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At least 163 records · Page 9Linked to original sources
PubMed1965

BACTERIOPHAGE DEOXYRIBONUCLEATE INFECTION OF COMPETENT BACILLUS SUBTILIS.

Reilly, Bernard E. (Western Reserve University, Cleveland, Ohio), and John Spizizen. Bacteriophage deoxyribonucleate infection of competent Bacillus subtilis. J. Bacteriol. 89:782-790. 1964.-Phenol extracts of the Bacillus subtilis bacteriophages phi1, phi25, and phi29 contained infectious deoxyribonucleic acid. The infectivity was destroyed by catalytic amounts of deoxyribonuclease but not by specific phage antiserum, ribonuclease, or trypsin. An infectivity of >10(6) infectious centers formed per mug of deoxyribonucleic acid (DNA) added was obtained. The stability of the infectious centers permitted an examination of a single cycle of phage replication in cells unable to adsorb the mature virus. A typical cycle was observed, although the latent period was increased and the burst size slightly reduced after DNA infection. The development of competence for bacterial transformation was strongly correlated with susceptibility to viral DNA infection. Both appeared and disappeared at the same phase of growth in the cell population. More than 4% of the viable cells in the competent population were infected by viral DNA. The kinetics of the transition of phi29 DNA infection to deoxyribonuclease insensitivity, and the relationship of infectivity to DNA dilution, were similar to the results obtained for bacterial transformation of a single marker. The doseresponse curve of phi1 and phi25 DNA was characteristic of that obtained in multiple transformation of unlinked genetic markers. Because of the low efficiency of infection, about 10(-4) per phage equivalent of DNA added, it was not possible to prove that DNA alone was sufficient to initiate infection.

Bacillus subtilis↗
PubMed1995

Acinetobacter calcoaceticus liberates chromosomal DNA during induction of competence by cell lysis.

A transformation assay was used to assay the amount of DNA present in the extracellular medium of a growing culture of Acinetobacter calcoaceticus. It was observed that small amounts of DNA were liberated during the entire exponential growth phase in a batch culture. Release of DNA could be fully accounted for by lysis of cells. Lysis was quantified via simultaneous measurement of beta-galactosidase activity of cells and supernatant, with a strain that contained a plasmid (pAPA100) with lacZ under control of a constitutive beta-lactamase promoter. In conclusion, no evidence could be obtained indicating that Acinetobacter calcoaceticus actively excretes DNA, to be used for DNA exchange.

Acinetobacter calcoaceticus↗
PubMed1984

Protoplast transformation of glutamate-producing bacteria with plasmid DNA.

A method for polyethylene glycol-induced protoplast transformation of glutamate-producing bacteria with plasmid DNA was established. Protoplasts were prepared from cells grown in the presence of penicillin by treatment with lysozyme in a hypertonic medium. The concentration of penicillin during growth affected the efficiency of formation, regeneration, and polyethylene glycol-induced DNA uptake of protoplasts. Regeneration of protoplasts was accomplished on a hypertonic agar medium containing sodium succinate and yeast extract. The spectinomycin and streptomycin resistance plasmid pCG4, originally from Corynebacterium glutamicum T250, could transform various glutamate-producing bacteria such as C. glutamicum, Corynebacterium herculis, Brevibacterium flavum, and Microbacterium ammoniaphilum. The plasmid was structurally unchanged and stably maintained in new hosts. The transformation frequency of most competent protoplasts with pCG4 DNA isolated from primary transformants was high (ca. 10(6) transformants per microgram of covalently closed circular DNA) but was still two orders of magnitude below the frequency of transfection with modified DNA of the bacteriophage phi CGI. The difference was ascribed to the involvement of regeneration in transformation.

Brevibacterium↗
PubMed1993

Natural Transformation of Acinetobacter calcoaceticus by Plasmid DNA Adsorbed on Sand and Groundwater Aquifer Material.

