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PubMedDate not supplied

Plasmid-bearing, plasmid-free organisms competing for two complementary nutrients in a chemostat.

A model of competition for two complementary nutrients between plasmid-bearing and plasmid-free organisms in a chemostat is proposed. A rigorous mathematical analysis of the global asymptotic behavior of the model is presented. The work extends the model of competition for a single-limited nutrient studied by Stephanopoulos and Lapidus [Chem. Engng. Sci. 443 (1988) 49] and Hsu, Waltman and Wolkowicz [J. Math. Biol. 32 (1994) 731].

Bacteria↗
PubMed1994

Polyamine-induced Z-DNA conformation in plasmids containing (dA-dC)n.(dG-dT)n inserts and increased binding of lupus autoantibodies to the Z-DNA form of plasmids.

Blocks of potential Z-DNA-forming (dA-dC)n.(dG-dT)n sequences are ubiquitous in eukaryotic genomes. We examined whether naturally occurring polyamines, putrescine, spermidine and spermine, could provoke the Z-DNA conformation in plasmids pDHf2 and pDHf14 with 23 and 60 bp inserts respectively of (dA-dC)n.(dG-dT)n sequences using an e.l.i.s.a. Spermidine and spermine could provoke Z-DNA conformation in these plasmids, but putrescine was ineffective. For pDHf2 and pDHf14, the concentration of spermidine at the midpoint of B-DNA to Z-DNA transition was 25 microM, whereas that of spermine was 16 microM. Polyamine structural specificity was evident in the ability of spermidine homologues to induce Z-DNA. Inorganic cations, Co(NH3)6(3+) and Ru(NH3)6(3+), were ineffective. Our experiments also showed increased binding of anti-DNA autoantibodies from lupus patients as well as autoimmune MRL-lpr/lpr mice to pDHf2 and pDHf14 in the presence of polyamines. These data demonstrate that small blocks of (dA-dC)n.(dG-dT)n sequences could assume the Z-DNA conformation in the presence of natural polyamines. Increased concentrations of polyamines in the sera of lupus patients might facilitate immune complex-formation involving circulating DNA and anti-Z-DNA antibodies.

Animals↗
PubMed1996

Plasmid transfer and expression of the transfer (tra) gene product of plasmid pIJ101 are temporally regulated during the Streptomyces lividans life cycle.

We previously have shown that a chromosomally integrated copy of the tra gene of plasmid pIJ101 under the control of the KorA repressor protein, which regulates transcription of tra as well as its own synthesis, can promote the conjugal transfer of both chromosomal and plasmid genes in Streptomyces lividans. Using an antibody generated against a fusion protein containing the C-terminal portion of Tra, we show here that this essential conjugation protein is present in membrane fractions of both surface-grown S. lividans, which mate readily, and of cells grown in liquid culture, where mating has not been found. Expression of Tra during the S. lividans life cycle was temporally regulated and was reduced late during vegetative growth so that little or no Tra protein was detected in cells as they began to differentiate morphologically and produce secondary metabolites. Comparison of the membrane concentration of Tra protein with tra mRNA concentration during the S. lividans life cycle indicated that the disappearance of Tra is post-transcriptionally controlled and thus is not mediated by KorA. The results of 'interrupted mating' experiments, together with the time of appearance of Tra in S. lividans membranes, indicate that the intermycelial transfer of pIJ101 in S. lividans is complete by the onset of cellular differentiation.

Bacterial Proteins↗
PubMed1999

Conjugative pili of IncP plasmids, and the Ti plasmid T pilus are composed of cyclic subunits.

TrbC propilin is the precursor of the pilin subunit TrbC of IncP conjugative pili in Escherichia coli. Likewise, its homologue, VirB2 propilin, is processed into T pilin of the Ti plasmid T pilus in Agrobacterium tumefaciens. TrbC and VirB2 propilin are truncated post-translationally at the N terminus by the removal of a 36/47-residue leader peptide, respectively. TrbC propilin undergoes a second processing step by the removal of 27 residues at the C terminus by host-encoded functions followed by the excision of four additional C-terminal residues by a plasmid-borne serine protease. The final product TrbC of 78 residues is cyclized via an intramolecular covalent head-to-tail peptide bond. The T pilin does not undergo additional truncation but is likewise cyclized. The circular structures of these pilins, as verified by mass spectrometry, represent novel primary configurations that conform and assemble into the conjugative apparatus.

