Form Of Plasmid Research Articles

Variable epitope library-based vaccines: Shooting moving targets

While the antigenic variability is the major obstacle for developing vaccines against antigenically variable pathogens (AVPs) and cancer, this issue is not addressed adequately in current vaccine efforts. We developed a novel variable epitope library (VEL)-based vaccine strategy using immunogens carrying a mixture of thousands of variants of a single epitope. In this proof-of-concept study, we used an immunodominant HIV-1-derived CD8+ cytotoxic T-lymphocyte (CTL) epitope as a model antigen to construct immunogens in the form of plasmid DNA and recombinant M13 bacteriophages. We generated combinatorial libraries expressing epitope variants with random amino acid substitutions at 2–5 amino acid positions within the epitope. Mice immunized with these immunogens developed epitope-specific CD8+ IFN-γ+ T-cell responses that recognized more than 50% of heavily mutated variants of wild-type epitope, as demonstrated in T-cell proliferation assays and FACS analysis. Strikingly, these potent and broad epitope-specific immune responses were long lasting: after 12 months of priming, epitope variants were recognized by CD8+ cells and effector memory T cells were induced. In addition, we showed, for the first time, the inhibition of T-cell responses at the molecular level by immune interference: the mice primed with wild-type epitope and 8 or 12 months later immunized with VELs, were not able to recognize variant epitopes efficiently. These data may give a mechanistic explanation for the failure of recent HIV vaccine trials as well as highlight specific hurdles in current molecular vaccine efforts targeting other important antigenically variable pathogens and diseases. These findings suggest that the VEL-based strategy for immunogen construction can be used as a reliable technological platform for the generation of vaccines against AVPs and cancer, and contribute to better understanding complex host–pathogen interactions.

Inhibition of Heterotrimeric G Protein Signaling by a Small Molecule Acting on Gα Subunit

The simultaneous activation of many distinct G protein-coupled receptors (GPCRs) and heterotrimeric G proteins play a major role in various pathological conditions. Pan-inhibition of GPCR signaling by small molecules thus represents a novel strategy to treat various diseases. To better understand such therapeutic approach, we have characterized the biomolecular target of BIM-46187, a small molecule pan-inhibitor of GPCR signaling. Combining bioluminescence and fluorescence resonance energy transfer techniques in living cells as well as in reconstituted receptor-G protein complexes, we observed that, by direct binding to the Galpha subunit, BIM-46187 prevents the conformational changes of the receptor-G protein complex associated with GPCR activation. Such a binding prevents the proper interaction of receptors with the G protein heterotrimer and inhibits the agonist-promoted GDP/GTP exchange. These observations bring further evidence that inhibiting G protein activation through direct binding to the Galpha subunit is feasible and should constitute a new strategy for therapeutic intervention.

Novel Methodology for Predicting Photogenotoxic Risk of Pharmaceutical Substances Based on Reactive Oxygen Species (ROS) and DNA-Binding Assay

Drug-induced phototoxic skin responses involves photoirritation, photoallergy, and photogenotoxicity. In the present study, we attempted to develop effective analytical tools to predict the photogenotoxic potential of pharmaceutical substances on the basis of their photobiological and DNA-binding properties. Photocleavage of plasmid DNA was assessed by capillary gel electrophoresis (CGE), and generation of reactive oxygen species (ROS) from photoirradiated photosensitizers was also assessed by spectrophotometrical determination. The affinity of drugs with DNA was determined by fluorescent titration or competitive binding assay using ethidium bromide. Structural transition from supercoiled form to open circular form of plasmid DNA were observed in the presence of nine irradiated-photosensitizers, however DNA damage by six phototoxic and five nonphototoxic drugs were negligible or weak even though some chemicals exhibited significant generation of ROS. DNA photocleavers tended to interact with DNA, binding constants of which were found to be less than 1 µM, whereas most nonphotogenotoxic chemicals failed to bind with DNA. The differences in DNA-binding properties might be attributed to a data discrepancy between ROS-generation and DNA photocleaving activity. The combination use of ROS assay and DNA-binding assay could be fast and reliable prediction for the photogenotoxic potential of a large number of drug candidates at the drug-discovery stage. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3647–3658, 2009

Immune responses to T-cell epitopes of SARS CoV-N protein are enhanced by N immunization with a chimera of lysosome-associated membrane protein