It is known that plasmid DNA and linear duplex DNA molecules adsorb to chemically purified mineral grains of sand and to particles of several clay fractions. It seemed desirable to examine whether plasmid DNA would also adsorb to nonpurified mineral materials taken from the environment and, particularly, whether adsorbed plasmid DNA would be available for natural transformation of bacteria. Therefore, microcosms consisting of chemically pure sea sand plus buffered CaCl(2) solution were compared with microcosms consisting of material sampled directly from a groundwater aquifer (GWA) plus groundwater (GW) with respect to the natural transformation of Acinetobacter calcoaceticus by mineral-associated DNA. The GWA minerals were mostly sand with inorganic precipitates and organic material plus minor quantities of silt and clay (illite and kaolinite). The amount of plasmid DNA which adsorbed to GWA (in GW) was about 80% of the amount which adsorbed to purified sand (in buffered CaCl(2) solution). Plasmid DNA adsorbed on sand transformed A. calcoaceticus significantly less efficiently than did plasmid DNA in solution. In contrast, the transformation by sand-adsorbed chromosomal DNA was as high as that by DNA in solution. In GWA/GW microcosms, the efficiency of transformation by chromosomal DNA was similar to that in sand microcosms, whereas plasmid transformation was not detectable. However, plasmid transformants were found at a low frequency when GWA was loaded with both chromosomal and plasmid DNA. Reasons for the low transformation efficiency of plasmid DNA adsorbed to mineral surfaces are discussed. Control experiments showed that the amounts of plasmid and chromosomal DNA desorbing from sand during incubation with a cell-free filtrate of a competent cell suspension did not greatly contribute to transformation in sand microcosms, suggesting that transformation occurred by direct uptake of DNA from the mineral surfaces. Taken together, the observations suggest that plasmid DNA and chromosomal DNA fragments which are adsorbed on mineral surfaces in a sedimentary or soil habitat may be available (although with different efficiencies for the two DNA species) for transformation of a naturally competent gram-negative soil bacterium.

Journal Article↗
PubMed1983

Plasmid marker rescue transformation in Bacillus subtilis.

We constructed an 18-megadalton plasmid (pBD221) carrying resistance determinants for kanamycin, chloramphenicol, and erythromycin, as well as the hisH determinant from the Bacillus licheniformis chromosome. This plasmid has a copy number of about one and can be stably maintained in Bacillus subtilis. Linear fragments of pBD221 DNA were used to transform competent cultures carrying mutant variants of the same plasmid. Rescue transformation did not proceed by recircularization and replication of the donor DNA. Rescue transformation exhibited first-order dependence on DNA concentration, and the concentration dependence curve was virtually identical to the curve obtained with chromosomal DNA. The donor DNA molecular weight dependence of plasmid marker rescue transformation obtained by using restriction fragments was not distinguishable from previously published data obtained by using fractionated sheared chromosomal DNA. Plasmid rescue transformation, like chromosomal transformation, was dependent on the recE, recA, recB, and recD gene products. Plasmid rescue transformation, like chromosomal transformation, proceeded with few exchanges. Linkage data obtained with the plasmid rescue system fit a quantitative model based on studies with chromosomal transformation. We conclude that plasmid marker rescue transformation probably proceeds by a mechanism similar to the mechanism used during the formation of chromosomal transformants and hence may be considered an appropriate general model for the study of transformational recombination.

Anti-Bacterial Agents↗
PubMed2006

Multiple phases of competence occur during the Helicobacter pylori growth cycle.

The gastric pathogen Helicobacter pylori undergoes genetic exchange at unusually high frequencies, primarily through natural transformation. Despite progress toward understanding the molecular mechanism of natural transformation in H. pylori, little is known about how competence is regulated or its relationship to DNA release. By measuring transformation incrementally throughout the growth curve, we show that H. pylori exhibits a novel pattern of competence with distinct peaks of transformation during both logarithmic and stationary growth phases. Furthermore, different H. pylori strains vary in the presence and timing of their competence peaks. We also examined the process of DNA release in relation to competence. Although extensive DNA release does not occur until late stationary phase, sufficient genomic DNA was present during the logarithmic phase to yield measurable transformants. These results demonstrate that the state of competence in H. pylori occurs in an unprecedented pattern during the growth curve with no clear relationship to DNA release.