Agrobacterium tumefaciens↗
PubMed2003

A specific region in the N terminus of a replication initiation protein of plasmid RK2 is required for recruitment of Pseudomonas aeruginosa DnaB helicase to the plasmid origin.

Broad host range plasmid RK2 encodes two versions of its essential replication initiation protein, TrfA, using in-frame translational starts spaced 97 amino acids apart. The smaller protein, TrfA-33, is sufficient for plasmid replication in many bacterial hosts. Efficient replication in Pseudomonas aeruginosa, however, specifically requires the larger TrfA-44 protein. With the aim of identifying sequences of TrfA-44 required for stable replication of RK2 in P. aeruginosa, specific deletions and a substitution mutant within the N terminus sequence unique to TrfA-44 were constructed, and the mutant proteins were tested for activity. Deletion mutants were targeted to three of the four predicted helical regions in the first 97 amino acids of TrfA-44. Deletion of TrfA-44 amino acids 21-32 yielded a mutant protein, TrfA-44Delta2, that had lost the ability to bind and load the DnaB helicase of P. aeruginosa or Pseudomonas putida onto the RK2 origin in vitro and did not support stable replication of an RK2 mini-replicon in P. aeruginosa in vivo. A substitution of amino acid 22 within this essential region resulted in a protein, TrfA-44E22A, with reduced activity in vitro, particularly with the P. putida helicase. Deletion of amino acids 37-55 (TrfA-44Delta3) slightly affected protein activity in vitro with the P. aeruginosa helicase and significantly with the P. putida helicase, whereas deletion of amino acids 71-88 (TrfA-44Delta4) had no effect on TrfA activity in vitro with either helicase. These results identify regions of the TrfA-44 protein that are required for recruitment of the Pseudomonas DnaB helicases in the initiation of RK2 replication.

Amino Acid Sequence↗
PubMed2001

Modulation of cellular responses by plasmid CD40L: CD40L plasmid vectors enhance antigen-specific helper T cell type 1 CD4+ T cell-mediated protective immunity against herpes simplex virus type 2 in vivo.

Engineering gene therapy vectors to modulate the immune response is an important goal. In this regard, costimulation of T cells is a critical determinant in immune activation. The costimulatory molecule CD40, expressed on antigen-presenting cells, is thought to interact with CD40 ligand (CD40L) expressed on activated CD4(+) or CD8(+) T cells to further drive interleukin-2 receptor (IL-2R) expression and antigen-specific T cell expansion necessary for both class II and class I responses. To compare the specific roles of these two costimulatory molecules in immune induction in a herpes simplex virus (HSV) model, we constructed plasmid DNAs expressing CD40 and CD40L, coimmunized these molecules with a gD plasmid vaccine, and then analyzed immune modulatory effects as well as protection against lethal HSV-2 challenge. We observed that gD-specific IgG production was unaffected by these molecules. However, a higher production of IgG2a isotype was induced by CD40L coinjection, suggesting that CD40L drives immune responses towards a helper T cell type 1 (Th1) phenotype. CD40L also enhanced Th cell proliferative responses and production of Th1-type cytokines (IL-2 and IFN-gamma) and beta-chemokines (RANTES and MIP-1alpha) from splenocytes. In contrast, CD40 showed slightly increasing effects on T cell proliferation responses and cytokine and chemokine production. When animals were challenged with a lethal dose of HSV-2, CD40L-coimmunized animals exhibited a significantly enhanced survival rate, as compared with CD40 coinjection or gD DNA vaccine alone. This enhanced protection appears to be mediated by Th1-type CD4(+) T cells, as determined by in vitro and in vivo T cell subset deletion. CD40L also promoted migration of CD4(+) T cells into the muscle sites. These studies demonstrate that CD40L can play an important role in protective antigen-specific immunity in a gene-based model system through increased expansion of the CD4(+) Th1 T cell subset in vivo.

