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PubMed1998

Formation of single-stranded DNA during DNA transformation of Neisseria gonorrhoeae.

Neisseria gonorrhoeae is naturally competent for DNA transformation. In contrast to other natural prokaryotic DNA transformation systems, single-stranded donor DNA (ssDNA) has not previously been detected during transformation of N. gonorrhoeae. We have reassessed the physical nature of gonococcal transforming DNA by using a sensitive nondenaturing native blotting technique that detects ssDNA. Consistent with previous analyses, we found that the majority of donor DNA remained in the double-stranded form, and only plasmid DNAs that carried the genus-specific DNA uptake sequence were sequestered in a DNase I-resistant state. However, when the DNA was examined under native conditions, S1 nuclease-sensitive ssDNA was identified in all strains tested except for those bacteria that carried the dud-1 mutation. Surprisingly, ssDNA was also found during transformation of N. gonorrhoeae comA mutants, which suggested that ssDNA was initially formed within the periplasm.

Bacterial Proteins↗
PubMed1975

Competitive inhibition of transformation in group H Streptococcus strain Challis by heterologous deoxyribonucleic acid.

Glucosylated deoxyribonucleic acid (DNA) from phages T4 and T6 competes poorly with homologous DNA causing only a slight decrease of transformation in Group H Streptococcus strain Challis. Other types of heterologous DNAs (Micrococcus luteus, Clostridium perfringens, Escherichia coli, calf thymus and non-glucosylated phage T6 DNA), in contrast to glucosylated T4 and T6 DNAs, compete with transforming DNA to the normal, high extent. These results indicate that as in transformation of Bacillus subtilis, the presence of glucose attached to 5-hydroxymethylcytosine in phage T6 DNA considerably decreases the interaction of such DNA with competent cells of the Challis strain. It also indicates that the guanine plus cytosine content of DNA is not decisive in determining its interaction with competent cells.

Animals↗
PubMed1992

Transformation of Bacillus subtilis by DNA bound on montmorillonite and effect of DNase on the transforming ability of bound DNA.

The equilibrium adsorption and binding of DNA from Bacillus subtilis on the clay mineral montmorillonite, the ability of bound DNA to transform competent cells, and the resistance of bound DNA to degradation by DNase I are reported. Maximum adsorption of DNA on the clay occurred after 90 min of contact and was followed by a plateau. Adsorption was pH dependent and was greatest at pH 1.0 (19.9 micrograms of DNA mg of clay-1) and least at pH 9.0 (10.7 micrograms of DNA mg of clay-1). The transformation frequency increased as the pH at which the clay-DNA complexes were prepared increased, and there was no transformation by clay-DNA complexes prepared at pH 1. After extensive washing with deionized distilled water (pH 5.5) or DNA buffer (pH 7.5), 21 and 28%, respectively, of the DNA remained bound. Bound DNA was capable of transforming competent cells (as was the desorbed DNA), indicating that adsorption, desorption, and binding did not alter the transforming ability of the DNA. Maximum transformation by bound DNA occurred at 37 degrees C (the other temperatures evaluated were 0, 25, and 45 degrees C). DNA bound on montmorillonite was protected against degradation by DNase, supporting the concept that "cryptic genes" may persist in the environment when bound on particulates. The concentration of DNase required to inhibit transformation by bound DNA was higher than that required to inhibit transformation by comparable amounts of free DNA, and considerably more bound than free DNase was required to inhibit transformation by the same amount of free DNA. Similarly, when DNA and DNase were bound on the same or separate samples of montmorillonite, the bound DNA was protected from the activity of DNase.

Adsorption↗
PubMed1984

Simian virus 40 large T-antigen point mutants that are defective in viral DNA replication but competent in oncogenic transformation.

The large T antigen of simian virus 40 (SV40) is a multifunctional protein that is essential in both the virus lytic cycle and the oncogenic transformation of cells by SV40. To investigate the role of the numerous biochemical and physiological activities of T antigen in the lytic and transformation processes, we have studied DNA replication-deficient, transformation-competent large T-antigen mutants. Here we describe the genetic and biochemical analyses of two such mutants, C2/SV40 and C11/SV40. The mutants were isolated by rescuing the integrated SV40 DNA from C2 and C11 cells (CV-1 cell lines transformed with UV-irradiated SV40). The mutant viral early regions were cloned into the plasmid vector pK1 to generate pC2 and pC11. The mutations that are responsible for the deficiency in viral DNA replication were localized by marker rescue. Subsequent DNA sequencing revealed point mutations that predict amino acid substitutions in the carboxyl third of the protein in both mutants. The pC2 mutation predicts the change of Lys----Arg at amino acid 516. pC11 has two mutations, one predicting a change of Pro----Ser at residue 522, and another predicting a Pro----Arg change at amino acid 549. The two C11 mutations were separated from each other to form two distinct viral genomes in pC11A and pC11B. pC2, pC11, pC11A, and pC11B are able to transform both primary and established rodent cell cultures. The C11 and C11A T antigens are defective in ATPase activity, suggesting that wild-type levels of ATPase activity are not necessary for the oncogenic transformation of cells by T antigen.