DNA, Bacterial↗
PubMed1982

Biological activity of cloned rat endogenous C-type virus DNA transferred by microinjection.

The biological activity of a molecularly cloned DNA of a rat endogenous C-type leukaemia helper virus, RHHV, was assessed by intranuclear microinjection into normal rat kidney cells (NRK153). Release of rat C-type leukaemia helper viruses by the microinjected cells was examined by superinfection of Kirsten-transformed non-producer cells (K-NRK). Immediate release of helper leukaemia viruses at a very low level was observed only in the NRK153 m3 . 5/cir cells microinjected with the supercoiled form of RHHV DNA in toto, suggesting that the circular form of the virus DNA might have expedited the replication and expression of virus particles. Genome rescue experiments were also performed by co-cultivating the microinjected NRK153 m cells carrying various linear RHHV DNAs, in toto or of subgenomic sizes, with K-NRK cells. The results indicated that both the total and the 5.8 to 6.2 kb DNA fragment proximal to the 5' terminus of the cloned RHHV 8.8 kb DNA were able to rescue successfully a transforming replication-competent pseudotype virus. Subgenomic DNA fragments derived from the centre or the 3' end of the RHHV DNA were ineffective in the genome rescue experiments.

Animals↗
PubMed2001

Evaluation of biological and physical protection against nuclease degradation of clay-bound plasmid DNA.

In order to determine the mechanisms involved in the persistence of extracellular DNA in soils and to monitor whether bacterial transformation could occur in such an environment, we developed artificial models composed of plasmid DNA adsorbed on clay particles. We determined that clay-bound DNA submitted to an increasing range of nuclease concentrations was physically protected. The protection mechanism was mainly related to the adsorption of the nuclease on the clay mineral. The biological potential of the resulting DNA was monitored by transforming the naturally competent proteobacterium Acinetobacter sp. strain BD413, allowing us to demonstrate that adsorbed DNA was only partially available for transformation. This part of the clay-bound DNA which was available for bacteria, was also accessible to nucleases, while the remaining fraction escaped both transformation and degradation. Finally, transformation efficiency was related to the perpetuation mechanism, with homologous recombination being less sensitive to nucleases than autonomous replication, which requires intact molecules.

Acinetobacter↗
PubMed2003

Nucleotide excision repair rates in rat tissues.

We have determined and compared nucleotide excision repair capability of several rat tissues by a method based on restoration of the transformation activity of UV-irradiated pBlueScript by incubation in repair-competent protein extracts. After 3 h of incubation, plasmid DNA was isolated and used to transform competent Escherichia coli cells. Damaged plasmids showed low transformation efficiency prior to incubation in repair-competent extracts. After incubation the transformation efficiency was restored to different extents permitting calculation of the repair capacity of the extracts. Our results showed that rapidly proliferating tissues such as liver, kidney and testis showed higher nucleotide excision repair capacity than slowly proliferating tissues such as heart, muscle, lung and spleen. When liver and splenocytes were stimulated to proliferation by partial hepatectomy and mitogen stimulation, their repair capability increased in parallel with the respective proliferative rates.

Animals↗
PubMed1985

Genetic transformation in Streptococcus sanguis. Effects on genetic transformation by culture filtrates of Streptococcus sanguis (serogroups H and W) and streptococcus mitis (mitior) with reference to identification.

Streptococcus sanguis (serogroups H and W) strains are frequently competent in genetic transformation. In a collection of 18 strains of S. sanguis isolated from blood cultures, 12 strains were spontaneously competent in transformation by streptomycin-resistant DNA, and three strains were induced to competence by the use of culture filtrates containing competence factor (CF). Culture filtrates from spontaneously competent S. sanguis strains only induced competence in transformation of strains of the same species. In addition to the three "constitutional" types of S. sanguis with regard to specificity of transformation previously reported (originally represented by the strain Challis, 13b and NCTC 7863), a possible fourth type, (represented by strain 21452) was found. Further studies are required to ascertain the role of these types. The "constitutional" types are characterized by the range of activity of their CF's. These specificities seem useful in identification of S. sanguis and its separation from S. mitis (mitior). Tests of spontaneously competent strains with culture filtrates from other competent strains showed variable effects on the transformation frequencies in different strains.

