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1.
Extracellular proteins produced by the four human commensal species of mutans streptococci were analysed. The organisms used were Streptococcus mutans, serotypes c, e and f, Streptococcus cricetus, serotype a, Streptococcus rattus, serotype b, and Streptococcus sobrinus, serotypes d and g. They were grown in continuous culture at different generation times and pH values in media containing either glucose or fructose to determine the extent of variation in extracellular protein production that could occur for an individual strain. The results for different organisms grown under the same conditions were then compared. The total amount of protein of molecular mass greater than or equal to 60 kDa varied considerably with the growth conditions and with the strain. Generally more protein was present at a higher pH, conditions under which the organisms also form more lipoteichoic acid. With respect to individual protein components SDS-PAGE proved better than isoelectric focusing for detecting phenotypic responses by a particular strain to environmental changes and differences between the different strains. Differences in the molecular masses of protein components were particularly pronounced in the regions designated P1 (185-200 kDa), P2 (130-155 kDa) and P3 (60-95 kDa). Every strain produced at least one component in the P1 region that cross-reacted with antiserum to the purified protein from S. mutans serotype c, a protein which is indistinguishable from antigens B and I/II. Two components in the P2 region were dominant in the case of S. cricetus and S. sobrinus strains and showed glucosyltransferase (GTF) activity. GTF activity was also detected in the P3 region, particularly with S. mutans strains.  相似文献   

2.
Streptococcus mutans Ingbritt (serotype c) was found to secrete basic glucosyltransferase (sucrose: 1,6-alpha-D-glucan 3-alpha and 6-alpha-glucosyltransferase). The enzyme preparation obtained by ethanol fractionation, DEAE Bio-Gel A chromatography, chromatofocusing and preparative isoelectric focusing was composed of three isozymes with slightly different isoelectric points (pI 8.1-8.4). The molecular weight was estimated to be 151000 by SDS-polyacrylamide gel electrophoresis. The specific activity of the enzyme was 9.8 IU per mg of protein and the optimum pH was 6.5. The enzyme was activated 2.4-fold by commercial dextran T10, and had Km values of 7.1 micro M for the dextran and 4.3 mM for sucrose. Glucan was de novo synthesized from sucrose by the enzyme and found to be 1,6-alpha-D-glucan with 17.7% of 1,3,6-branching structure by a gas-liquid chromatography-mass spectroscopy.  相似文献   

3.
In addition to the 1,3-alpha-D-glucan synthetase (pI 4.9) and the highly-branched 1,6-alpha-D-glucan synthetase (pI 3.9-4.1), Streptococcus mutans 6715 (serotype g) was found to secrete the third glucosyltransferase in multiple forms (pI 5.5-7.0), which exhibited 87% 1,6-alpha-bond-, 6% 1,3-alpha-bond- and 7% 1,3,6-branch-forming activities. The production of this enzyme was extremely enhanced when the organism was grown in Tween 80-supplemented medium. The 3 glucosyltransferases from the same organism were enzymatically and immunologically distinct from each other, and they were commonly found among the serotype g strains.  相似文献   

4.
Glucan-binding proteins of Streptococcus mutans serotype c.   总被引:13,自引:0,他引:13  
Three glucan-binding proteins have been isolated from the extracellular fluid cultures of Streptococcus mutans serotype c. These proteins were adsorbed to glucans containing 1,3-alpha or 1,6-alpha bonds and linked to various chromatographic supports: they were eluted from columns by a dextran solution. Glucosyltransferase activity was associated with two of the glucan-binding proteins.  相似文献   

5.
An extracellular glucosyltransferase (sucrose: 1,6-, 1,3-alpha-D-glucan 3-alpha- and 6-alpha-D-glucosyltransferase, EC 2.4.1.-) of Streptococcus mutans HS6 (serotype a) was purified from culture supernatant by DEAE-Sepharose chromatography and preparative isoelectric focusing. The molecular weight measured by SDS-PAGE was 159 000 and the isoelectric point was pH 4.9. The specific activity was 89.7 i.u. (mg protein)-1 and the optimum pH was 6.0. The Km value for sucrose was 4.9 mM and the enzyme activity was not stimulated by exogenous dextran T10. Glucan was synthesized de novo from sucrose by the purified enzyme and consisted of 49.1 mol% 1,6-alpha-linked glucose and 33.9 mol% 1,3-alpha-linked glucose, with 13.6 mol% terminal glucose and 3.3 mol% 1,3,6-alpha-branched glucose.  相似文献   

