trisaccharide alpha-D-GlcNAc-(1-->4)-[alpha-L-Fuc-(1-->6)]-D-GlcNAc (3), from chitobiose (2) and GDP-Fucose (1). After purification, the trisaccharide 3 was obtained in a 84% overall yield. In order to elucidate the structural requirements for the acceptors, the specificity of the enzyme was studied towards mono-, di- and trisaccharides, which are structurally related to chitobiose. The enzyme uses, among others, the disaccharide N-acetyl lactosamine as a good substrate; the monosaccharide GlcNAc is a weak acceptor. Finally, several racemic polyhydroxylated indolizidines have been tested as potential inhibitors of the enzyme. Indolizidine 21 was the best inhibitor with an IC50 of 4.5 x 10(-5) M. Interestingly, this compound turned out to be the best mimic for the structural features of the fucose moiety in the presumed transition state.

Acetylglucosaminidase↗

PubMed1988

Role of antibodies in the opsonization of Yersinia spp.

We have determined the opsonic capacity of specific antibodies in patient sera obtained after Yersinia infection. The results indicate that Yersinia antibodies lead to complement activation through the classical pathway, thus overcoming the inhibition of complement-mediated opsonization in the absence of specific antibodies provided by the virulence plasmid in Yersinia enterocolitica and Yersinia pseudotuberculosis. Further, antibodies against plasmid-encoded structures, the Yersinia outer membrane proteins (YOPs), are not necessary in this effect. This is indicated by two facts. (i) Monoclonal antibodies directed against the O polysaccharide of Y. enterocolitica O:3 are capable of opsonizing the plasmid-containing bacteria through C1q binding. (ii) Rabbit antisera show opsonic activity when obtained by immunization both with plasmid-containing Y. enterocolitica expressing the YOPs and a plasmid-cured variant not expressing these proteins.

Antibodies, Bacterial↗

PubMed2004

Plasmid-DNA loaded chitosan microspheres for in vitro IL-2 expression.

Interleukin-2 (IL-2) expression plasmid (pCXWN-hIL-2) loaded chitosan microspheres were evaluated for using in gene-based immunotherapy. Chitosan microspheres containing pCXWN-hIL-2 were prepared by using a precipitation technique. In addition, the effects of different factors such as the concentration (0.35-0.70%) and the molecular weight of chitosan (low and medium molecular weights), the plasmid amount (5-10 microg/ml) and the presence of glutaraldehyde during the encapsulation process, on microsphere characteristics were investigated. The size of microspheres changed between 1.45 and 2.00 microm. All the formulation factors affected the size of microspheres. The structure of plasmid remained unchanged during the encapsulation process and the release studies. Plasmid encapsulation efficiency of chitosan microspheres was high (82-92%). The zeta potential values of microspheres was approximately +5.2 to +12.4 mV. In vitro release properties of microspheres changed with formulation variables. In vitro release of DNA changed with the concentration and molecular weight of chitosan and initial plasmid amount. Addition of glutaraldehyde is not necessary for a formulation. MAT-LyLu, the rat prostate adenocarcinoma cell line, was used for the determination of the in vitro transfectional activity of IL-2 encoding plasmid DNA loaded chitosan microspheres and the level of IL-2 expressed into the cells was assayed using a ELISA kit. High level of IL-2 expression was obtained with plasmid-loaded chitosan microspheres. Microspheres showed similar IL-2 production as lipofectin. The molecular weight of chitosan used and the amount of plasmid influenced the in vitro IL-2 production in the cells. Encapsulation of IL-2 encoding gene into chitosan microspheres might be a useful strategy to increase the expression and to control the delivery of cytokine gene to cells.

Animals↗

PubMed1996

Identification of multiple plasmids released from recombinant genomes of Hansenula polymorpha by transformation of Escherichia coli.

Total DNAs isolated from two Hansenula polymorpha (Pichia angusta) strains having chromosomal single or tandem multiple integrations of a pUC18-derived expression plasmid produced Escherichia coli transformants which contained plasmids of different size and/or organization than that of the expression plasmid. Evidence that plasmid-like structures are formed in H. polymorpha and that their formation is stimulated by DNA damage is presented in this study.

Escherichia coli↗

PubMed1992

Existence in Escherichia coli of a mechanism that generates 'staggered' head-to-head dimers of plasmid DNA; possible involvement of the Tn3 transposase.

