[Action of EDTA on sporulation, competence and potential transformants of Bacillus subtilis].
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Bacillus subtilis↗
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Results for “DNA Transformation Competence”
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High efficiency introduction of plasmid DNA into glycine treated Enterococcus faecalis by electroporation.
A highly efficient electroporation system for Enterococcus faecalis was developed by systematically optimizing different parameters. One parameter found to be particularly critical for electroporation was cultivation of E. faecalis in medium containing a high glycine concentration, prior to electroporation. Osmotic stabilization of cells with 0.5 M sucrose was also found to be critical during glycine treatment. 10(6) transformants per microgram of plasmid DNA were consistently obtained within 48 h. Electrocompetent preparations of E.-faecalis could be stored at - 70 degrees C without loss of competence.
DNA, Bacterial↗
Competence for deoxyribonucleic acid uptake and deoxyribonuclease action external to cells in the genetic transformation of Diplococcus pneumoniae.
A mutant of Diplococcus pneumoniae that apparently does not require activator can become competent for uptake of deoxyribonucleic acid (DNA) when grown in dilute cultures or in the presence of trypsin. Development of competence in both mutant and wild strains is temperature dependent, being 10-fold greater at 30 C than at 37 C. Induction of competence on a shift from 37 to 30 C requires protein synthesis and the presence of Mg(2+) and Ca(2+); uptake of DNA does not require protein synthesis. Competence decays exponentially at higher temperatures. As well as taking up DNA, competent cells release oligonucleotide fragments of donor DNA in the medium external to the cells. Normal strains release fragments comparable in amount to the DNA taken up; but, in a mutant selected for inability to degrade DNA in agar, the amount of fragments formed external to the cells is only 40% of DNA uptake. Requirements for external deoxyribonuclease action are identical to those for DNA uptake: prior development of competence and the presence during treatment with DNA of Mg(2+) ions and a source of energy.
Calcium↗
Cloning and expression of the 75 kDa DnaK-like protein of Chlamydia psittaci and the evaluation of the recombinant protein by immunoblotting and indirect ELISA.
The 75 kDa dnaK-like gene of Chlamydia psittaci ovine abortion strain S26/3 was isolated from an EMBL 3 chlamydial DNA library. A 7 kb DNA fragment containing the gene was subcloned into Bluescribe (M13+) plasmid and used to transform competent E. coli. These cells were found to express a cytoplasmic protein of 75 kDa. Monospecific antibodies against the protein prepared by antibody elution reacted with the native 75 kDa protein. Recombinant clones did not adhere to McCoy cell monolayers in cell adhesion studies. The 75 kDa protein purified by ion-exchange chromatography was used in immunoblotting and indirect enzyme-linked immunosorbant assay (ELISA) studies using sera previously screened for chlamydial antibodies by an indirect ELISA incorporating solubilised chlamydial elementary bodies and by microimmunofluorescence. Immunoblotting identified 6/11 sera from infected ewes that had either typical placental lesions or had been found positive on examination of stained placental smears and 1/11 sera from ewes that had no typical placental lesions. The ELISA gave positive reactions with 29 of 65 known positive sera and 15 of the 76 negative sera. It is concluded that the 75 kDa DnaK-like protein is unsuitable as an antigen for antibody detection but its potential as a component for a sub-unit vaccine against ovine enzootic abortion warrants further study.
Abortion, Veterinary↗
Application of electroporation for transfer of plasmid DNA to Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Bacillus, Staphylococcus, Enterococcus and Propionibacterium.
Plasmid DNA was introduced by electroporation into Bacillus, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Propionibacterium and Staphylococcus as an alternative to competent-cell or protoplast transformation. Plasmid-containing transformants were recovered in these recipients at frequencies ranging from 10(1) to 10(5) transformants micrograms-1 of pGK12. Several parameters of the protocol, including DNA concentration, voltage, plating regimen and electroporation buffers were evaluated to determine conditions that improved transformation frequencies for Lactobacillus acidophilus. Using optimized conditions, the following plasmids were introduced into L. acidophilus: pAMB1, pC194, pGB354, pGKV1, pSA3, pTRK13, pTV1 and pVA797. The ability to transfer plasmid DNA via eletroporation will greatly facilitate the application of recombinant DNA methodology and transposon technology to Gram-positive bacteria for cloning and analysis of significant genes.
