Membrane location of a deoxyribonuclease implicated in the genetic transformation of Diplococcus pneumoniae.

The cellular localization of enzymes in Diplococcus pneumoniae was examined by fractionation of spheroplasts. A deoxyribonuclease implicated in the entry of deoxyribonucleic acid (DNA) into the cell during genetic transformation was located in the cell membrane. This enzyme, the major endonuclease of the cell (endonuclease I), which is necessary for the conversion of donor DNA to single strands inside the cell and oligonucleotides outside, thus could act at the cell surface. Another enzyme, the cell wall lysin (autolysin), was also found in the membrane fraction. Other enzymes, including amylomaltase, two exonucleases, and adenosine triphosphate-dependent deoxyribonuclease, and a restriction type endonuclease, were located in the cytosol within the cell. None of the enzymes examined were predominantly periplasmic in location. Spheroplasts were obtained spontaneously on incubation of pneumococcal cells in concentrated sugar solutions. The autolytic enzyme appears to be involved in this process. Cells that were physiologically competent to take up DNA formed osmotically sensitive spheroplasts two to three times faster than cells that were not in the competent state. Although some genetically incompetent mutants also formed spheroplasts more slowly, other such mutants formed them at the faster rate.     

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).     

Binding of Deoxyribonucleic Acid by Cell Walls of Transformable and Nontransformable Streptococci

Cell walls isolated from competent streptococci (group H strain Challis) were shown to bind more homologous and heterologous deoxyribonucleic acid (DNA) than noncompetent walls. Heat- and alkali-denatured DNA was not bound by either wall preparation. Pretreatment of cell walls with cetyltrimethylammonium bromide sharply increased the binding of DNA but did not increase transformation of whole cells. Pretreatment of the walls with either sodium dodecylsulfate, deoxyribonuclease and ribonuclease, or with crude competence-provoking factor did not affect the binding of DNA. Antiserum prepared against whole competent cells completely blocked transformation and also inhibited DNA binding to competent cell walls. Adsorption of this antiserum with competent Challis cells removed its blocking action for both binding and transformation. Pretreatment of walls with trypsin and Pronase destroyed their ability to bind DNA. Trypsin treatment also blocked transformation in whole cells. The transforming activity of DNA bound to cell walls was found to be protected from deoxyribonuclease action. Significant differences were observed in the arginine, proline, and phenylalanine content of competent and noncompetent walls. With few exceptions, the amino acids released from competent cell walls by trypsin were several-fold greater than from noncompetent walls. The results indicate that (i) two binding sites exist, one in competent cells only and essential for subsequent transformation, and a second, present in all cells, which is not involved in transformation; (ii) both sites are protein in nature; (iii) the transformation site is blocked by antibody; and (iv) the competent cell wall possesses tryptic-sensitive protein not present in the noncompetent wall.     

Inhibition of the Development of Competence in Streptococcus sanguis (Wicky) by Reagents that Interact with Sulfhydryl Groups: Discernment of the Competence Process

Reagents that interact with sulfhydryl groups are shown to inhibit competence factor (CF)-induced competence development in Streptococcus sanguis (Wicky) strain WE4 (Wicky 4 Ery(R)). Inhibition is correlated with specific inhibition of either the function or biosynthesis of three competent cell-related proteins and is reversed by either 2-mercaptoethanol or dithiothreitol. Mercuric chloride (5 muM) or N-ethylmaleimide (NEM; 50 muM) inhibited (i) the function but not the biosynthesis or activation of the competent cell-associated autolysin; (ii) the biosynthesis of a competent cell-associated protein of unknown function, demonstrated by polyacrylamide gel electrophoresis of acidified phenol extracts; and (iii) the biosynthesis or activation of distinct deoxyribonucleic acid (DNA)-binding sites. Neither reagent at the indicated concentration interfered with the uptake of CF by cells or with the uptake and expression of DNA by competent cells. Neither reagent inactivated CF or genetic markers coded by the transforming DNA, nor did they inhibit cell growth or viability appreciably. The data reveal that either mercuric chloride or NEM can differentially inhibit induced protein synthesis and, in addition, conclusively show that some autolytic activity is essential for the onset of the competent state.     

Early stages in Bacillus subtilis transformation: association between homologous DNA and surface structures.