The structures of two plasmids, one found in one of the transformants with putative products of an in vitro DNA rearrangement reaction containing the Tn3 transposase, and another found as a spontaneous tnpA negative derivative of an overproducer of the Tn3 transposase, have been elucidated. They both had shown non-conventional results in restriction enzyme analysis. For the determination of the structures, we used restriction enzyme analysis, denaturation and renaturation experiments, and DNA nucleotide sequencing. The structures turned out to be 'staggered' head-to-head dimers of the original monomer plasmids, containing gigantic inverted repeats separated by two identical spacers which are also in inverted orientations themselves. Two alternative models for the mode of origin of such a structure, a bimolecular model and a unimolecular one, are discussed. The circumstances in which these two plasmids occurred suggest possible involvement of the Tn3 transposase in their generation.

DNA Transposable Elements↗

PubMed1997

Microbial linear plasmids.

While plasmids were originally considered to be generally circular until almost two decades ago, linear elements were reported to exist as well. They are now known to be common genetic elements in both, pro- and eukaryotes. Two types of linear plasmids exist, the so-called hairpin plasmids with covalently closed ends and those with proteins bound to their 5' termini. Hairpin plasmids are common in human-pathogenic Borrelia spirochetes, in which they are instrumental in escape from the immunological response; cryptic hairpin elements are present in mitochondria of the plant pathogenic fungus Rhizoctonia solani. Plasmids with 5' attached proteins constitute the largest group. In actinomycetous bacteria they are conjugative and usually confer advantageous phenotypes, e.g. formation of antibiotics, degradation of xenobiotics, heavy-metal resistance and growth on hydrogen as the sole energy source. In contrast, the majority of linear plasmids from eukaryotes are cryptic, with only a few exceptions. In some yeasts a killer phenotype may be associated, the most thoroughly investigated elements being those from Kluyveromyces lactis killer strains. In Neurospora spp. and in Podospora anserina, senescence and longevity respectively are correlated with linear plasmids. This review focuses on the biology of linear plasmids, their environmental significance and their use as tools in molecular and applied microbiology.

Bacteria↗

PubMed1990

[Pili determined by the tra genes of F-like plasmids].

The study of structural and functional features of plasmid-specific pili synthetized by E. coli cells under control of 27 F-like plasmids was performed. All the plasmids determined the pili of "flexible" type which were classified into 3 groups on the basis of difference in cell sensitivity to pili-specific phages f1, f2, Q. The possible role of transposons in the variation of pili functional properties was supposed.

Conjugation, Genetic↗

PubMed1991

Comparative analysis of the replication regions of IncB, IncK, and IncZ plasmids.

Minireplicons from the I-complex plasmids R387 (IncK) and pIE545 (IncZ) were constructed, and the nucleotide sequences of their replication regions were compared with that of the B plasmid, pMU720. The coding sequence of the putative replication protein, RepA, of each plasmid was located. RepA of K and B plasmids were homologous, whereas RepA of Z resembled RepA1 of FII plasmid. Sequences upstream of RepA were conserved in the three I-complex plasmids. Group B and Z plasmids were incompatible.

Bacterial Proteins↗

PubMed1982

Conservative integration of bacteriophage Mu DNA into pBR322 plasmid.

In order to clarify the first step in Mu integrative recombination, we have infected a bacterial strain harboring the plasmid pBR322 and isolated Mu DNA in a supercoiled form associated with this plasmid. These structures show an association of Mu with PBR322 without any preliminary replication.

Bacteriophage mu↗

PubMed2005

Construction of plasmid-based expression vectors for Bacillus subtilis exhibiting full structural stability.

A series of plasmid-based expression vectors have been constructed allowing stable intracellular expression of recombinant proteins in Bacillus subtilis strains. These expression vectors are based on the recently described Escherichia coli-B. subtilis shuttle vector pMTLBS72 which replicates as theta circles. Besides the weak constitutive promoter P(lepA), we inserted three different controllable promoters: P(gsiB) which can be induced by heat and acid shock, and by ethanol, P(xylA) and P(spac) which respond to the addition of xylose and IPTG, respectively. The versatility of these expression vectors was demonstrated by fusing their promoters to a reporter gene and by overexpression of the HtpG protein with three of them. All recombinant vectors exhibited full structural stability.

Bacillus subtilis↗