Fate of heterospecific transforming DNA bound to Streptococcus sanguis.

The fate of 3H-labeled str-r fus-s DNA from Streptococcus pneumoniae, bound after a 1-min uptake to 14C-labeled str-s fus-r S. sanguis recipients, was followed by techniques previously developed for analyzing the fate of homospecific DNA. Heterospecific S. pneumoniae DNA was bound and formed complexes with recipient protein in a manner similar to that of homospecific DNA but transformed relatively poorly. The rate at which complexed heterospecific DNA becomes physically associated with recipient DNA, and at which donor markers are integrated into the chromosome, was slower than in the case of homospecific DNA. In addition, about half of the heterospecific donor counts initially bound in trichloracetic acid-insoluble form were gradually solubilized and released from the cell. The association of heterospecific DNA with the recipient chromosome was more unstable than that involving homospecific DNA, since only associations of the former type were largely dissociated by isolation and resedimentation. The donor DNA-containing material so dissociated had the same sedimentation properties as complexed heterospecific DNA before association, indicating that the complex of single-stranded donor DNA and recipient protein formed on uptake moves as a whole from its site of formation to synapse with the chromosome.     

Specific inactivation of heterospecific transforming DNA by a factor derived from Streptococcus sanguis lysates.

Summary A heat-sensitive factor obtained from lysates of competent Streptococcus sanguis cells reacts specifically with native DNA of heterospecific (S. pneumoniae or calf thymus) origin. In vitro it does not alter the double or single strand length of the DNA, nor does it affect uptake of the DNA by competent S. sanguis or S. pneumoniae cells in DNase I-resistant form. Following uptake, however, DNA previously exposed to the factor loses over 90% of its biological activity. Reaction of heterospecific DNA with the factor is cometitive, suggesting a competition for binding to the factor. Heating treated DNA prior to its reaction with recipient cells, apparently by irreversibly dissociating the factor, restores to the DNA its original potential transforming activity. Specific activity of the factor can be increased in cells grown under certain conditions; this increase is blocked by erythromycin.     

Transformation of Streptococcus sanguis Challis with Streptococcus lactis plasmid DNA.

Streptococcus lactis plasmid DNA, which is required for the fermentation of lactose (plasmid pLM2001), and a potential streptococcal cloning vector plasmid (pDB101) which confers resistance to erythromycin were evaluated by transformation into Streptococcus sanguis Challis. Plasmid pLM2001 transformed lactose-negative (Lac-) mutants of S. sanguis with high efficiency and was capable of conferring lactose-metabolizing ability to a mutant deficient in Enzyme IIlac, Factor IIIlac, and phospho-beta-galactosidase of the lactose phosphoenolpyruvate-phosphotransferase system. Plasmid pDB101 was capable of high-efficiency transformation of S. sanguis to antibiotic resistance, and the plasmid could be readily isolated from transformed strains. However, when 20 pLM2001 Lac+ transformants were analyzed by a variety of techniques for the presence of plasmids, none could be detected. In addition, attempts to cure the Lac+ transformants by treatment with acriflavin were unsuccessful. Polyacrylamide gel electrophoresis was used to demonstrate that the transformants had acquired a phospho-beta-galactosidase characteristic of that normally produced by S. lactis and not S. sanguis. It is proposed that the genes required for lactose fermentation may have become stabilized in the transformants due to their integration into the host chromosome. The efficient transformation into and expression of pLM2001 and pDB101 genes in S. sanguis provides a model system which could allow the development of a system for cloning genes from dairy starter cultures into S. sanguis to examine factors affecting their expression and regulation.     

DNA base sequence homologies among strains of Streptococcus sanguis.

DNA was isolated from 19 strains and substrains of Streptococcus sanguis and analysed for guanine plus cytosine (GC) contents and base sequence homologies. Three groups could be discerned: group 1 strains had 40-8 to 42-8 mol % GC; group 2, 42-7 to 44-0 mol % GC; group 3, 43-8 to 46-4 mol % GC. DNA homologies between groups 1 and 3 were 40 to 60% at 67 degrees C and 40% at 72 degrees C. The homologies of group 2 towards groups 1 and 3 were much lower. Strains in groups 1 and 3 hydrolysed arginine and aesculin and fermented inulin; group 2 strains did not. Groups 1 and 3 could be considered subspecies of S. sanguis. Group 2 should not be considered S. sanguis.     