In our previous study by Gupta et al, dominant T-cell epitopes of SARS CoV-N(N) protein were predicted by software. The spectrum of interferon (IFN)-γ responses of Balb/c mice immunized against two different forms of SARS CoV-N plasmid was then analyzed. A cluster of dominant T-cell epitopes of SARS CoV-N protein was found in the N-terminus (amino acids 76–114). On the basis of this study, four different plasmids were constructed: (i) DNA encoding the unmodified N (p-N) or N70−122 (p-N70−122) as an endogenous cytoplasmic protein or (ii) DNA encoding a lysosome-associated membrane protein (LAMP) chimera with N (p-LAMP/N) or N70−122 (p-LAMP/N70−122). The immune responses of mice to these four constructs were evaluated. The results showed marked differences in the responses of the immunized mice. A single priming immunization with the p-LAMP/N construct was sufficient to elicit an antibody response. Enzyme-linked immunospot (ELISpot) assay indicated that p-LAMP/N70−122 and p-LAMP/N plasmids both elicited a greater IFN-γ response than p-N. p-N and p-N70−122 constructs induced low or undetectable levels of cytokine secretion. We also found that the p-LAMP/N70−122 construct promoted a long-lasting T-cell memory response without an additional boost 6 months after three immunizations. These findings show that DNA vaccines, even epitope-based DNA vaccines using LAMP as chimera, can elicit both humoral and cellular immune responses.

Effect of the atmospheric pressure nonequilibrium plasmas on the conformational changes of plasmid DNA

The cold atmospheric pressure plasma, which has been widely used for biomedical applications, may potentially affect the conformation of DNA. In this letter, an atmospheric pressure plasma plume is used to investigate its effects on the conformational changes of DNA of plasmid pAHC25. It is found that the plasma plume could cause plasmid DNA topology alteration, resulting in the percentage of the supercoiled plasmid DNA form decreased while that of the open circular and linearized form of plasmid DNA increased as detected by agrose gel electrophoresis. On the other hand, further investigation by using polymerase chain reaction method shows that the atmospheric pressure plasma jet treatments under proper conditions does not affect the genes of the plasmid DNA, which may have potential application in increasing the transformation frequency by genetic engineering.

Gene Therapy for Allergic Diseases

Allergic diseases, such as allergic asthma, allergic rhinitis, atopic dermatitis, conjunctivitis, urticaria, food allergy, and/or anaphylaxis, are associated with the skewing of immune responses towards a T helper 2 (TH2) phenotype, resulting in eosinophilic inflammation. TH2 cytokines, such as interleukin (IL)-4, IL-5 and IL-13, promote IgE production, mast cell differentiation, and eosinophil growth, migration and activation which then lead to the pathologic abnormalities in allergic diseases. Moreover, the impaired function of regulatory T cells has been noted in allergic diseases. To date, treatments for allergic diseases, such as antihistamines, corticosteroids, bronchodilators and some allergen-specific immunotherapy, are effective but costly and require long-term and recurrent drug administration. Gene therapy has been shown to be an easy, effective, and convenient treatment by delivering the allergen or the therapeutic protein in the form of plasmid DNA in vivo to modulate allergic immune responses. We summarize here the recent advances of gene therapy in allergic diseases and discuss the challenges in clinical application.

Gene transfer efficiency of high primary amine content, hydrophobic, alkyl-oligoamine derivatives of polyethylenimine

In this study, a series of alkyl-oligoamine derivatives of low-toxicity 10 kDa polyethylenimine (PEI) were synthesized to enhance the hydrophobicity of PEI while preserving most of its primary amine content. PEI was reacted with a series of ω-bromoalkylcarboxylic acids with different chain lengths (2-bromoacetic, 6-bromohexanoic, 10-bromodecanoic and 16-bromohexadecanoic acids) to modify hydrophobicity followed by coupling to various oligoamines (spermine, spermidine, ethylendiamine or diethylentriamine) to partially restore primary amine density. These modifications were designed to influence hydrophobic–hydrophilic balance as well as maintain the proton sponge effect in order to create an efficient vector with low toxicity. Ethidium bromide exclusion assays and dynamic light scattering studies showed that the modified PEIs could bind to plasmid DNA and form nanoparticles in the range of 100 nm. The transfection efficiency of modified PEIs complexed with a luciferase reporter gene (pCMV-luc) in N2A murine neuroblastoma cells was increased to a level comparable to that of 25,000 Da PEI. These results indicate that hydrophobic modification of low-toxicity PEI without reduction in primary amine content is an effective strategy for improving transfection efficiency of polycation-based non-viral vectors while maintaining low toxicity.