Adjuvants, Immunologic↗
PubMed2005

II. Cationic lipid-formulated plasmid DNA-based Bacillus anthracis vaccine: evaluation of plasmid DNA persistence and integration potential.

Several formulated plasmid DNA (pDNA)-based vaccines are being evaluated for safety and efficacy in healthy human subjects. A safety concern for any vaccine that contains genetic material, be it whole organism, live-attenuated, or gene-based, is the potential for integration into genomic DNA (gDNA). To address this concern, a preclinical pDNA persistence/integration study was conducted in rabbits to determine the level of pDNA in muscle 2, 28, and 64 days after intramuscular injection of DMRIE:DOPE-formulated pDNAs encoding Bacillus anthracis detoxified LF and PA proteins (VCL-AB01 vaccine). Total DNA was extracted from day 64 muscle tissue and fractionated by column agarose gel electrophoresis (CAGE). Plasmid copy number (PCN) in muscle 64 days after injection (geometric mean, 2808 PCN/microg of total DNA or 150,000 diploid genomes) was determined by quantitative polymerase chain reaction. Analysis of total DNA from five VCLAB01- injected rabbits revealed that two of five samples had no detectable PCN in the high molecular weight fraction after one round of CAGE, two samples had PCN under the lower limit of quantitation, and the remaining sample had 123 PCN/microg. All PCN in the latter sample cleared after an additional round of CAGE. It appears, therefore, that persisting PCN fractionate as low molecular weight material and are most likely not integrated into gDNA. Even if the worst-case assumption is made that the highest PCN found associated with gDNA represented covalently integrated pDNA inserts, the frequency of mutation would still be 500-fold lower than the autosomal spontaneous mutation rate.

Animals↗
PubMed1986

Deletion and rearrangement of plasmid DNA during transformation of Escherichia coli with linear plasmid molecules.

When E. coli was transformed with linearized pBR322 DNA, many transformants contained recircularized plasmids bearing deletions and other rearrangements. Most aberrant molecules were less than monomeric length and had lost the restriction site used for linearization, with the deleted region extending mono- (type Ia) or bi-directionally (type Ib). Type II deletants were greater than monomeric but less than dimeric and contained the pBR322 sequence in direct repeat with deletion at one or both junctions (type IIa) or in inverted repeat with loss of sequence at both junctions (type IIb). Type III deletants were greater than dimeric but less than trimeric, consisting of pBR322 sequences in both direct and inverse repeat with deletions at two or more junctions. Transformation frequencies for linear DNA were drastically reduced in xth-1- bacteria with type IIb deletants predominating in transformants. This indicates that exonuclease III is important for perfect recyclization of plasmids and the generation of type I deletants. In vivo recyclization of in vitro ligation products explains many of the aberrant DNA molecules that are encountered during gene cloning.

Chromosome Deletion↗
PubMed1997

Transcription from plasmid expression vectors is increased up to 14-fold when plasmids are transfected as concatemers.

A protocol for increasing transcription from plasmid expression vectors is presented. A vector containing chloramphenicol acetyltransferase (CAT) gene was digested leaving the transcription cassette intact. Heat inactivation of restriction enzymes followed by ligation of the digestion products yielded concatemers which migrated as a single band in agarose gel electrophoresis. Mouse fibroblasts transfected with the concatemers gave a CAT activity that was 14-fold greater than that of cells transfected with a similar mass (equimolar gene number) of the native plasmid. The effect was independent of promoter type, restriction enzyme, number of restriction sites and with a noted exception, cell line.

Animals↗
PubMed1979

Plasmid replication functions. IV. Promoters in the replication region of plasmid R6-5.

Eight RNA polymerase binding sites have been shown to map within the EcoRI fragment E-2 (replication region RepA EcoRI fragment) of plasmid R6-5 and all but one have been shown to contain active promoters of transcription. Three of the identified promoters are located within a 2.7 kb region essential for controlled, autonomous plasmid replication and may be involved in the functional expression of the three R6-5 replication determinants that have thus far been identified, namely the origin of vegetative replication, oriV, the replication control gene, cop, and the determinant of an essential, positive-acting element, designated RepA.

Base Composition↗
PubMed1987

Characterization and localization of plasmid functions involved in pock formation and pock resistance of plasmid pSK3 of Streptomyces kasugaensis MB273.