6.
By using acid-urea polyacrylamide gel electrophoresis, two cationic proteins were found in the isolated outer membranes of Salmonella typhimurium SH5014. Also, all the other enterobacterial strains studied (five additional strains of S. typhimurium, one strain of Salmonella minnesota, and three strains of Escherichia coli K12) had those proteins. The most abundant (OMB2) was purified in preparative acid-urea polyacrylamide gel electrophoresis and reversed-phase high pressure liquid chromatography (HPLC). It had a molecular mass of 16 kDa, a pI above 10.0, and was rich in arginine and lysine. 72% of the total amino acid sequence was determined by sequencing several HPLC-purified proteolytic fragments and 55 amino acids from the NH2 terminus. Furthermore, we isolated by molecular cloning the corresponding gene, named it ompH, and determined its nucleotide sequence. By combining protein and nucleotide sequence data, we determined the primary structure of the entire OmpH protein. It consists of 141 amino acids, possesses regions very rich in basic amino acids, and has a molecular mass of 15,862 kDa.  相似文献   

7.
Characterization of common cell surface-bound antigens inCandida albicans strains, particularly those expressed in the walls of mycelial cells might be useful in the diagnosis of systemic candidiasis. Hence, antigenic similarities among wall proteins and mannoproteins fromC. albicans clinical serotype A and B isolates, were studied using polyclonal (mPAbs) and monoclonal (MAb 4C12) antibodies raised against wall antigens from the mycelial form of a commonC. albicans serotype A laboratory strain (ATCC 26555). Zymolyase digestion of walls isolated from cells of the different strains studied grown at 37°C (germination conditions), released, in all cases, numerous protein and mannoprotein components larger than 100 kDa, along with a 33–34 kDa species. The pattern of major antigens exhibiting reactivity towards the mPAbs preparation was basically similar for all the serotype A and B isolates, though minor strain-specific bands were also observed. The immunodeterminant recognized by MAb 4C12 was found to be absent or present in very low amounts inC. albicans isolates other than the ATCC 26555 strain, yet high molecular weight species similar in size (e.g., 260 kDa) to the wall antigen against which MAb 4C12 was raised, were observed, particularly in wall digests from serotype A strains. Cell surface hydrophobicity, an apparently important virulence factor inC. albicans, of the cell population of each serotype B strain was lower than that of the corresponding serotype A counterparts, which is possibly due to the fact that the former strains exhibited a reduced ability to form mycelial filaments under the experimental conditions used.Abbreviations CSH cell surface hydrophobicity - IIF indirect immunofluorescence  相似文献   

8.
The single glucosyltransferase (GTF) of Streptococcus gordonii Challis CH1 makes alpha 1,3- and alpha 1,6-linked glucans from sucrose. The GTF carboxyl-terminal region has six direct repeats thought to be involved in glucan binding. Strains with defined mutations in this region have been described recently (M. M. Vickerman, M. C. Sulavik, P. E. Minick, and D. B. Clewell, Infect. Immun. 64:5117-5128, 1996). Strain CH107 GTF has three internal direct repeats deleted; the 59 carboxyl-terminal amino acids are identical to those of the parental strain. This deletion resulted in decreased enzyme activity but did not affect the amount of cell-associated GTF protein. The GTFs of strains CH2RPE and CH4RPE have six and eight direct repeats, respectively, but are both missing the 14 carboxyl-terminal amino acids. Strain CH2RPE had significantly decreased levels of cell-associated GTF; this decrease was not obviated by the increased number of direct repeats in strain CH4RPE. Thus, the carboxyl-terminal amino acids appeared to influence the amount of cell-associated GTF more than the direct repeats. The qualitative and quantitative differences in the GTFs did not affect the abilities of these strains to accumulate on hydroxyapatite beads in the absence of sucrose. However, when sucrose was added as a substrate for GTF, the mutant strains were unable to accumulate on these surfaces to the same extent as the parent. These differences in sucrose-associated accumulation may be due to changes in the nature of the glucans produced by the different enzymes and/or cohesive interactions between these glucans and the GTF on the surfaces of the growing streptococci.  相似文献   