The addition of ethylenediaminetetraacetate to competent cultures of Bacillus subtilis irreversibly inhibited the transformability as well as the cellular binding of DNA. Our results show that the inhibition of DNA binding by ethylenediaminetetraacetate in whole cells, protoplasts, and membrane vesicles is mainly due to a permanent alteration of the DNA receptors. Transformation absolutely requires free magnesium ions, whereas DNA binding is a magnesium-independent step. In contrast to ethylenediaminetetraacetate, the absence of Mg2+ does not irreversibly affect the capacity of the competent cells to be transformed DNA-binding receptors located at the cell surface remain associated with the plasma membrane after protoplasting and after isolation of membrane vesicles. A Mg2+-dependent endonucleolytic activity associated with the membrane appears to be responsible for the lower levels of binding by protoplasts in the presence of this ion.     

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.     

Interactions of competent Streptococcus sanguis (Wicky) cells with native or denatured, homologous or heterologous deoxyribonucleic acids.

Competent cell-deoxyribonucleic acid (DNA) interactions were examined using tritium-labeled homologous or heterologous native or denatured DNAs and competent Streptococcus sanguis Wicky cells (strain WE4). The DNAs used were extracted from WE4 cells, Escherichia coli B cells, and E. coli bacteriophages T2, T4, T6, and T7. The reactions examined were: (i) total DNA binding, (ii) deoxyribonuclease-resistant DNA binding, and (iii) the production of acid-soluble products from the DNA. Optimal temperatures for the reactions were as follows: reaction (i), between 30 and 40 degrees C; reaction (ii), 30 degrees C; and reaction (iii), greater than 40 degrees C. The rates for the reactions (expressed as molecules of DNA that reacted per minute per colony-forming unit) did not vary greatly from one DNA source to another. With a constant competent cell concentration and differing DNA concentrations below a saturation level (from a given source), a different but constant fraction of the added DNA was cell bound, deoxyribonuclease resistant, and degraded to acid-soluble products. In experiments where the number of competent cells was varied and the DNA concentration was held constant, again essentially the same result was obtained. The extent of reactions (i), (ii), and (iii) depended upon the numbers as well as the source of DNA molecules applied to competent cells. Calcium ion essential for native DNA-cell reactions was also found essential for denatured DNA-cell reactions. Data obtained from competition experiments lead to the conclusion that competent WE4 cells contain specific sites for native as well as denatured DNAs.     

Nucleolytic degradation of homologous and heterologous deoxyribonucleic acid molecules at the surface of competent pneumococci.

Competent pneumococci can catalyze the rapid and quantitative degradation of extracellular deocyribonucleic acid (DNA) molecules through the activity of surface-located nucleases (endo- and, possibly, exonucleases as well). Both homologous and heterologous DNAs are degraded by a mechanism that seems to involve a cyclic process: (i) attachment of DNA to the cell surface followed by (ii) nucleolytic attack, and (iii) release to the medium. Processes (ii) and (iii) are both inhibited by ethylenediaminetetraacetate. Whereas surface nuclease activity is specific for competent cells, the bulk of this activity is not coupled to irreversible DNA uptake (deoxyribonuclease-resistant binding). Pneumococcal DNA treated with ultraviolet irradiation or nitrous acid (cross-linking?) is selectively impaired in the ability to irreversibly bind to competent cells, whereas reversible binding is normal.     

Role of Streptococcus sanguis (strain Wicky) cell surface-located deoxyribonucleic acid-binding factor in transformation of a homologous strain

In a previous report we demonstrated the presence of a factor binding deoxyribonucleic acid (DNA) in vitro (BF) in cell leakage fluids from transformable Streptococcus sanguis strains Wicky, Challis, and Blackburn. BF originating from strain Wicky was purified to homogeneity, and its properties are described. In this work, it was found that BF occurs at the surface of Wicky cells in two forms, loosely bound (LB-BF) and strongly bound to the cell envelope. It was demonstrated that LB-BF formed fast-sedimenting complexes with exogenous DNA at the surface of Wicky cells. About 10-fold-more DNA became associated as a fast-sedimenting complex in competent than in incompetent cells. Thus, LB-BF is a cell receptor for exogenous DNA. However, the comparison of the effects of some agents on the transformation yield and the formation of LB-BF-DNA complexes, showed that the influence of these agents on both observed phenomena is not parallel and may be even opposite. These results are interpreted to mean that the LB-BF-DNA complexes do not take part in transformation. The problem of participation of BF strongly bound with the cell membrane fraction remains to be elucidated.     