Cationic Polyrotaxanes as Gene Carriers: Physicochemical Properties and Real-Time Observation of DNA Complexation, and Gene Transfection in Cancer Cells

Cationic polymers have been studied as promising nonviral gene delivery vectors. In contrast to the conventional polycations with long sequences of covalently bonded repeating units, we have developed a series of novel cationic polyrotaxanes consisting of multiple oligoethyleneimine-grafted beta-cyclodextrin rings threaded on a poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol) triblock copolymer chain. In this study, these cationic polyrotaxanes with different oligoethyleneimine chain lengths were investigated for DNA binding ability, cytotoxicity, and gene transfection efficiency in cancer cells. Fluorescent titration assay results indicated that all the polyrotaxanes could completely condense plasmid DNA and form stable complexes at N/P ratio of 2, where the N/P ratio is the molar ration of amine groups in the cationic molecule to phosphate groups in the DNA. Particularly, tapping mode AFM imaging in aqueous environment was conducted to observe the morphology of the polyrotaxane/DNA complexes and their formation processes in real time. In both SK-OV-3 and PC3 cancer cells, these polyrotaxanes showed low cytotoxicity and high transfection efficiency which is comparable to or significantly higher than that of high molecular weight branched polyethylenimine (25 kDa), one of the most effective gene-delivery polymers studied to date. In addition, the synthesized polyrotaxanes displayed sustained gene delivery capability in PC3 cells in the presence or absence of serum. Therefore, these cationic polyrotaxanes with strong DNA binding ability, low cytotoxicity, and high and sustained gene delivery capability have a high potential as novel nonviral gene carriers in clinical cancer gene therapy.

Nanotechnology in Vaccine Development: A Step Forward

The study of nanotechnology for epicutaneous delivery of pharmaceuticals and vaccines is burgeoning. Topically applied nanomaterials have been shown to enter tape-stripped skin and reach draining lymph nodes in an inbred strain of mice. Nanomaterials in the form of plasmid DNA, proteins, and virus particles accumulate in hair follicles, diffuse via dendritic cells to draining lymph nodes, and elicit antigen-specific humoral and cell-mediated immunity. Topically immunized mice have also demonstrated resistance to infection with live virus. Advantages of nanotechnology include uniformity, reproducibility, and precision in the synthesis and manufacture of candidate compounds. Combined with novel pharmacokinetics and the possibility of targeted therapy, nanotechnology-based vaccines may prove superior to existing vaccines and have the potential to open therapeutic avenues for treating infectious disease and malignancy.

P17: Ethanol-induced ultrastructural changes in rat cerebellum after gene therapy

Ethanol-induced oxidative impairment of tissues is in the focus of interest for gene therapy. The one of the most important antioxidant enzyme is superoxiddismutase (SOD), especially mitochondrial Mn-SOD. Previous experiments on rats have shown that tissues sensitive to ethanol-induced oxidative stress are cerebellar cortex and Purkinje cells (Celec et al., Life Sci, 2003). Male Wistar rats ( n =40) were randomly divided into four groups: control, alcohol, SOD, alcohol+SOD. Plasmid pcDNA3 containing the gene for mitochondrial Mn-SOD (300 μg in 100 μl TE buffer) or the same volume of saline was administered intramuscularly into the musculus biceps femoris of left hind limb once a week based information on the expression after non viral gene transfer from literature. Morphological analysis by TEM showed swollen mitochondria and impairment of myelin sheaths of nerve fibres in the brain in all groups except the control (water) group. Our antioxidative therapy has revealed no protective effect on ethanol-induced cerebellum impairment. In addition to SOD supplement in the form of plasmid, pcDNA3 caused pathological changes in mitochondria and myelin structure. This result indicates an elevated activity of SOD without appropriately elevated activity of catalase or glutathione peroxidase – enzymes that detoxify SOD product – hydrogen peroxide. It is possible, that hydrogen peroxide decomposes nonenzymatically into hydroxyl radical. It is more reactive than superoxide anion. The approach of using SOD gene therapy for antioxidant treatment of alcohol-induced tissue damage failed to show a clear positive effect. Further studies using a combination of antioxidant genes should follow. This work was supported by grant of Slovak Research and Development Agency No. APVT-20-003104.