Animals↗
PubMed1998

All seven comG open reading frames are required for DNA binding during transformation of competent Bacillus subtilis.

The seven proteins encoded by the comG operon of Bacillus subtilis exhibit similarity to gene products required for the assembly of type 4 pili and for the secretion of certain proteins in gram-negative bacteria. Although polar transposon insertions in comG result in the loss of transformability and in the failure of cells grown through the competence regimen to bind DNA, it was not known whether the ComG proteins are all required for competence. We have constructed strains missing each of these proteins individually and found that they are all nontransformable and fail to bind transforming DNA to the cell surface. The implications of these findings are discussed.

Amino Acid Sequence↗
PubMed1984

Degradation of Escherichia coli chromosomal and plasmid DNA in serum.

Incubation of serum-sensitive [3H]thymidine labelled Escherichia coli PC2166 (RSF1030) and E. coli AM1281 (pBR322) harbouring small plasmids (mol. wt 5.5 X 10(6) and 2.6 X 10(6] in serum resulted in killing of 99.9% of the bacteria within 15 min and in the release of 85% of the radioactivity into the medium after 1 h incubation. The fate of chromosomal and plasmid DNA during incubation of the bacteria in serum was analysed by measurement of the amount of DNA-associated radioactivity, by TCA precipitation, by agarose gel electrophoresis and by the capacity of DNA to transform competent acceptor bacteria. Chromosomal DNA and high molecular weight plasmid DNA were rapidly degraded after 1 h incubation of bacteria in serum. However, low molecular weight plasmid DNA was virtually unaffected and remained physicochemically as well as biologically intact during up to 4 h of incubation of bacteria in serum.

Blood Bactericidal Activity↗
PubMed2000

DNase I induced DNA degradation is inhibited by neomycin.

Preparations of antimicrobials from biotechnological sources containing nucleic acids may serve as vector for the dissemination of resistance genes. An essential prerequisite for the acquisition of a new resistance phenotype in a transformational scenario is the availability of physically intact DNA molecules capable of transforming competent microorganisms. DNA is thought to be an easy target for catabolic processes when present in the natural habitat of bacteria (e.g. gastrointestinal tract, soil) due to the overall presence of nucleolytic enzymes. Aminoglycoside antibiotics are known to display a strong affinity to nucleic acids rendering these compounds to be primary candidates for exerting DNA protective functions in the gastrointestinal tract when applied orally during antibiotic chemotherapy. Using a DNase I protection assay it could be demonstrated that neomycin B at a concentration of 2 mM completely inhibited degradation of plasmid DNA in vitro. No inhibition of degradation was observed with streptomycin and kanamycin and the non-aminoglycoside antibiotics oxytetracycline and ampicillin under identical assay conditions. Thus, neomycin preparations may be able to promote structural integrity of contaminating DNA-fragments in DNase-rich environments.

Ampicillin↗
PubMed1999

ComEA is a DNA receptor for transformation of competent Bacillus subtilis.

Competent cells of Bacillus subtilis efficiently bind and internalize DNA. ComEA and the seven proteins encoded by the comG operon are required in vivo for the binding step. We show here that ComEA, a bitopic membrane protein, is itself capable of high-affinity DNA binding. A domain necessary for DNA binding is located at the C-terminus of ComEA. Proteins with similar 60-80 amino acid residue domains are widespread among bacteria and higher organisms. ComEA shows a marked preference for double-stranded DNA and can bind to oligomers as small as 22 bp in length. DNA binding by ComEA exhibits no apparent base sequence specificity. Using a membrane vesicle DNA-binding assay system we show that in the absence of cell wall, ComEA is still required for DNA binding, whereas the requirement for the ComG proteins is bypassed. We conclude that the ComG proteins are needed in vivo to provide access of the binding domain of ComEA to exogenous DNA. Possible specific roles for the ComG proteins are discussed.

Amino Acid Sequence↗
PubMed1983

[Transformation of competent Bacillus subtilis cells by chromosomal and plasmid DNA incorporated in liposomes].

Transformation with chromosomal and plasmid DNAs comprised in liposomes of different compositions was studied on competent cells of Bacillus subtilis. Transformation with chromosomal DNA comprised in liposomes appeared to constitute 1.1 to 1.5% of the control, and transformation with plasmid DNA in liposomes reaches 8 to 11%, as compared to the control. It has been revealed that absorbtion of chromosomal or plasmid DNA comprised in liposomes by competent cells is 1-2 orders higher than that of chromosomal or plasmid DNAs which are not contained in liposomes. Besides, chromosomal DNA in liposomes was found to be transferred to competent cells in the double-stranded form, while during common transformation without liposomes, the DNA transferred is single-stranded.

Bacillus subtilis↗
PubMed1982

Mutant of simian virus 40 large T-antigen that is defective for viral DNA synthesis, but competent for transformation of cultured rat cells.