9.
An enzyme capable of dehalogenating vicinal haloalcohols to their corresponding epoxides was purified from the 3-chloro-1,2-propanediol-utilizing bacterium Arthrobacter sp. strain AD2. The inducible haloalcohol dehalogenase converted 1,3-dichloro-2-propanol, 3-chloro-1,2-propanediol, 1-chloro-2-propanol, and their brominated analogs, 2-bromoethanol, as well as chloroacetone and 1,3-dichloroacetone. The enzyme possessed no activity for epichlorohydrin (3-chloro-1,2-epoxypropane) or 2,3-dichloro-1-propanol. The dehalogenase had a broad pH optimum at about 8.5 and a temperature optimum of 50 degrees C. The enzyme followed Michaelis-Menten kinetics, and the Km values for 1,3-dichloro-2-propanol and 3-chloro-1,2-propanediol were 8.5 and 48 mM, respectively. Chloroacetic acid was a competitive inhibitor, with a Ki of 0.50 mM. A subunit molecular mass of 29 kDa was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With gel filtration, a molecular mass of 69 kDa was found, indicating that the native protein is a dimer. The amino acid composition and N-terminal amino acid sequence are given.  相似文献   

10.
Wall-associated protein antigens of Streptococcus mutans.   总被引:64,自引:0,他引:64  
When heat-killed whole organisms of Streptococcus mutans strain Ingbritt (serotype c) were injected into rabbits, antibodies to at least 12 antigens were detectable by crossed immunoelectrophoresis. In contrast, when rabbits were immunized with organisms which had been subjected to extraction with the detergent sodium dodecyl sulphate (SDS), antibodies to only two protein antigens were found. These two proteins (A and B), while existing in a form apparently closely associated with peptidoglycan, could also be recovered from homogenates of whole organisms after sonication and from culture filtrates. Antigenic material was excreted throughout growth. SDS-polyacrylamide gradient gel electrophoresis showed A to have a molecular weight of 29 000, while B had a molecular weight of 190 000. Antigen B was purified to apparent homogeneity as judged by SDS-polyacrylamide gel electrophoresis and isoelectric focusing. All of six strains of serotype c examined produced antigen B. Strains of serotypes e and f also produce antigenically identical proteins and strains of serotypes d and g produce proteins which cross-reacted with antigen B. Antigen B was specifically precipitated by rabbit antiserum to human heart tissue.  相似文献   

11.
Streptococcus mutans Ingbritt (serotype c) was shown to have a significant amount of cell-associated glucosyltransferase activity which synthesizes water-insoluble glucan from sucrose. The enzyme was extracted from the washed cells with SDS, renatured with Triton X-100, adsorbed to 1,3-alpha-D-glucan gel, and then eluted with SDS. The enzyme preparation was electrophoretically homogeneous, and the specific activity was 7.3 i.u. (mg protein)-1. The enzyme had an Mr of 158,000 as determined by SDS-PAGE, and was a strongly hydrophilic protein, as judged by its amino acid composition. The enzyme gradually aggregated in the absence of SDS. The enzyme had an optimum pH of 6.5 and a Km value of 16.3 mm for sucrose. Activity was stimulated 1.7-fold by dextran T10, but was not stimulated by high concentrations of ammonium sulphate. Below a sodium phosphate buffer concentration of 50 mm, activity was reduced by 75%. This enzyme synthesized an insoluble D-glucan consisting of 76 mol% 1,3-alpha-linked glucose and 24 mol% 1,6-alpha-linked glucose.  相似文献   

12.
Listeria monocytogenes serotypes 4a, 4b and 7, and L. ivanovii, all grown at 20 degrees C, were negatively stained and examined by electron microscopy. Crude extracts of the cell surface of L. monocytogenes serotypes 1/2b, 3b, 3c, 4a, 4b, 4d and 7 and of L. ivanovii (all grown at 20 degrees C) were examined by SDS-PAGE and Western blotting using (i) affinity-purified polyclonal monospecific antibody, and (ii) monoclonal antibody, each raised against 29 kDa flagellin of serotype 4b. No flagella were seen on serotype 7 by electron microscopy and no flagellin was detected in crude cell surface extracts of serotype 7 either in silver-stained gels or in Western blots. The monospecific polyclonal antibody detected flagellins of approximate molecular mass 29 kDa in each of the seven flagellate strains including L. ivanovii. The monoclonal antibody detected 29 kDa flagellin in serotypes 1/2b, 3b, 4a, 4b and 4d, but not the flagellins of serotype 3c or L. ivanovii, which had a slightly lower molecular mass. Following prolonged electrophoresis of crude flagellar extracts the 29 kDa complex was resolved into three closely migrating bands. In a heterologous system using serotype 1/2b crude flagellar extract, all three bands were detected using the polyclonal antibody whereas only two bands were detected by the monoclonal antibody. It is concluded that polyclonal anti-flagellin antibodies are not useful tools with which to distinguish serotypes of L. monocytogenes sensu lato in immunoblotting, but that differences can be determined using a monoclonal antibody directed against particular components of the flagellar complex. These differences did not fully correspond to those anticipated from results of agglutination tests.  相似文献   