Buffers↗
A rapid and simple procedure for high-efficiency lithium acetate transformation of cryopreserved Schizosaccharomyces pombe cells.
A rapid, simple, convenient, and highly efficient transformation of the fission yeast Schizosaccharomyces pombe has been developed. Freezing fission yeast cells in glycerol, a permeating cryoprotectant, with lithium acetate improved remarkably the transformation efficiency by one to two orders of magnitude. The optimum concentration of glycerol was found to be 30%, which is higher than that (10-15%) in the conventional cryopreservation of yeast cells. Glycerol not only played a role in cryopreserving the competent cells but also improved the transformation efficiency of the process. The thawed cell suspension with glycerol and lithium acetate was immediately mixed with carrier DNA, plasmid DNA and polyethylene glycol. Next, the mixture was heat shocked and directly spread on a selection plate. This simple procedure yielded more than 10(6) transformants/microg plasmid DNA, reducing the time required to only 20 min in total, including the thawing time. Furthermore, the frozen competent cells were stored long-term for more than 3 months without any significant loss of efficiency.
Acetates↗
Immune condition in patients with Hodgkin's disease.
In 40 cases of Hodgkin's disease (24 untreated patients and 16 patients in remission) the immunological humoral and cell-mediated competence was studied using test of hypersensitivity of DNCB and PPD, blastic transformation and the ability of 3H-DNA synthesis in lymphocytes stimulated with PHA and PWM, and in some cases, with PPD. In all patients the concentration of serum immunoglobulins was determined as well. Impairment of immunological reactivity was found in about 40% of patients, on the average, in various stages of the disease and during remissions following treatment with cytostatic agents and radiotherapy. The most frequent abnormality found in 52.5% of cases was impairment of blastic transformation of lymphocytes stimulated with PHA in vitro. In 60% of cases negative results of intradermal test with PPD and in 75% of patients the DNCB test was negative. No correlation could be found between impairment of the immune competence in this disease and the clinical stage and/or type of histological changes in lymph nodes.
Cells, Cultured↗
Effects of exposure to high and low osmotic pressure upon Neisseria meningitidis transformation.
Transformation efficiency in competent variants of the Neisseria meningitidis Strain M1 increased more than three-fold after exposure of the recipient cells to approximately 35 atmospheres osmotic pressure for 20 min. Growth of the recipient cells at increased osmotic pressure contantly reduced the transformation efficiency. Exposure of the cells to hypotonic conditions reduced transformability significantly and attempts to restore transformability by means of supernatant solutions from competent cultures were unsuccessful. Incompetent variants of the Strain M1 could not be rendered competent by exposure to increased tonicity. The findings have been discussed in relation to the effects of tonicity variation on N. meningitidis competence variants.
DNA, Bacterial↗
The regulation of genetic competence in Bacillus subtilis.
Genetic competence develops as a global response of Bacillus subtilis to the onset of stationary phase, in glucose-minimal salts-based media. The onset of competence is accompanied by the expression of several late gene products that are required for the binding, processing and uptake of transforming DNA. A number of regulatory genes have been identified that are needed for the appropriate synthesis of the late gene products. The regulatory gene products include a number of known transcription factors, as well as several members of the bacterial two-component regulatory system. Genetic analysis has suggested a scheme for the flow of regulatory information signalling the onset of competence. Most of these regulatory products appear to be involved in the response to nutritional status, while the components responsible for growth stage and cell-type-specific control remain unknown. The general implications of this scheme for post-exponential expression are discussed.
Bacillus subtilis↗
Genetic transformation in freshwater: Escherichia coli is able to develop natural competence.
Until now, Escherichia coli was thought to be unable to develop natural competence, i.e., genetic transformation could be achieved only artificially with the aid of nonphysiological concentrations of calcium ions or by other treatments. We have tested the competence development of E. coli through transformation under natural conditions in river water, springwater, and mineral water which contained between 0 and 11 mM Ca2+, using pUC18 DNA. The presence of calcium ions at concentrations as low as 1 to 2 mM was sufficient to obtain transformants. Variations in the temperature of incubation were not required for competence development but had an influence on the transformation frequency. Using water from mineral springs originating from calcareous regions, we have obtained transformation frequencies with laboratory strains of E. coli similar to those reported for other gram-negative bacteria known to develop natural competence. The competence development of E. coli is most probably internally regulated (as for the other gram-negative bacteria), and inadequate conditions chosen for the transformation tests in the laboratory might impair the detection of higher natural transformation frequencies. The results will enhance our knowledge about the fate of laboratory or production strains of E. coli cells reaching natural aquatic ecosystems.