A mutant was isolated which demonstrates that the transforming activity of simian virus 40 large T-antigen is separable from its function in viral DNA replication. The mutant, SVR9D, is nonconditionally defective for viral DNA synthesis, but competent at wild-type level for morphological transformation of cultured rat cells. The lytic growth defect in SVR9D is complemented by the simian virus 40 A gene product present in the transformed CV1 cell line, COS1. The lesion in SVR9D DNA was mapped genetically by marker rescue of plaque formation and localized to a 214-base-pair segment of the viral genome bounded by nucleotide numbers 4100 and 4314. DNA sequence analysis showed the mutation to be an adenine-to-guanine transition at nucleotide number 4178. This change predicts a lysine-to-glutamic acid amino acid change at residue number 214 of the mutant large T-antigen polypeptide.

Animals↗
PubMed1971

Fate of transforming DNA after uptake by competent Bacillus subtilis: failure of donor DNA to replicate in a recombination-deficient recipient.

The fate of radioactively-labeled transforming DNA was studied in a recombination-deficient strain of Bacillus subtilis that carried the recB2 mutation (Rec(-)) and was sensitive to radiation. Experiments performed with extracts of this strain after transformation showed that the recovery of donor transforming activity and the appearance of recombinant transforming activity occurred to the same extent as in the Rec(+) strain. Sucrose gradient analyses revealed that donor-recipient complex is also formed to the same extent in the Rec(-) and Rec(+) strains, but that 80-90% of the donor genetic material in the complex failed to replicate in the Rec(-) mutant.

Bacillus subtilis↗
PubMed1966

Further evidence concerning the configuration of transforming deoxyribonucleic acid during entry into Bacillus subtilis.

Strauss, Norman (State University of New York at Buffalo, Buffalo, N.Y.). Further evidence concerning the configuration of transforming deoxyribonucleic acid during entry into Bacillus subtilis. J. Bacteriol 91:702-708. 1966.-The appearance of linked, unselected traits with selected markers was followed as a function of time after the exposure of competent cells to transforming deoxyribonucleic acid (DNA). It was found that the per cent cotransfer of a linked, unselected trait with a single selected trait increased sharply soon after the lag period characterizing the appearance of the selected trait. Similar results were obtained when cotransfer of a linked unselected trait with a pair of selected traits was examined. The results are taken as an unequivocal demonstration that the entry of transforming DNA into competent Bacillus subtilis occurs in longitudinal fashion. The nature of the linkage between try(2) and his(9) was characterized. It was found that, although these two traits had been found to be unlinked on the basis of recombination tests, the saturation curves showed these two traits to be present on the same fragment of DNA.

Bacillus subtilis↗
PubMed1999

HP0333, a member of the dprA family, is involved in natural transformation in Helicobacter pylori.

Helicobacter pylori is naturally competent for DNA transformation, but the mechanism by which transformation occurs is not known. For Haemophilus influenzae, dprA is required for transformation by chromosomal but not plasmid DNA, and the complete genomic sequence of H. pylori 26695 revealed a dprA homolog (HP0333). Examination of genetic databases indicates that DprA homologs are present in a wide variety of bacterial species. To examine whether HP0333 has a function similar to dprA of H. influenzae, HP0333, present in each of 11 strains studied, was disrupted in two H. pylori isolates. For both mutants, the frequency of transformation by H. pylori chromosomal DNA was markedly reduced, but not eliminated, compared to their wild-type parental strains. Mutation of HP0333 also resulted in a marked decrease in transformation frequency by a shuttle plasmid (pHP1), which differs from the phenotype described in H. influenzae. Complementation of the mutant with HP0333 inserted in trans in the chromosomal ureAB locus completely restored the frequency of transformation to that of the wild-type strain. Thus, while dprA is required for high-frequency transformation, transformation also may occur independently of DprA. The presence of DprA homologs in bacteria known not to be naturally competent suggests a broad function in DNA processing.

Amino Acid Sequence↗
PubMed2003

A technique permitting selection of penicillin-susceptible pneumococcal strains following transformation of a penicillin-resistant strain with a penicillin-susceptibility gene.

We report a technique useful for transformation experiments involving bacteria naturally competent for DNA transformation. It allows the selection of antibiotic-susceptible transformants following the transformation of a resistant strain with an antibiotic susceptibility gene. We show the effectiveness of this technique through the selection of penicillin-susceptible (MIC, 0.03 microg/ml) transformants following the transformation of a penicillin-resistant (MIC, 16 microg/ml) pneumococcal strain with a penicillin-susceptibility gene.

Carrier Proteins↗
PubMed1972

Number of deoxyribonucleic acid uptake sites in competent cells of Bacillus subtilis.

Two direct methods are presented for estimating the average number of deoxyribonucleic acid (DNA) uptake sites in competent cells of Bacillus subtilis from measurement of (14)C- or (3)H-thymine-labeled DNA uptake by competent culture. Advantage is taken of two facts: (i) effective contact between competent cells and transforming DNA molecules is established within a short time after mixing them together, and (ii) DNA molecules enter the competent B. subtilis cells in a linear fashion at a finite speed. From the number of DNA molecules initially attached to competent cells by brief exposure to transforming DNA in the first method or from the rate of DNA uptake by competent culture in the second method, the average number of DNA uptake sites is calculated to be 20 to 53 per competent cell.

Bacillus subtilis↗