Bacterial Proteins↗
PubMed1999

Microevolution through DNA exchange among strains of Neisseria meningitidis isolated during an outbreak in the Czech Republic.

Neisseria meningitidis is a highly variable bacterium. Indeed, N. meningitidis is naturally competent for transformation, and horizontal DNA exchange between strains may lead to mosaic genetic loci in N. meningitidis. We studied such an exchange in nature during an epidemic provoked by N. meningitidis. This epidemic started in the Czech Republic in 1993 and the original epidemic clone was shown to have the antigenic formula (serogroup:serotype:serosubtype) C:2a:P1.2,5. We analysed 145 meningococcal strains isolated in the Czech Republic between 1993 and 1997 using serological and genetic typing methods (multilocus enzyme electrophoresis and polymorphism of pilA and pilD genes). This analysis showed that genetic exchange between epidemic and endemic strains had occurred. Exchanges involved mostly surface-exposed structures such as the capsule, giving rise to new meningococcal variants. The expansion of these variants should be kept under close surveillance.

Alleles↗
PubMed1977

The effect of Waring Blendor treatment on transformation in Bacillus subtilis.

Treatment of competent Bacillus subtilis in a Waring Blendor for 10 s increases transformability of the culture about twofold while reducing the attachment of DNA to competent cells by 80%. The effectiveness of attached DNA in producing transformants is increased 10-fold by this treatment. The uptake of transforming DNA into a DNase-resistant state is progressively reduced by 70% during a 120-s blending treatment. Blending for 30-45 s diminishes transformability to about 10% of the original nonblended value without affecting the viable cell titer. No effect is produced by 30 s of blending on transformability if the irreversible uptake of DNA has been completed. Thus, the inhibition occurs at an early step in the transformation sequence. Treatment of the competent culture for 60 s or longer in the Waring Blendor reduces both the number of transformants obtained and the total number of viable cells.

Bacillus subtilis↗
PubMed2000

Regulation of competence for genetic transformation in Streptococcus pneumoniae: expression of dpnA, a late competence gene encoding a DNA methyltransferase of the DpnII restriction system.

The chromosomal DpnII gene cassette of Streptococcus pneumoniae encodes two methyltransferases and an endonuclease. One methyltransferase acts on double-stranded and the other on single-stranded DNA. Two mRNAs are transcribed from the cassette. One, a SigA promoter transcript, includes all three genes; the other includes a truncated form of the second methyltransferase gene (dpnA) and the endonuclease gene. The truncated dpnA, which is translated from the second start codon in the full gene, was shown to produce active enzyme. A promoter reporter plasmid for S. pneumoniae was devised to characterize the promoter for the second mRNA. This transcript was found to depend on a promoter that responded to the induction of competence for genetic transformation. The promoter contains the combox sequence recognized by a SigH-containing RNA polymerase. As part of the competence regulon, the dpnA gene makes a product able to methylate incoming plasmid strands to protect them from the endonuclease and allow plasmid establishment. Its function differs from most genes in the regulon, which are involved in DNA uptake. Comparison of R6 and Rx strains of S. pneumoniae showed the temperature dependence of transformation in R6 to result from temperature sensitivity of the uptake apparatus and not the development of competence.

Base Sequence↗
PubMed1985

Differential behavior of plasmids containing chromosomal DNA insertions of various sizes during transformation and conjugation in Haemophilus influenzae.