Cytotoxic Activity of Isoliquiritigenin towards CCRF‐CEM Leukemia Cells and its Effect on DNA Damage

The cytotoxic activity of isoliquiritigenin (ISL) towards CCRF-CEM human T-cell leukemia cells and DNA damage induction were investigated in the present study. The anti-proliferative effect of ISL on CCRF-CEM cell line IN VITRO was analyzed. In order to further explore the underlying mechanism of cell growth inhibition of ISL towards CCRF-CEM cell line, the cell cycle distribution and mitochondrial membrane potential (DeltaPsim) disruption were measured. ISL exerted significant activity towards CCRF-CEM cells. The growth inhibitory ratio was concentration- and time-dependent with an IC(50) value of 18.38 microM. The cell cycle was arrested in the G2/M phase upon treatment with low doses of ISL. Mitochondrial membrane potential (DeltaPsim) disruption was observed at low ISL doses. The effect of ISL on DNA damage was analyzed by agarose gel electrophoresis and atomic force microscopy (AFM). Either ISL or Cu2+ failed to cause pBR322 DNA damage. However, plasmid DNA was damaged after treatment with ISL in the presence of Cu2+. Two forms of plasmid DNA closed circular and linear forms were visualized by AFM.

The Supervirulence Plasmid pToK47 from Agrobacterium tumefaciens A281Improves Transformation Efficiency of Hevea brasiliensis

Problem statement: The present study investigates the ability of the pTok47 supervirulence plasmid form Agrobacterium tumefaciens A281 to enhance genetic transformation in Hevea brasiliensis. Approach: Hevea anther callus was transformed via Agrobacterium-mediated genetic transformation using two strains of Agrobacterium (GV2260 and GV3850) harboring the human serum albumin cDNA and the supervirulent plasmid pToK47 from Agrobacterium tumefaciens A281. The transformed callus was selected using kanamycin as the selection agent Results: The Agrobacterium strain GV2260 benefited from the presence of the supervirulence plasmid in giving a higher frequency of 7.4% transformed callus, 344.8% embryogenesis and 11.6% plantlet production compared to the corresponding strain on its own giving 0.9% transformed callus, 204.5% embryogenesis and 4.4% plantlet production. Similarly, Agrobacterium strain GV3850 conferred a higher frequency using the supervirulent plasmid, resulting in 3.5% transformed callus, 138.5% embryogenesis and 3.5% plantlet production compared to the corresponding strain on its own giving 0.7% transformed callus, 137.5% embryogenesis and 9.0% plantlet production. These findings were confirmed by the Wilcoxon Signed Rank Test that compared the effectiveness of the supervirulence plasmid in increasing the rate of genetic transformation in the calli surviving in kanamycin growth medium for GV2260 (p<0.001) and for GV3850 (p<0.05). Conclusion: This study showed that both Agrobacterium strains benefited from the presence of the supervirulence plasmid in giving a higher frequency of transformed callus, embryoids and plantlets. These results suggest that a highly virulent binary vector system might prove especially useful in generating high frequency transformation of Hevea.

Novel 7-mer peptide mediates nuclear targeting in MCF7 breast cancer cells.

Abstract Abstract #6023 Nonviral gene therapy strives to eliminate cancer by correcting the molecular cause of the disease. Corrective DNA sequences, in the form of plasmids (pDNA), must localize to the cell nucleus to obtain transgene expression. This low probability event contributes to inefficient transgene expression following nonviral gene delivery. Previous studies using a bacteriophage (phage) display approach modified for the discovery of intracellular ligands yielded a panel of 7-mer peptides associated with the nuclei of MCF7 human mammary epithelial cells. The phage clone with the highest frequency of nuclear recovery displayed the QPSPSPT peptide. This study confirms the nuclear targeting capability of QPSPSPT and suggests the potential for QPSPSPT as a novel nonviral gene delivery ligand to modulate transgene expression in human breast cancer cells.&amp;#x2028; The capacity for nuclear localization mediated by QPSPSPT was confirmed by image analysis of in vitro cell cultures exposed to fluorescently-labeled M13 phage displaying the heptapeptide. Control, wild-type M13 phage and phage displaying the QPSPSPT heptapeptide were labeled using an AlexaFluor 488 through conjugation to the pVIII coat proteins. Fluorescence microscopy revealed that 12.2% of MCF7 cell nuclei were associated with fluorescently labeled phage displaying the QPSPSPT peptide, but that wild-type M13 phage failed to localize to the cell nucleus.&amp;#x2028; Quantitative analysis of confocal fluorescence micrographs enabled determination of intracellular and intranuclear phage localization. The number of phage per MCF7 nucleus was 3.3-fold greater for samples displaying the QPSPSPT peptide relative to the wild type M13 population. The increase in nuclear localization was mediated by both an increase in the amount of QPSPSPT-phage per cell (1.7-fold) and the intracellular translocation of QPSPSPT-phage to the nucleus (1.9-fold) as compared to the wild-type M13 phage. The equivalent spherical diameter of an M13 phage is approximately 40 nm, suggesting that the QPSPSPT heptapeptide has the capacity to mediate the cellular entry and nuclear localization of payloads consistent with the size of pDNA or formulated nonviral gene delivery vehicles.&amp;#x2028; A commercial pDNA (pDualGC, Stratagene) was modified to enable the expression of fusion products consisting of a green fluorescent protein (GPF) bearing an N-terminus triple repeat of either the QPSPSPT or the positive control YSPTSPS. Transfection of these new constructs was performed independently in cultured MCF7 cells using Lipofectamine (Invitrogen). Six hours after transfection, the GFP-QPSPSPTx3 and GFP-YSPTSPSx3 fusion proteins appeared predominately in the cell nucleus in comparison with the cytosolic distribution of wild-type GFP. QPSPSPT has the capacity to preferentially localize proteins and phage to the cell nucleus, suggesting a potential role for this novel heptapeptide as a nonviral gene delivery adjuvant in breast cancer therapy.&amp;#x2028; This work was funded by the BCRP-CDMRP under awards BC023276 and BC044010. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6023.