Transformation of Streptococcus sanguis Challis with Streptococcus lactis plasmid DNA

Streptococcus lactis plasmid DNA, which is required for the fermentation of lactose (plasmid pLM2001), and a potential streptococcal cloning vector plasmid (pDB101) which confers resistance to erythromycin were evaluated by transformation into Streptococcus sanguis Challis. Plasmid pLM2001 transformed lactose-negative (Lac-) mutants of S. sanguis with high efficiency and was capable of conferring lactose-metabolizing ability to a mutant deficient in Enzyme IIlac, Factor IIIlac, and phospho-beta-galactosidase of the lactose phosphoenolpyruvate-phosphotransferase system. Plasmid pDB101 was capable of high-efficiency transformation of S. sanguis to antibiotic resistance, and the plasmid could be readily isolated from transformed strains. However, when 20 pLM2001 Lac+ transformants were analyzed by a variety of techniques for the presence of plasmids, none could be detected. In addition, attempts to cure the Lac+ transformants by treatment with acriflavin were unsuccessful. Polyacrylamide gel electrophoresis was used to demonstrate that the transformants had acquired a phospho-beta-galactosidase characteristic of that normally produced by S. lactis and not S. sanguis. It is proposed that the genes required for lactose fermentation may have become stabilized in the transformants due to their integration into the host chromosome. The efficient transformation into and expression of pLM2001 and pDB101 genes in S. sanguis provides a model system which could allow the development of a system for cloning genes from dairy starter cultures into S. sanguis to examine factors affecting their expression and regulation.     

Superhelical DNA in Streptococcus sanguis: role in recombination in vivo.

Summary Competent Streptococcus sanguis treated with non-lethal doses of coumermycin Al immediately before or after uptake of radioactive transforming DNA were reduced in their capacity to yield transformants. This treatment did not alter bacterial ability to bind DNA in DNase I-resistant form, nor did it prevent the single-stranded donor DNA-recipient protein complexes formed upon uptake at the surface of the bacteria from translocating to chromosomal sites. Inhibition of transformation by heterospecific DNA was greater than that by homospecific DNA. The reduction in transformant yield was not accompanied by any loss of donor counts incorporated into the recipient chromosome, but rather by a loss of genetic activity of incorporated donor material indicating a failure of genetic integration and degradation of donor DNA as a consequence of coumermycin treatment. The inhibitory effect of coumermycin on transformation was associated with in vivo loss of chromosomal DNA superhelicity. The chromosomal DNA remained intact, however, indicative of inhibition of a gyrase-like enzyme responsible for the maintenance of negative supercoiling of the S. sanguis chromosome. Upon treatment with the drug, a coumermycin-resistant mutant strain showed neither loss of chromosomal superhelicity nor any inhibitory effect on genetic integration of donor DNA. The evidence supports the idea that chromosomal superhelicity promotes genetic recombination in vivo.     

Transformation in Bacillus subtilis. Fate of newly introduced transforming DNA

Summary Donor deoxyribonucleic acid (molecular weight 5-8×10⁷) introduced into competent cells of Bacillus subtilis is converted to molecules with a weight average molecular weight of 9×10⁶. These molecules, having little transforming activity, constitute in all probability eclipse phase DNA. At least part of the DNA is transiently complexed with a cellular component, changing its buoyant behaviour in CsCl gradients. When shortly after uptake of donor DNA the total DNA extracted from recipient cells is sheared to a molecular weight of 8×10⁶ or less, no eclipse phase is discernable. Donor marker frequencies in sheared, reisolated DNA mixtures decrease by a factor of 4 as a function of time of incubation of the transforming cells. This indicates that only 25% of the irreversibly absorbed DNA is finally integrated into the recipient genome.     

Transformation of Streptococcus sanguis to intrinsic penicillin resistance

A series of step-level penicillin-resistant derivatives of Streptococcus sanguis V288 (Challis) were obtained through successive genetic transformations. The DNA donor used was a laboratory-derived, penicillin-resistant multistep mutant of the recipient strain. Detection of the penicillin-binding proteins (PBPs) of wild-type and transformants revealed five major PBPs. While it was found that S. sanguis can acquire intrinsic resistance in a stepwise manner and the mechanism was similar to those of some other organisms (changes in penicillin-binding protein affinity and/or in extent of penicillin binding), multiple-PBP changes accompanied a single step-level of resistance. All of the PBPs showed varying degrees of decreased affinity for [3H]benzylpenicillin with increasing penicillin resistance. Of these, the consistent, dramatic and progressive decrease of PBP 4 binding was most notable. After an initial decrease at the first step-level of resistance, PBP 5 was restored to wild-type levels, indicating a possible important role in survival. Genetic linkage of the first two step-levels of resistance was demonstrated by examination of transformation frequencies and by hit-kinetics experiments. A convenient method is described for the quantitative comparison of fluorographs containing PBPs with a wide range of affinities for penicillin.     

Return of Streptococcus faecalis DNA cloned in Escherichia coli to its original host via transformation of Streptococcus sanguis followed by conjugative mobilization.