13.
Extracellular enzymes with glucanase activities are an important component of actinomycete-fungus antagonism. Streptomyces sp. EF-14 has been previously identified as one of the most potent antagonists of Phytophthora spp. A beta-1,6-glucanase (EC 3.2.1.75; glucan endo-1,6-beta-glucosidase) was purified by four chromatographic steps from the culture supernatant of strain EF-14 grown on a medium with lyophilized cells of Candida utilis as main nutrient source. The glucanase level in this medium followed a characteristic pattern in which the rise of beta-1,6-glucanase activity always preceded that of beta-1,3-glucanase. The molecular mass of the enzyme was estimated to be 65 kDa and the pI approximately 5.5. It hydrolyzed pustulan by an endo-mechanism generating gentiobiose and glucose as final products. Laminarin was not hydrolyzed indicating that the enzyme does not recognize beta-1,6-links flanked by beta-1,3-links. No significant clearing of yeast cell walls in liquid suspensions or in agar plates was observed indicating that this beta-1,6-glucanase is a non-lytic enzyme. This is the first beta-1,6-glucanase characterized from an actinomycete.  相似文献   

14.
Extracellular 1,3-alpha-D-glucan synthase (sucrose: 1,3-alpha-D-glucan 3-alpha-D-glucosyltransferase, EC 2.4.1.-) of Streptococcus mutans HS6 (serotype a) was purified from culture supernatant by ultrafiltration, DEAE-Sepharose chromatography and preparative isoelectric focusing. The enzyme had a molecular weight of 158 000 by SDS-PAGE and an isoelectric point of pH 5.2. The specific activity of the enzyme was 48.3 i.u. (mg protein)-1. The Km for sucrose was 1.2 mM and the activity was optimal at pH 6.0. The enzyme activity was stimulated about 20-fold in the presence of dextran T10. Glucan was synthesized de novo from sucrose by the enzyme and characterized as a linear 1,3-alpha-D-glucan by GC-MS.  相似文献   

15.
A (1,3)-beta-D-glucanase [(1,3)-beta-D-glucan-3-glucanohydrolase] from Ruminococcus flavefaciens grown on milled filter paper was purified 3,700-fold (19% yield) and appeared as a single major protein and activity band upon polyacrylamide gel electrophoresis. The enzyme did not hydrolyze 1,6-beta linkages (pustulan) or 1,3-beta linkages in glucans with frequent 1,6-beta-linkage branch points (scleroglucan). Curdlan and carboxymethylpachyman were hydrolyzed at 50% the rate of laminarin. The enzyme had a Km of 0.37 mg of laminarin per ml, a pH optimum of 6.8, and a temperature optimum of 55 degrees C and was stable to heating at 40 degrees C for 60 min. The molecular mass of the enzyme was estimated to be 26 kDa by gel filtration and 25 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was completely inhibited by 1 mM Hg2+, Cu2+, and KMnO4, 75% by 1 mM Ag2+, and Ni2+, and 50% by 1 mM Mn2+ and Fe3+. In a 2-h incubation with laminaridextrins (seven to nine glucose units) or curdlan and excess enzyme, the major products were glucose (30 to 37%), laminaribiose (17 to 23%), laminaritriose (18 to 28%), laminaritetraose (13 to 21%), and small amounts of large laminarioligosaccharides. With laminarihexaose and laminaripentaose, the products were equal quantities of laminaribiose and glucose (30%) and laminaritetraose and laminaritriose (18 to 21%). Laminaribiose or laminaritriose were not hydrolyzed, indicating a requirement for at least four contiguous 1,3-beta-linked glucose units for enzyme activity. The enzyme appeared to have the properties of both an exo- and an endoglucanase.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

