Interactions of homologous and heterologous deoxyribonucleic acids and competent Bacillus subtilis cells.

Glucosylated and nonglucosylated bacteriophage T4 deoxyribonucleic acids (DNAs) are able to bind to competent cells of Bacillus subtilis, although the former does so in a rather unstable fashion, probably because of the glucosylation. Several heterologous DNAs compete with homologous DNA for the same receptors in binding and in transformation. A different pattern in competition for DNA binding was observed for homologous and T4 glucosylated DNAs in intact cells as compared with protoplasts or membrane vesicles. The results are consistent with the existence of two types of receptor sites on the membrane of competent B. subtilis cells.     

Selective release of a deoxyribonucleic acid-binding factor from the surface of competent pneumococci.

Methods are described that resulted in the selective release of deoxyribonucleic acid (DNA)-binding factor from the surface of competent pneumococci. The same methods caused a parallel inactivation of the DNA-binding capacity of the extracted bacteria. Genetically or physiologically incompetent pneumococci did not yield binding factor upon exposure to the same methods. The solubilized binding factor appeared to be a protein; it could be assayed by a membrane filter binding procedure. The binding factor had properties reminiscent of those of the DNA receptors of transformable pneumococci (Seto et al., 1975).     

Cell surface-located deoxyribonucleic acid receptors in transformable pneumococci.

We studied deoxyribonucleic acid (DNA) binding in transformable pneumococci. The relevant findings are as follows. (i) At least half of the DNA Molecules adsorbed to competent cells in the growth medium are attached to sites on the protoplast membrane. (ii) Most of the DNA bound to live competent cells in the presence of glucose is not released by moderate shear or by autolysin treatment. In contrast, most of the DNA adsorbed to competent cells in the absence of glucose is shear and autolysin sensitive. (iii) The presence of binding sites resembling in properties the sites in live competent cells can be demonstrated in wall-membrane complexes. Most of these sites are lost during preparation of cell walls and protoplasts. It is suggested that the DNA-binding site is a membrane component (protein?) Stabilized by polysaccharide (cell Wall) material. (IV) Mechanical or enzymatic damage to the cell wall or change in the ionic conditions can induce DNA binding (and surface-nuclease activity) in the incompetent pneumococci. However, such cells still show neither genetic transformation nor extensive nuclease-resistant binding of DNA. It is suggested that both competent and incompetent cells contain a large number of sequestered DNA-binding sites that can be unmasked by several experimental conditions. Induction of the competent state by the competence activator protein may involve an endogenous unmasking process.     

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.     

Transformation of leucine and rifampin traits in Neisseria gonorrhoeae with deoxyribonucleic acid from homologous and heterologous origins.

A leucine-requiring, rifampin-sensitive strain of Neisseria gonorrhoeae was transformed to a leucine-nonrequiring, rifampin-resistant phenotype with deoxyribonucleic acid (DNA) obtained from both N. meningitidis and N. gonorrhoeae. The transforming efficiency of the meningococcal DNA was about 10- to 100-fold less than that of the homologous gonococcal DNA. A chemically defined medium that would support growth of most gonococcal isolates was used as a complete medium. A minimal medium was used for selection of Leu+ transformants. N-methyl-N'-nitro-N-nitrosoguanidine was used as a mutagen for isolating leucine prototrophs from leucine-requiring isolates of N. gonorrohoeae.     

Fate of heterologous deoxyribonucleic acid in Bacillus subtilis.