Deletion derivatives and recombinants of the plasmid pSK3, which is a cointegrate of pSK1 and pSK2 in Streptomyces kasugaensis, were constructed and analysed for their ability to transfer and 'pock' on strains carrying pSK1 or pSK2. Various deletions in the pSK1 and/or pSK2 regions of pSK3 were grouped into nine classes on the basis of their pock-forming ability and pock resistance. Analysis of these deletions and insertions provided tentative locations of DNA regions for two pock-resistance determinants (Por1 and Por2), two pock-forming determinants (Poc1 and Poc2) consisting of plasmid transfer and spread determinants (Tra/Spr), and two replication determinants (Rep1 and Rep2) corresponding to the pSK1 and pSK2 regions of pSK3. In particular, the Por2 function in the pSK2 region was determined to be located in a 1.35 kb segment.

DNA, Bacterial↗
PubMed1991

Partial deletion of the Rhizobium phaseoli CFN23 symbiotic plasmid implies a concomitant amplification of plasmid DNA sequences.

Rhizobium leguminosarum biovar phaseoli CFN23 loses its ability to nodulate beans at a high frequency because of a deletion of part of its symbiotic (pSym) plasmid (Soberón-Chávez et al., 1986). We report here that at least 80 kb of pSym are deleted upon loss of the symbiotic phenotype; the deletion removes the nitrogenase structural nifHDK and the common nodABC genes. The size of the deleted pSym is not reduced, since it is accompanied by an amplification of other pSym plasmid sequences. This genetic rearrangement is similar to the deletion and amplification of yeast mitochondrial DNA leading to 'petite' mutations.

Blotting, Southern↗
PubMed1992

The copy number of plasmid pLS1 is regulated by two trans-acting plasmid products: the antisense RNA II and the repressor protein, RepA.

The promiscuous plasmid pLS1 encodes two transacting elements that regulate its copy number: protein RepA and antisense RNA II. In vitro transcription showed that RNAs for both repressors are synthesized from two promoters, PAB and PII. From PAB, genes encoding RepA (transcriptional repressor) and RepB (initiator of replication) are cotranscribed, the target of RepA being located within PAB. Mutants in repA or in PAB are still sensitive to RepA. However, cloning of the repA gene in a compatible replicon did not result in incompatibility towards pLS1. From PII, the 50-nucleotide RNA II is synthesized. The main incompatibility determinant towards pLS1 corresponds to the coding sequence for RNA II. The RNA II target could be reduced to 21 nucleotides, including the RepB initiation of translation signals. We propose that plasmids of the pLS1 family (pE194, pADB201, and pLB4) share functional and structural characteristics for the regulation of their copy numbers.

Bacterial Proteins↗
PubMed1994

Mutations that affect regulation of the korB gene of Streptomyces lividans plasmid plJ101 alter plasmid transmission.

Using the catechol dehydrogenase gene as a reporter, we isolated random mutations in the plJ101 korB gene operator/promoter (OP) region that affect korB expression and regulation. We mapped these mutations to inverted repeat sequences within the promoter and studied their effects on binding of the KorB repressor protein to the OP, on expression of the korB gene, and on plasmid transmission during mating. Additionally, we investigated the biological effects of KorB binding to a locus (sti, for strong incompatibility) adjacent to the korB OP and implicated in plJ101 replication. Our results identify sites that influence the synthesis and autoregulation of KorB; they also show that interaction of KorB with sti affects repression of korB and transmission of plasmids to spores of recipients.

Bacterial Proteins↗
PubMed1975

Plasmid-determined resistance to erythromycin: comparison of strains of streptococcus faecalis and streptococcus pyogenes with regard to plasmid hmology and resistance inducibility.

Streptococcus faecalis strains DS-5 and Streptococcus pyogenes strain AC-1 both have a 17 million dalton plasmid that determines resistance to erythromycin, lincomycin, and vernamycin B(alpha). The results of deoxyribonucleic acid-deoxyribonucleic acid hybridization experiments indicate that the two plasmids are about 95% homologous. It was also shown that erythromycin resistance is inducible in AC-1 and constitutive in DS-5.

Drug Resistance, Microbial↗