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.     

Fate of homospecific transforming DNA bound to Streptococcus sanguis.

The fate of [3H]DNA from Streptococcus sanguis str-r43 fus-s donors in [14C]S. sanguis str-s fus-r1 recipients was studied by examining the lysates prepared from such recipients at various times after 1 min of exposure to DNA. The lysates were analyzed in CsCl and 10 to 30% sucrose gradients; fractions from the gradients were tested for biological activity and sensitivity to nucleases, subjected to various treatments and retested for nuclease sensitivity, and run on 5 to 20% neutral and alkaline sucrose gradients. The results demonstrate that donor DNA bound to S. sanguis cells in a form resistant to exogenous deoxyribonuclease is initially single stranded and complexed to recipient material. Donor DNA can be removed from the complex upon treatment of the complex with Pronase, phenol, or isoamyl alcohol-chloroform. Within the complex, donor DNA is relatively insensitive to S1 endonuclease but can regain its sensitivity by treatment with phenol. With time the complex moves as a whole to associate physically with the recipient chromosome. After a noncovalent stage of synapsis, donor material is covalently bonded to and acquires the nuclease sensitivity of recipient DNA, while donor markers regain transforming activity and become linked to resident markers.     

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.     

Enhanced transformability with heterospecific deoxyribonucleic acid in a Streptococcus sanguis mutant impaired in ribonucleic acid polymerase activity.

We have induced with nitrosoguanidine in Streptococcus sanguis a mutation conferring inability to grow and synthesize ribonucleic acid (RNA) at 42 C, the optimal temperature for growth and RNA synthesis in the parental strain. The mutation (ts) is transferable via transforming deoxyribonucleic acid (DNA) and is replaceable by its wild-type allele with fairly high efficiency in transformation reactions. The ts mutation is unlinked to the sites of mutation conferring resistance of rifampin (rifr) and streptolydigin (stgr), known to affect the beta subunit of DNA-dependent RNA polymerase. Extracts from strains carrying the ts mutation are more sensitive to elevated temperatures than are parental extracts when assayed for DNA-dependent RNA polymerase. The conclusion that the mutation causes a temperature-sensitive defect in some component of this enzyme (other than beta) is supported by the finding that the polymerase activity of a heat-inactivated ts stgr extract cannot be increased by addition of an unheated ts stgs extract, which is itself inactivated by streptolydigin. S. sanguis recipients carrying the ts mutation are highly transformable with heterospecific DNA, especially at the restrictive temperature.     

Transformation of Streptococcus sanguis Challis by plasmid deoxyribonucleic acid from Streptococcus faecalis.

Plasmid deoxyribonucleic acid (DNA) from Streptococcus faecalis, strain DS5, was transferred to the Challis strain of Streptococcus sanguis by transformation. Two antibiotic resistance markers carried by the beta plasmid from strain DS5, erythromycin and lincomycin, were transferred to S. sanguis at a maximum frequency of 1.8 x 10-5/colony-forming unit. Approximately 70% of the covalently closed circular DNA isolated from transformant cultures by dye buoyant density gradients was shown to be hybridizable to beta plasmid DNA. Two major differences were observed between the beta plasmid from S. faecalis and the plasmid isolated from transformed S. sanguis: (i) the beta plasmid from strain DS5 sedimented in velocity gradients at 43S, whereas the covalently closed circular DNA from transformed Challis sedimented at 41S, suggesting a 1.5-Mdal deletion from the beta plasmid occurred; (ii) although the 43S beta plasmid remained in the supercoiled configuration for several weeks after isolation, the 41S plasmid was rapidly converted to a linear double-stranded molecule. Attempts to transform S. sanguis with the alpha plasmid from S. faecalis, strain DS5, were unsuccessful.     

Specific coaggregation and the cell wall of Streptococcus sanguis

Sacculi prepared from Streptococcus sanguis 34 by extensive extraction of cells with hot sodium dodecyl sulfate-2-mercaptoethanol retained the ability to coaggregate with Actinomyces viscosus T14V. When S. sanguis 34 was disrupted by homogenization with glass beads and fractionated by differential centrifugation, only the cell wall fraction agglutinated A. viscosus T14V. When strain 34 was treated with lysozyme, the coaggregating capability of the cells was essentially unaltered. Sacculi prepared from lysozyme-treated strain 34 and additionally purified by electrophoresis were agglutinated by strain T14V.     

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.     

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.     

Inactivation of D-glucosyltransferases from oral Streptococcus mutans and Streptococcus sanguis by photochemical oxidation

Cell-free D-glucosyltransferase of D-glucose-grown Streptococcus mutans AHT was completely inactivated in the presence of 0.002% of Methylene Blue at 25 degrees and pH 7.0 after illumination with a 150-W incandescent lamp. The rate of inactivation was decreased at pH values less than 7.0. Histidine was the only amino acid residue modified to a significant extent, and the rates of oxidation of histidine residues and loss of enzyme activity closely agreed. Production of both water-insoluble and -soluble D-glucan fractions from sucrose by the oxidized D-glucosyltransferase preparations was significantly inhibited. Photooxidation with 0.002% of Rose Bengal at pH 7.0 or higher also induced complete inactivation of the D-glucosyltransferase. These results strongly suggest that the imidazole portion of histidine may function as part of the active sites of both D-glucosyltransferase isozymes of S. mutans AHT, which are responsible for the synthesis of (1 goes to 3)- and (1 goes to 6)-alpha-D-glucosidic linkages. The D-glucosyltransferases from S. mutans 6715 and AHT-mutant M1, and Streptococcus sanguis ATCC 10558 were also almost completely inactivated by Methylene Blue-sensitized photooxidation.     