Plasmids with chromosomal insertions were constructed by removal of a 1.1-kilobase-pair piece from the 9.8-kilobase-pair vector plasmid pDM2 by EcoRI digestion and inserting in its place various lengths of chromosomal DNA (1.7, 3.4, and 9.0 kilobase pairs) coding for resistance to novobiocin. A fourth plasmid was constructed by insertion of the largest piece of chromosomal DNA into the SmaI site of pDM2. The plasmids without inserts were taken up poorly by competent cells and thus were considered not to contain specific DNA uptake sites. The presence of even the smallest insert of chromosomal DNA caused a large increase in transformation of Rec+ and Rec- strains. The frequency of plasmid establishment in Rec+ cells by transformation increased exponentially with increasing insert size, but in Rec- cells there was less transformation by the larger plasmids. Conjugal transfer of these plasmids was carried out with the 35-kilobase-pair mobilizing plasmid pHD147. The frequency of establishment of plasmids by this method not only was not markedly affected by the presence of the insertions, but also decreased somewhat with increase in insert size and was independent of rec-1 and rec-2 genes. Recombination between plasmid and chromosome was readily detected after transformation, but could not be detected after transconjugation even when the recipient cells were Rec+ and made competent. These data suggested that there is a special processing of plasmid DNA that enters the competent cells in transformation that makes possible recombination of homologous regions of the plasmid with the chromosome and pairing with the chromosome that aids plasmid establishment.

Ampicillin↗
PubMed1975

Single-strand regions in the deoxyribonucleic acid of competent Haemophilus influenzae.

The deoxyribonucleic acid (DNA) of competent wild-type Haemophilus influenzae and rec1 mutant cells contains single-strand regions, as judged by alkaline sucrose sedimentation, benzoylated naphthoylated diethylaminoethyl-cellulose fractionation, and digestion with an enzyme specific for single-strand regions in DNA. In contrast, the DNA of competent rec2 cells does not contain single-strand regions. Since transforming DNA does not associate with recipient DNA in the rec2 mutant as it does in wild type and rec1, it is concluded that the single-strand regions in the DNA of the competent cells are important for an early step in recombination between cell DNA and transforming DNA.

Bromodeoxyuridine↗
PubMedDate not supplied

[Genetic transformation of Bacillus subtilis cells in the presence of clay minerals montmorillonite and kaolinite].

The effect of the clay minerals montmorillonite and kaolinite on the transformation of competent Bacillus subtilis cells with chromosomal DNA was studied. Clay particles were found to substantially increase the transformation frequency of competent cells, as well as the rate of their spontaneous chromosomal and plasmid transformation. The effect was ascribed to the adsorption of bacterial cells on the surface of mineral particles.

Antidiarrheals↗
PubMed1973

Relationship between prophage induction and transformation in Haemophilus influenzae.

The interaction between transformation and prophages of HP1c1, S2, and a defective phage of Haemophilus influenzae has been investigated by measurement of (i) the effect of prophage on transformation frequency and (ii) the effect of transformation on phage induction. The presence of any of the prophages does not appreciably alter transformation frequencies in various Rec(+) and Rec(-) strains. However, exposure of competent lysogens to transforming deoxyribonucleic acid (DNA) may induce phage but only in Rec(+) strains, which are able to integrate transforming DNA into their genome. Transformation of Rec(+) lysogens with DNA irradiated with ultraviolet (UV) light causes the production of even more phage than results from unirradiated DNA, but this indirect UV induction is not as effective as direct induction by UV irradiation of lysogens. Both types of UV induction are influenced by the repair capacity of the host. Wild-type cells contain a prophage and can be induced by transformation to produce a defective phage, which kills a small fraction of the cells. Defective phage in wild-type cells are also induced by H. parainfluenzae DNA, and a much larger fraction of the cells is killed. Strain BC200, which is highly transformable but is not inducible for defective phage, is not killed by H. parainfluenzae DNA, suggesting that wild-type cells are killed by killed by this DNA because of phage induction. A minicell-producing mutant, LB11, has been isolated. Some phage induction occurs in this strain when the cells are made competent, unlike the wild type. A large majority of LB11 cells surviving the competence regime are killed by exposure to transforming DNA.

Bacteriophages↗