CsCl density gradient fractionation of cell lysates was employed to follow the fate of Escherichia coli, phage T6, and non-glucosylated phage T6 deoxyribonucleic acid (DNA) after uptake by competent cells of Bacillus subtilis 168 thy minus trp minus. Shortly after uptake, most of the radioactive Escherichia coli or non-glucosylated T6 DNA was found in the denatured form; the remainder of the label was associated with recipient DNA. Incubation of the cells after DNA uptake led to the disappearance of denatured donor DNA and to an increase in the amount of donor label associated with recipient DNA. These findings are analogous to those previously reported with homologous DNA. By contrast, T6 DNA, which is poorly taken up, appeared in the native form shortly after uptake and was degraded on subsequent incubation. The nature of the heterologous DNA fragments associated with recipient DNA was investigated with Escherichia coli 2-H and 3-H-labeled DNA. Association of radioactivity with recipient DNA decreased to one-fourth in the presence of excess thymidine; residual radioactivity could not be separated from recipient DNA by shearing (sonic oscillation) and/or denaturation, but was reduced by one-half in the presence of a DNA replication inhibitor. Residual radioactivity associated with donor DNA under these conditions was about 5% of that originally taken up. Excess thymidine, but not the DNA replication inhibitor, also decreased association of homologous DNA label with recipient DNA; but, even in the presence of both of these, the decrease amounted to only 60%. It is concluded that most, or all, of the Escherichia coli DNA label taken up is associated with recipient DNA in the form of mononucleotides via DNA replication.     

Role of polyadenylic acid in a deoxyribonucleic acid-membrane fraction extracted from pneumococci.

After the addition of radioactive polyadenylic acid to cell suspensions of pneumocci, part of the radioactivity becomes associated with a deoxyribonucleic acid (DNA)-membrane fraction extracted from the cells. A variety of techniques show that a portion of this associated radioactivity may represent oligoadenylates complexed to DNA, probaby as part of a ribonucleic acid (RNA) component. Polyadenylic acid, which had previously been shown to enhance DNA synthesis in cell suspensions (Firshein and Benson, 1968), also enhances the extent of DNA synthesis by the DNA-membrane fraction in vitro under specific conditions of concentration and conformation. The mechanism of action of this enhancement may be related to the ability of oligoadenylates to increase the number of initiation sites for DNA replication by stimulating the production of an RNA primer, thus providing additional 3'-OH groups with which DNA polymerase can react.     

Role of deoxyribonucleic acid ligase in a doxyribonucleic acid membrane fraction extracted from pneumococci.

Deoxyribonucleic acid (DNA) ligase has been detected in a DNA membrane fraction extracted from Pneumococcus. The specific activity of the enzyme in this fraction is 10-fold greater than in the remaining cell extract. It remains firmly bound (with other enzymes) to the complex after a purification procedure in which a considerable percentage of the macromolecules are dissociated. The ligase acts in two ways in the DNA membrane fraction in vitro. One, it catalyzes the linkage of small-molecular-weight pieces of newly synthesized DNA into heavier-molecular-weight DNA strands as shown by others (M Gellert, 1976; R. Okazaki, A. Sugino, S. Hirose, T. Okazaki, Y. Imae, R. Kainuma-Kuroda, T. Ogawa, M. Arisawa, and Y. Kurosowa, 1973; B. Olivera and I. Lehman, 14; and A. Sugino, S. Hirose, and R. Okazaki, 1972) and, two, it protects DNA from degradation by deoxyribonucleases. This latter effect is due to a competition between the ability of the nucleases to degrade DNA and the ability of DNA ligase to seal the nicks produced by these degradative enzymes. The ligase acts cooperatively with other enzymes in the DNA membrane fraction to synthesize DNA.     

Heterologous deoxyribonucleic acid uptake and complexing with cellular constituents in competent Bacillus subtilis.