血链球菌细菌素抑菌活性研究

检测分离纯化的血链球菌细菌素对牙周可疑致病菌的抑制作用。通过羟基磷灰石(HA)柱层析、SephadexG-150凝胶柱层析、中空纤维柱超滤脱盐、浓缩纯化提取血链球菌细菌素,以具核梭杆菌为指示菌,洞平板法检测细菌素的抑菌活性。经HA柱层析得到4个相互分离的组分,经洞平板法检测,第Ⅱ峰的蛋白具有抑菌活性,冻干后得纯化后的细菌素,终产率为0.082%;1mg/mL的细菌素溶液可形成17mm的抑菌环,最小抑菌浓度为62.5μg/mL。血链球菌细菌素对牙周可疑致病菌具有较强的拮抗作用。     

Photolabeling of dextransucrase from Streptococcus sanguis with p-azidophenyl alpha-D-glucopyranoside.

Dextransucrase from Streptococcus sanguis ATCC 10558 was photolabeled using p-azidophenyl alpha-D-glucopyranoside with an apparent rate constant of inactivation of 1.40 min-1. The dissociation constant for this compound, which acts as an acceptor molecule in the enzymatic reaction, is 90 microM. Apparently two acceptor binding sites exist on dextransucrase as shown by (i.) photolabeling the enzyme with p-azidophenyl-alpha-D-[5,6-3H]glucopyranoside and (ii.) fluorescence titration experiments.     

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.     

The inactivation ofBacillus subtilis transforming DNA by mutagenic agents

The inactivation of three genetic markers inBacillus subtilis transforming DNA by UV rays, nitrous acid and hydroxylamine was studied. The inactivation of markers by UV and HNO₂ was marker specific, whereas the hydroxylamine inactivates the markers approximately to the same degree.     

Isolation of a coaggregation-inhibiting cell wall polysaccharide from Streptococcus sanguis H1.

Coaggregation between Streptococcus sanguis H1 and Capnocytophaga ochracea ATCC 33596 cells is mediated by a carbohydrate receptor on the former and an adhesin on the latter. Two methods were used to release the carbohydrate receptor from the gram-positive streptococcus, autoclaving and mutanolysin treatment. The polysaccharide released from the streptococcal cell wall by either treatment was purified by ion-exchange chromatography; this polysaccharide inhibited coaggregation when preincubated with the gram-negative capnocytophaga partner. After hydrolysis of the polysaccharide by hydrofluoric acid (HF), the major oligosaccharide of the polysaccharide was purified by high-performance liquid chromatography. By analysis of the HF hydrolysis of the polysaccharide and the purified oligosaccharide, this major oligosaccharide appeared to be the repeating unit of the polysaccharide, with minor components resulting from internal hydrolysis of the major oligosaccharide. Gas chromatography results showed that the oligomer was a hexasaccharide, consisting of rhamnose, galactose, and glucose, in the ratio of 2:3:1, respectively. By weight, the purified hexasaccharide was a fourfold-more-potent inhibitor of coaggregation than the native polysaccharide. Resistance to hydrolysis by sulfuric acid alone and susceptibility to hydrolysis by HF suggested that oligosaccharide chains of the polysaccharide are linked by phosphodiester bonds. Studies with a coaggregation-defective mutant of S. sanguis H1 revealed that the cell walls of the mutant contained neither the polysaccharide nor the hexasaccharide repeating unit. The purification of both a polysaccharide and its constituent hexasaccharide repeating unit, which both inhibited coaggregation, and the absence of this polysaccharide or hexasaccharide on a coaggregation-defective mutant strongly suggest that the hexasaccharide derived from the polysaccharide functions as the receptor for the adhesin from C. ochracea ATCC 33596.     

Saliva-binding protein (SsaB) from Streptococcus sanguis 12 is a lipoprotein.

Two lipoprotein consensus sequences (Leu-X-X-Cys) are found in the presumptive signal peptide region (positions 12 to 15 and 17 to 20) of saliva-binding protein (SsaB) from Streptococcus sanguis 12. Three analogs of SsaB containing Cys-->Gly mutations were constructed by site-directed mutagenesis of pSA2, the recombinant plasmid expressing SsaB. [3H]palmitate was incorporated into SsaB only when the native Cys-20 residue was present. These data show that SsaB is a lipoprotein and that Cys-20 is the critical site for acylation.     

Uptake of transforming DNA in Gram-positive bacteria: a view from Streptococcus pneumoniae

In a working model for the uptake of transforming DNA based on evidence taken from both Bacillus subtilis and Streptococcus pneumoniae, the ComG proteins are proposed to form a structure that provides access for DNA to the ComEA receptor through the peptidoglycan. DNA would then be delivered to the ComEC-ComFA transport complex. A DNA strand would be degraded by a nuclease, while its complement is pulled into the cell by ComFA through an aqueous pore formed by ComEC. The nuclease is known in S. pneumoniae only as EndA. We have examined the processing (i.e. binding, degradation and internalization) of DNA in S. pneumoniae strains lacking candidate uptake proteins. Mutants were generated by transposon insertion in endA, comEA/C, comFA/C, comGA and dprA. Processing of DNA was abolished only in a comGA mutant. As significant binding was measured in comEA mutants, we suggest the existence of two stages in binding: surface attachment (abolished in a comGA mutant) required for and preceding deep binding (by ComEA). Abolition of degradation in comGA and comEA mutants indicated that, despite its membrane location, EndA cannot access donor DNA by itself. We propose that ComEA is required to deliver DNA to EndA. DNA was still bound and degraded in comEC and comFA mutants. We conclude that recruitment of EndA can occur in the absence of ComEC or ComFA and that EndA is active even when the single strands it produces are not pulled into the cell. Finally, inactivation of dprA had no effect on the internalization of DNA, indicating that DprA is required at a later stage in transformation.