Calcium↗
Efficient cDNA cloning method using solid phase DNA probe.
We are now developing a novel and efficient method using solid phase DNA probe to isolate a particular recombinant cDNA from single stranded cDNA library. Target clone coding metapyrocatechase (MPC) and cDNA library constructed from mRNA of U-937 (human lymphoma cell line) were converted to single stranded form by superinfection of helper phage (M13KO7). Probe DNA (25 mer) composed of a portion of the target cDNA was synthesized, attached to an HPLC gel and used as a solid phase DNA probe. Hybridization between probe DNA and target clone was performed in an Eppendorf tube within a few hours. Competent cell (JM109) was transformed with about one-twentieth of hybridized and eluted fraction by Hanahan's method. From the mixture of 1 ng of MPC vector and 5 micrograms of cDNA library, we obtained 50 colonies containing MPC gene out of 63 transformed colonies.
Catechol 2,3-Dioxygenase↗
Identification and characterization of Helicobacter pylori genes essential for gastric colonization.
Helicobacter pylori causes one of the most common, chronic bacterial infections and is a primary cause of severe gastric disorders. To unravel the bacterial factors necessary for the process of gastric colonization and pathogenesis, signature tagged mutagenesis (STM) was adapted to H. pylori. The Mongolian gerbil (Meriones unguiculatus) was used as model system to screen a set of 960 STM mutants. This resulted in 47 H. pylori genes, assigned to 9 different functional categories, representing a set of biological functions absolutely essential for gastric colonization, as verified and quantified for many mutants by competition experiments. Identification of previously known colonization factors, such as the urease and motility functions validated this method, but also novel and several hypothetical genes were found. Interestingly, a secreted collagenase, encoded by hp0169, could be identified and functionally verified as a new essential virulence factor for H. pylori stomach colonization. Furthermore, comB4, encoding a putative ATPase being part of a DNA transformation-associated type IV transport system of H. pylori was found to be absolutely essential for colonization, but natural transformation competence was apparently not the essential function. Thus, this first systematic STM application identified a set of previously unknown H. pylori colonization factors and may help to potentiate the development of novel therapies against gastric Helicobacter infections.
Animals↗
DNA uptake during genetic transformation and the growing zone of the cell envelope.
Continuous cellular incorporation of choline molecules is essential for DNA binding to competent pneumococci. The choline molecules are incorporated into the cell surface at the equatorial region of the cocci. As a working hypothesis, it is proposed that DNA molecules enter these bacteria at the growing zone(s) of the cell envelope.
Amino Alcohols↗
The transfer and stable integration of the HSV thymidine kinase gene into mouse cells.
Treatment of mutant mouse cells (Ltk-) deficient in thymidine kinase with Bam I restriction endonuclease-cleaved HSV-1 DNA results in the appearance of numerous surviving colonies which stably express thte tk+ phenotype. Through a series of electrophoretic fractionations in concert with transfection assays, we isolated a 3.4 kb fragment which contains the thymidine kinase gene and which alone is competent in the biochemical transformation of Ltk- cells. In this report, we have examined the distribution of tk sequences in the DNA of several transformed clones following stable gene transfer. A series of complementary experiments involving reassociation kinetics in solution and annealings with tk DNA to restriction-cleaved cellular DNA following electrophoresis and transfer to filters allow us to make the following general conclusions concerning the fate of the tk gene in all clones examined: the tk gene is present in all cells at a frequency of one copy per chromosomal complement; the tk gene is stably integrated in the DNA of all transformants; and integration is not site-specific and occurs at different loci in the DNA of all transformants examined. The existence of a single active tk gene in tk+ transformants now facilitates an analysis of the sequence organization of tk- mutant cells and provides a useful model system for studies on the transfer of cellular genes.
Cell Line↗
Regulation of DNA synthesis in division-arrested mouse C127 cells permissive for bovine papillomavirus DNA amplification.