DNA-hydrolyzing activity of sIgA-abzymes of human milk is differentially inhibited by nucleoside triphosphates

It is known that human milk contains secretory immunoglobulines A (sIgA-abzymes) possessing affinity to mammalian DNA and capacity to cleave plasmid DNA. Regulation of DNA-hydrolyzing activity of those sIgA-abzymes is poorly studied. Here we investigate the effect of nucleoside triphosphates towards the DNase activity of sIgA-abzymes which were purified from human milk by a sequential chromatography on protein A-agarose, DEAE-Fractogel, and DNA-cellulose. By using DNA-hydrolysing assay, we revealed that 1 mM ATP and 1 mM dATP markedly reduced the cleavage of linear form of plasmid DNA by sIgA-abzymes, while the effect of GTP, CTP, TTP and dGTP was much weaker. dCTP and dTTP did not influence DNase activity of the sIgA-abzymes. Possible mechanisms of nucleotide-mediated inhibition of the DNase activity of sIgA-abzymes are discussed. Keywords: abzymes, secretory immunoglobulin A, human milk, DNA-hydrolyzing activity.

Enhanced DNA dynamics due to cationic reagents, topological states of dsDNA and high mobility group box 1 as probed by PicoGreen

We report a study of dynamics with a dsDNA-specific dye called PicoGreen bound to plasmid DNA (3.4 kb), and show that at low dye/DNA phosphate ratios (1 : 100 and below), PicoGreen dynamics reflect the motional dynamics of dsDNA. We further evaluated the usefulness of this probe by measuring the time-resolved fluorescence dynamics of PicoGreen bound to dsDNA in the presence of cationic reagents that affect DNA dynamics [MgCl2 and polyethyleneimine (PEI)] and also with plasmid DNA in different topological states. Among these conditions, MgCl2, PEI and the supercoiled form of plasmid resulted in increases in the very short component (0.2-0.4 ns) of the rotational correlation time. Separately, HMGB1 protein enhanced DNA dynamics, as observed from the rotational correlation times of very short (0.2-0.4 ns) and short (2-4 ns) rotational correlation timescale components. By studying the effect of specific deletion mutants HMGB1-DeltaA (deletion of 98 N-terminal amino acids) and HMGB1-DeltaC (deletion of 30 C-terminal amino acids), we show that the acidic C-terminal tail is required for enhancement of DNA dynamics. We then discuss the possible mechanisms and implications of HMGB1-mediated flexibility of DNA in the context of formation of large nucleoprotein complexes.

Lagging-Strand DNA Replication Origins Are Required for Conjugal Transfer of the Promiscuous Plasmid pMV158

The promiscuous streptococcal plasmid pMV158 is mobilizable by auxiliary plasmids and replicates by the rolling-circle mechanism in a variety of bacterial hosts. The plasmid has two lagging-strand origins, ssoA and ssoU, involved in the conversion of single-stranded DNA intermediates into double-stranded plasmid DNA during vegetative replication. Transfer of the plasmid also would involve conversion of single-stranded DNA molecules into double-stranded plasmid forms in the recipient cells by conjugative replication. To test whether lagging-strand origins played a role in horizontal transfer, pMV158 derivatives defective in one or in both sso's were constructed and tested for their ability to colonize new hosts by means of intra- and interspecies mobilization. Whereas either sso supported transfer between strains of Streptococcus pneumoniae, only plasmids that had an intact ssoU could be efficiently mobilized from S. pneumoniae to Enterococcus faecalis. Thus, it appears that ssoU is a critical factor for pMV158 promiscuity and that the presence of a functional sso plays an essential role in plasmid transfer.