16.
A (1,3)-beta-D-glucanase [(1,3)-beta-D-glucan-3-glucanohydrolase] from Ruminococcus flavefaciens grown on milled filter paper was purified 3,700-fold (19% yield) and appeared as a single major protein and activity band upon polyacrylamide gel electrophoresis. The enzyme did not hydrolyze 1,6-beta linkages (pustulan) or 1,3-beta linkages in glucans with frequent 1,6-beta-linkage branch points (scleroglucan). Curdlan and carboxymethylpachyman were hydrolyzed at 50% the rate of laminarin. The enzyme had a Km of 0.37 mg of laminarin per ml, a pH optimum of 6.8, and a temperature optimum of 55 degrees C and was stable to heating at 40 degrees C for 60 min. The molecular mass of the enzyme was estimated to be 26 kDa by gel filtration and 25 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was completely inhibited by 1 mM Hg2+, Cu2+, and KMnO4, 75% by 1 mM Ag2+, and Ni2+, and 50% by 1 mM Mn2+ and Fe3+. In a 2-h incubation with laminaridextrins (seven to nine glucose units) or curdlan and excess enzyme, the major products were glucose (30 to 37%), laminaribiose (17 to 23%), laminaritriose (18 to 28%), laminaritetraose (13 to 21%), and small amounts of large laminarioligosaccharides. With laminarihexaose and laminaripentaose, the products were equal quantities of laminaribiose and glucose (30%) and laminaritetraose and laminaritriose (18 to 21%). Laminaribiose or laminaritriose were not hydrolyzed, indicating a requirement for at least four contiguous 1,3-beta-linked glucose units for enzyme activity. The enzyme appeared to have the properties of both an exo- and an endoglucanase.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

17.
A filamentous phage, 'lvpf5,' of Vibrio parahaemolyticus O3:K6 strain LVP5 was isolated and characterized. The host range was not restricted to serotype O3:K6, but 7 of 99 V. parahaemolyticus strains with a variety of serotypes were susceptible to the phage. The phage was inactivated by heating at 80 C for 10 min and by treating with chloroform. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the phage exhibited a 3.8 kDa protein. The amino-terminal amino acid sequence of the coat protein was determined as AEGGAADPFEAIDLLGVATL. The phage genome consisted of a single-stranded DNA molecule. The activity of the phages was inhibited by anti-Na2 pili antibody.  相似文献   

18.
The gene encoding a glucosyltransferase which synthesized water-insoluble glucan, gtfI, previously cloned from Streptococcus sobrinus strain MFe28 (mutans serotype h) into a bacteriophage lambda vector, was subcloned into the plasmid pBR322. The recombinant plasmid was stable in Escherichia coli and gtfI was efficiently expressed. The GTF-I expressed in E. coli was compared to the corresponding enzymes in S. sobrinus strains MFe28 (serotype h), B13 (serotype d) and 6715 (serotype g) and shown to resemble them closely in molecular mass and isoelectric point. The insoluble glucan produced by GTF-I from recombinant E. coli consisted of 1,3-alpha-D-glycosyl residues (approximately 90%). An internal fragment of the gtfI gene was used as a probe in hybridization experiments to demonstrate the presence of homologous sequences in chromosomal DNA of other streptococci of the mutans group.  相似文献   

19.
A beta-1,6-glucanase was purified to apparent homogeneity from a commercial yeast digestive enzyme prepared from Streptomyces rochei by a series of column chromatographies. The molecular mass of the purified enzyme was 60 kDa by SDS-PAGE. The purified enzyme had an optimum pH range from 4.0 to 6.0 and was stable in the same pH range. The enzyme was stable under 50 degrees C but lost almost all activity at 60 degrees C. The enzyme was specific to beta-1,6-glucan and had little activity towards beta-1,3-glucan and beta-1,4-glucan. When the beta-1,6-glucan was hydrolyzed with the purified enzyme for 5 h, the reaction products contained 20% glucose, 36% gentiobiose, and 44% other oligosaccharides, suggesting that the enzyme is an endo-type glucanase. When the purified enzyme was used for the digestion of the cell wall of Saccharomyces cerevisiae, cell-wall proteins covalently bound to the cell-wall glucan were recovered as soluble forms, suggesting that this enzyme is useful for analysis of yeast-cell wall proteins.  相似文献   

20.
Leuconostoc mesenteroides UL5 was found to produce a bacteriocin, referred as mesenterocin 5, active against Listeria monocytogenes strains but with no effect on several useful lactic acid bacteria. The antimicrobial substance is a protein, since its activity was completely destroyed following protease (pronase) treatment. However, it was relatively heat stable (100 degrees C for 30 min) and partially denaturated by chloroform. The inhibitory effect of the bacteriocin on sensitive bacterial strains was determined by a critical-dilution micromethod. Mutants of L. mesenteroides UL5 which had lost the capacity to produce the bacteriocin were obtained. The mutant strain was stable and phenotypically identical to parental cells and remained resistant to the bacteriocin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to detect bacteriocin activity corresponding to an apparent molecular mass of about 4.5 kDa.  相似文献   

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