Spontaneous amplification of bovine papillomavirus type 1 DNA occurs following a prolonged period of serum starvation of wild-type virus-transformed C127 cell lines and is associated with abundant viral E2 protein synthesis and a concomitant induction of viral oncogene (E5 and E6) expression. We show here that a subpopulation of the permissive cells incorporate bromo-deoxyuridine under conditions of cell growth arrest (serum starvation), whereas DNA synthesis is suppressed in the resting population of nonpermissive cells. Flow cytometric measurements of the cellular DNA content of the permissive cell population indicated that it contained predominantly a 4n DNA content, suggesting that these cells were blocked in the G2 phase of the cell cycle. In keeping with the hypothesis that viral DNA amplification is associated with the induction of a cellular S phase, we observed a specific induction of expression of two cell proliferation-related cellular antigens (PCNA and Ki67) in a subpopulation of permissive cells. C127 cell lines transformed by an E5-minus bovine papillomavirus type 1 mutant, which was competent for autonomous plasmid replication in mitotic cells, were completely defective for the induction of DNA synthesis and mutant viral DNA amplification under conditions of serum starvation. Moreover, the E5 protein is shown by immunofluorescence analysis to be expressed at a high level specifically in the permissive cell population. These results imply a dual role for the viral E5 protein in the C127 model system, both as a transforming protein and as a factor required for the induction of viral DNA amplification in postmitotic cells. We suggest that E5 acts at an early step in the induction of this process in C127 cells and may be required to turn on host cell DNA synthesis as a prerequisite for viral DNA amplification.
Animals↗
Mutational analysis of genes involved in pilus structure, motility and transformation competency in the unicellular motile cyanobacterium Synechocystis sp. PCC 6803.
The relevance of pilus-related genes to motility, pilus structure on the cell surface and competency of natural transformation was studied by gene disruption analysis in the unicellular motile cyanobacterium Synechocystis: sp. PCC 6803. The genes disrupted in this study were chosen as related to the pil genes for biogenesis of the type IV pili in a Gram-negative bacterium Pseudomonas aeruginosa. It was found that motility of Synechocystis cells was lost in the mutants of slr0063, slr1274, slr1275, slr1276, slr1277 and sll1694 together with a simultaneous loss of the thick pili on the cell surface. Competency of the natural transformation was lost in the mutants listed above and slr0197-disruptant. The gene slr0197 was previously predicted as a competence gene by a search with sequence-independent DNA-binding structure [Yura et al. (1999) DNA Res. 6: 75]. It was suggested that both DNA uptake for natural transformation and motility are mediated by a specific type IV-like pilus structure, while a putative DNA-binding protein encoded by slr0197 is additionally required for the DNA uptake. Based on the homology with the pil genes in P: aeruginosa, slr0063, slr1274, slr1275, slr1276, slr1277 and sll1694 were designated pilB1, pilM, pilN, pilO, pilQ and pilA1, respectively. The gene slr0197 was designated comA.
Amino Acid Sequence↗
OVERNIGHT INCUBATION TECHNIQUE FOR OBTAINING TRANSFORMABLE BACILLUS SUBTILUS CELLS OF REPRODUCIBLE COMPETENCY.
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Bacillus↗
Bacteriophage-associated gene transfer in pneumococcus: transduction or pseudotransduction?
Lysates of pneumococcal phage PG24 transferred genes from one host to another in a process with many of the properties of generalized transduction, in that the host genes were packaged in DNase-resistant particles that closely resembled infectious phage in physical properties, adsorbed to the recipient cells like phage, and were inhibited by antisera to the phage and by trypsin. However, phage processes did not complete the transfer of host DNA as they did phage DNA. Instead, gene transfer required development of competence and entry of the host DNA by the endonuclease-dependent pathway used for transforming and transfecting DNA. This process often occurred on the assay plate hours after adsorption of the particles to the cells, and the transfer was DNase sensitive if challenged at this time. Phenotypic expression was therefore also delayed. The product of entry was like that in transformation, a single strand of DNA that integrates by formation of a hex-sensitive donor-recipient heteroduplex. Whether this gene transfer process is unique to this system or is only the first one described is not clear. The term "pseudotransduction" may be useful in calling attention to its unexpected features. The DNA of PG24 phage has anomalous physical properties reflecting unusual bases.
Bacteriophages↗