It is possible to select transmembrane potential (delta psi)-altered mutants in Streptococcus pneumoniae on the basis of their resistance to the antifolate methotrexate. Comparison of such a mutant strain ( amiA9 ) with its parent was used to evaluate the role of delta psi in the uptake of certain amino acids. The delta psi-dependent uptake of isoleucine, leucine, valine, and asparagine showed a reduced maximum velocity of uptake, and decrease in the transport constant of the energy-dependent, delta psi-independent uptake of lysine, methionine, and glutamine was observed. No reduction of the intracellular pool of ATP or of lactate excretion could be detected in the mutant strain. Moreover, studies on membrane preparations suggest that the phenotype expressed by the amiA mutation is not a consequence of alteration of its ATPase activity or susceptibility to N,N'-dicyclohexylcarbodiimide. Therefore, it is unlikely that the amiA mutation affects the H+ F1F0 ATPase which is involved in the establishment of the proton motive force in anaerobic bacteria. We propose that another function contributes to delta psi in S. pneumoniae. The amiA gene may be the structural gene of that function.     

Isolation and analysis of sacculi from Streptococcus sanguis.

Sacculi were prepared from Streptococcus sanguis 34 by exhaustive extraction of bacteria with hot 1% sodium dodecyl sulfate-0.5% 2-mercaptoethanol. Lyophilized residue was dissociated by brief sonication to single bodies closely resembling streptococci in phase-contrast microscopic density, staining properties, and morphology. Electron micrographs revealed bodies that contained variable amounts of cellular contents and were bounded by intact cell walls. Chemical analyses of sacculi demonstrated the presence of peptidoglycan, carbohydrate, protein, and phosphate. The hexose content of sacculi varied 10-fold depending upon the composition of the growth medium. When sacculi were subjected to treatment with 5 M LiCl, 8 M urea, 40% phenol (25 degrees C), or dimethyl sulfoxide most of the nitrogen and carbohydrate present was recovered in the insoluble fraction. These data suggest that sacculi contain the cell wall fraction of the extracted bacteria and that most of the carbohydrates and proteins of sacculi are firmly bound to the insoluble fraction, which contains the peptidoglycan matrix.     

Biosynthesis of glycosylated glycerolphosphate polymers in Streptococcus sanguis.

Two types of glycosylated glycerolphosphates were synthesized when a particulate enzyme prepared from Streptococcus sanguis was incubated with [3H]-phosphatidylglycerol and uridine diphosphate-[14C]glucose in the presence of MgCl2. The first type was extractable with saline and contained no fatty acid. The second type was pellet bound and could be extracted with 0.1% sodium dodecyl sulfate. Both types of polymers were purified and partially characterized. The first type of polymer was fractionated into five polymers, peaks 2a, 2b, 2c, 3a, and 3b. All except peak 2a, which contained only [3H]glycerol, contained both [3H]glycerol and [14C]glucose. [3H]NaBH4 reduction of acid hydrolysates of the polymers revealed that all of the polymers contained glucose as the major sugar componenta nd xylose as the minor sugar component. The second type of polymer was fractionated into three polymers, P-1, P-2, and P-3. All contained [3H]-glycerol, [14C]glucose, and fatty acids. P-1 appeared to be pure, whereas P-2 and P-3 contained two polymers each, as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Energetics of the initial phase of adhesion of Streptococcus sanguis to hydroxylapatite.

The initial adhesion of Streptococcus sanguis 10556 to artificial salivary pellicle and to bare hydroxylapatite was studied at several temperatures between 18 and 37 degrees C. When the natural logarithms of rate constants for adsorption and desorption were plotted against reciprocal temperatures in Arrhenius plots, curved lines were obtained, indicating that the thermodynamic quantities of enthalpy and entropy of activation were temperature dependent. For the bare hydroxylapatite system, the heat capacity (delta Cp = dH/dT) was large and negative. delta Cp was also negative for adhesion to saliva-coated hydroxylapatite, although its value was lower. Negative heat capacities, when coupled with favorable entropy, are often indicative of either electrostatic or hydrophobic interactions. When electrolyte (100 mM ammonium sulfate) was added to the cell-hydroxylapatite bead mixture, the rate and extent of adhesion were decreased. Addition of nonpolar p-dioxane (10% [vol/vol], final concentration) to the mixture enhanced binding. This suggests that electrostatic linkages participate in the primary adhesion of streptococci to both substrata. The strongly positive entropy values and the lesser temperature dependence of the saliva-coated hydroxylapatite system suggest that another entropy-driven process is imposed on the electrostatic linkages. This supports a role for hydrophobicity, suggesting that a combination of electrostatic and hydrophobic forces mediate the initial adhesion of S. sanguis to the salivary pellicle.     

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.