ComEA Is Essential for the Transfer of External DNA into the Periplasm in Naturally Transformable Vibrio cholerae Cells

The DNA uptake of naturally competent bacteria has been attributed to the action of DNA uptake machineries resembling type IV pilus complexes. However, the protein(s) for pulling the DNA across the outer membrane of Gram-negative bacteria remain speculative. Here we show that the competence protein ComEA binds incoming DNA in the periplasm of naturally competent Vibrio cholerae cells thereby promoting DNA uptake, possibly through ratcheting and entropic forces associated with ComEA binding. Using comparative modeling and molecular simulations, we projected the 3D structure and DNA-binding site of ComEA. These in silico predictions, combined with in vivo and in vitro validations of wild-type and site-directed modified variants of ComEA, suggested that ComEA is not solely a DNA receptor protein but plays a direct role in the DNA uptake process. Furthermore, we uncovered that ComEA homologs of other bacteria (both Gram-positive and Gram-negative) efficiently compensated for the absence of ComEA in V. cholerae, suggesting that the contribution of ComEA in the DNA uptake process might be conserved among naturally competent bacteria.     

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.     

Properties of the DNA-binding domain of the simian virus 40 large T antigen.

T antigen (Tag) from simian virus 40 binds specifically to two distinct sites in the viral origin of replication and to single-stranded DNA. Analysis of the protein domain responsible for these activities revealed the following. (i) The C-terminal boundary of the origin-specific and single-strand-specific DNA-binding domain is at or near amino acid 246; furthermore, the maximum of these DNA-binding activities coincides with a narrow C-terminal boundary, spanning 4 amino acids (246 to 249) and declines sharply in proteins with C termini which differ by a few (4 to 10) amino acids; (ii) a polypeptide spanning residues 132 to 246 of Tag is an independent domain responsible for origin-specific DNA binding and presumably for single-stranded DNA binding; and (iii) a comparison of identical N-terminal fragments of Tag purified from mammalian and bacterial cells revealed differential specificity and levels of activity between the two sources of protein. A role for posttranslational modification (phosphorylation) in controlling the DNA-binding activity of Tag is discussed.     

Binding and degradation of proteoglycans by cultured arterial smooth muscle cells. II. Binding sites of proteoglycans on the cell surface

Exogenous proteoglycans stained for electron microscopy with colloidal gold and/or cuprolinic blue bind to the surface of cultured arterial smooth muscle cells at two different sites. (I) About 20% of the proteoglycans adsorbed to the cells from the culture medium interact as monomeric and multimeric proteoglycans with smooth or coated membrane areas. (II) The bulk of exogenous proteoglycans exhibits high affinity binding to cell membrane-associated 10 nm fibrils containing or being closely associated with fibronectin and to collagen. It is suggested that the self association of proteoglycans and their binding to the cell membrane and to cell surface-associated fibronectin and collagen are important for maintaining an appropriate micro-environment for the cultured cells.     

Mg2+-dependent interaction of DNA with eukaryotic cells

Interaction of DNA with eukaryotic cells under conditions similar to those providing DNA adsorption onto liposomes was studied. It was revealed that mouse fibroblasts (line A9) and myeloma cells bind phage and plasmid DNA in 0.3 M sucrose solution containing Mg2+-ions. Additional pretreatment of the cells by trypsin did not affect DNA adsorption efficiency. The major part of the adsorbed DNA recovered by salt treatment of the cells, but 10-15% of DNA was found to be irreversible. Up to 50% of the irreversibly bound DNA molecules retain their linear size after treatment of cells with DNAse I. Efficiencies of DNA adsorption and irreversibly binding depend on the concentration of Mg2+ in the medium. The process of DNA irreversible binding is not inhibited by drugs affecting cell metabolism. It is assumed that DNA adsorbs onto the phospholipid domains of the cell membrane, and part of the adsorbed DNA is taken up into the interior of the cells.