The molecular and cellular basis of infection—Perspectives from the first advanced summer school in Africa

The molecular and cellular basis of infection—Perspectives from the first advanced summer school in Africa

High Frequency Plasmid Recombination Mediated by 28 bp Direct Repeats

The stability in Escherichia coli of a mammalian expression vector (pCIneo) and its derivative candidate DNA vaccine (pGPV-PV) is described. These multicopy pMB1-type plasmids were found to recombine in several recA E. coli strains due to the presence of two 28 bp direct repeats flanking intervening sequences of 1.6 kb (pCIneo) and 3.2 kb (pGPV-PV). In this recombination event, one of the direct repeats and the intervening sequence were deleted or duplicated, originating monomeric or/and hetero-dimeric plasmid forms, respectively. Additionally, the plasmid rearrangement led to the acquisition of a kanamycin resistance phenotype. Recombination frequencies between 7.8 x 10(-7) and 3.1 x 10(-5) were determined for DH5alpha and JM109(DE3) strains, respectively. Higher recombination frequencies were found in cells previously grown up to stationary growth phase being the monomeric plasmid form the prevalent one. Real-time PCR quantification revealed the presence of approximately 1.5 x 10(4) recombined molecules per 2 x 10(5 )cells pre-kanamycin exposure. Under selective pressure of this antibiotic, the number of recombined molecules increased approximately 2,000-fold in a 48-h period replacing the original plasmid form. The high frequency, at which deletion-duplication occurred in the absence of kanamycin selective pressure, should be regarded as a safety concern. This work highlights the impact of mutational hot spots on expression and cloning plasmid vectors and the need to carefully design plasmid vectors.

Variations of Plasmid Content in Rickettsia felis

BackgroundSince its first detection, characterization of R. felis has been a matter of debate, mostly due to the contamination of an initial R. felis culture by R. typhi. However, the first stable culture of R. felis allowed its precise phenotypic and genotypic characterization, and demonstrated that this species belonged to the spotted fever group rickettsiae. Later, its genome sequence revealed the presence of two forms of the same plasmid, physically confirmed by biological data. In a recent article, Gillespie et al. (PLoS One. 2007;2(3):e266.) used a bioinformatic approach to refute the presence of the second plasmid form, and proposed the creation of a specific phylogenetic group for R. felis.Methodology/Principal FindingsIn the present report, we, and five independent international laboratories confirmed unambiguously by PCR the presence of two plasmid forms in R. felis strain URRWXCal2 T, but observed that the plasmid content of this species, from none to 2 plasmid forms, may depend on the culture passage history of the studied strain. We also demonstrated that R. felis does not cultivate in Vero cells at 37°C but generates plaques at 30°C. Finally, using a phylogenetic study based on 667 concatenated core genes, we demonstrated the position of R. felis within the spotted fever group.SignificanceWe demonstrated that R. felis, which unambiguously belongs to the spotted fever group rickettsiae, may contain up to two plasmid forms but this plasmid content is unstable.

A multimer resolution system contributes to segregational stability of the prototypical staphylococcal conjugative multiresistance plasmid pSK41

The 46-kb plasmid pSK41 is the prototype of a family of staphylococcal conjugative multiresistance plasmids. Sequence analyses have revealed the presence of a putative resolvase gene, res, on pSK41, and identical or related genes carried by other staphylococcal multiresistance plasmids. Carriage of the res region was found to ameliorate the accumulation of multimeric plasmid forms, and recombinant plasmids encoding a wild-type res gene exhibited greater plasmid segregational stability than counterparts carrying a nonfunctional mutant, irrespective of whether the cognate or a heterologous replication system and host was utilized. In vitro DNA-binding studies demonstrated that purified Res protein binds within the intergenic region upstream of the res coding sequence. Six copies of an imperfect 11-bp repeat sequence were identified within DNA sequences protected by Res in DNAseI footprinting studies, in an arrangement that suggests a typical resolution site organization consisting of three subsites.