With competent cultures of Bacillus subtilis the uptake of Escherichia coli deoxyribonucleic acid (DNA) is about 50% that for homologous DNA. Uptake of phage T6 DNA, if any, is of the order of 7%, while nonglucosylated phage T6 (T6) DNA is taken up almost as effectively as homologous DNA. Both T6 and T4 DNA interfere only minimally with uptake of homologous DNA; by contrast, T6 DNA competes with homologous DNA as effectively as the latter itself. These results indicate that the glucose residues in the T-even phage DNA, located in the large groove of the DNA helix, reduce affinity for cellular receptors, leading to low binding of T6 DNA. The latter DNA is considerably less degraded by extracellular nucleases than homologous DNA, thus excluding enzymatic hydrolysis as the source of poor uptake. Affinity of DNA for competent cells was also evaluated by the formation, and detection in a CsCl density gradient, of complexes of DNA with cellular constituent(s). Such comlexes, similar to those previously observed with transforming DNA, are formed by E. coli DNA and T6 DNA; in reconstruction experiments the denatured forms of these same DNA samples form complexes when added to the cells before lysis. T6 DNA, on the other hand, does not form such a complex. The possible role of such complexes in transport of DNA to the cell interior is discussed.     

Single-stranded regions in transforming deoxyribonucleic acid after uptake by competent Haemophilus influenzae.

About 15% of donor deoxyribonucleic acid (DNA) is single stranded immediately after uptake into competent Haemophilus influenzae wild-type cells, as judged by its sensitivity to S1 endonuclease. This amount decreases to 4 to 5% by 30 min after uptake. Mutants which are defective in the covalent association of recipient and donor DNA form little or no S1 endonuclease-sensitive donor. At 17 C donor DNA taken up by the wild type contains single-stranded regions although there is no observable association, either covalent or noncovalent. The single-stranded regions are at the ends of donor DNA molecules, as judged by the unchanged sedimentation velocity after S1 endonuclease digestion. The amount of single-stranded donor remains constant at 17 C for more than 60 min after uptake, suggesting that the decrease observed at 37 C is the result of association of single-stranded ends with single-stranded regions of recipient cell DNA. Three sequential steps necessary for the integration of donor DNA into recipient DNA are proposed: the synthesis of single-stranded regions in recipient DNA, the interaction of donor DNA with recipient DNA resulting in the production of single-stranded ends on donor DNA, and the stable pairing of homologous single-stranded regions.     

Heterogeneity of deoxyribonucleic acid molecules isolated from Mycobacterium smegmatis.

Bulk chromosomal deoxyribonucleic acids (DNAs) of Mycobacterium smegmatis strains 607+ (wild type) and 607-1 (Strr) and orange-red pigmented variants (OR) were separated into two distinct bands (types 1 and 2) by cesium chloride density gradient centrifugation. Thermal denaturation analyses showed that type 1 and 2 DNA fragments of these strains possessed guanine plus cytosine contents averaging 69.2% and 60.8%, respectively. Type 1 and 2 DNAs from all strains tested were recovered in relatively equal quantities upon isolation and were found to have similar molecular weights (3.0 x 10(7)). Spectrophotometric assay of DNA reassociation showed that homology between any type 1 and 2 DNA fragments was always very low (29 to 33%), even within the same strain. Homologies among type 1 DNAs isolated from any strain were always high (92 to 98%), whereas homologies between type 2 DNA isolated from OR strains and that from their parental strain 607-1 were lower (51 to 55%). Transformation experiments revealed that methionine, leucine, folic acid, and streptomycin markers were found exclusively in type 1 DNA fragments. In addition to the two types of chromosomal DNA, plasmid DNA possessing a molecular weight of about 4 x 10(6) was found in strain 607-1.     

Interactions between exogenous deoxyribonucleic acid and membrane vesicles isolated from competent and noncompetent Bacillus subtilis.

Competent cultures of Bacillus subtilis 168 have been fractionated into a high-competent and a low-competent fraction by a large-scale separation technique. Membrane vesicles isolated from both cell fractions are equally active in the transport of L-glutamate. Both membrane vesicle preparations seem to have similar endo- and exonuclease activities. Also, both preparations are capable of binding deoxyribonucleic acid. However, especially at low deoxyribonucleic acid concentrations (1 mug or less per ml), vesicles obtained from competent cells bind significantly more deoxyribonucleic acid (up to sixfold) than vesicles from noncompetent cells.