Purification and Properties of Lytic Enzymes from Streptomyces globisporus 1829

Two lytic enzymes capable of lysing Streptococcus mutans have been purified to give a single band on disc-gel electrophoresis, respectively. The M–1 and M–2 enzymes were both proved to be N-acetylmuramidases. However, these enzymes were entirely different on their enzymatic properties. The molecular weights were about 20,000 and 11,000 for M–1 and M–2 enzymes, respectively, The maximal lytic activity of M–1 enzyme was obtained at ionic strength 0.05, while lytic activity of M–2 enzyme did not change within the ionic strength range of 0 to 0.05. The M–1 enzyme constituted the majority of the total lytic activity against the cell walls of Streptococcus mutans BHT of cultured filtrate. The M–2 enzyme showed less specific lytic activity on the cell walls of Streptococcus mutans BHT than M–1 enzyme.     

Characterization of cross-reactive polysaccharide antigen with serotype a and d strains from Streptococcus sobrinus 6715.

The polysaccharide antigen (designated SI) from Streptococcus sobrinus 6715 (serotype g) which cross-reacts with serotype a and d strains was purified by a specific anti-cross-reactive g-a antibody-Sepharose 4B affinity column. By a double immunodiffusion analysis, the SI antigen was found to lack the serotype-specific g site, but contained the cross-reactive sites g-a, g-d and g-(a-d) on a single molecule. Polysaccharide SI was composed of galactose, glucose and rhamnose in a molar ratio of 4.79:1.52:1. The results of the test on the inhibition of the precipitin reaction and methylation analysis suggested that the cross-reactive site g-a of the SI antigen appeared to have two regions, one containing galactose residues and the other, beta-linked glucose residues.     

Covalent linkage of the type- and group-specific antigens to the peptide moiety of the peptidoglycan of serotype III group BStreptococcus

The attachment sites for the two major cell wall polysaccharides, the type-and group-specific antigens of a serotype III group B streptococcus (GBS) were investigated with [14C]lysine to label the peptide portion of the peptidoglycan and [3H]acetate to label both polysaccharide antigens as well as the glycan backbone of the peptidoglycan. Mutanolysin-treated cell walls were subjected to trypsin digestion, followed by exhaustive beta-elimination with 6N ammonium hydroxide at 37°C. The resulting products were purified by column chromatography prior to chemical, immunological, and high-voltage electrophoresis analyses. Data from these studies indicated that both cell wall polymers are covalently attached to the peptidoglycan via the peptide unit. Additionally, during synthesis and assembly both antigens attached only to nascent peptidoglycan.     

A Role for Lewis a antigens on salivary agglutinin in binding to Streptococcus mutans

Streptococcus mutans is a major etiological agent in dental caries. Salivary agglutinin is one of the main salivary components binding to S.mutans. To learn more about the interaction of salivary agglutinin with S.mutans, parotid, submandibular, sublingual and palatal saliva samples were incubated with S. mutans suspension. Both depleted saliva samples and bacterial extracts were analyzed by SDS-PAGE and immunoblotting. Salivary agglutinin was present in all types of glandular saliva and in all cases bound to S.mutans, also to PC337C, a P1^– mutant of S.mutans. Agglutinin was separated by SDS-PAGE under reducing and non-reducing conditions and then transferred to nitrocellulose. Non-reduced agglutinin bound S.mutans, but reduced agglutinin did not. Adhesion of S.mutans to agglutinin-coated microplates was inhibited by amine-containing components, 1 M NaCl or KCl and EDTA. Adhesion decreased with decreasing pH with no adhesion below pH 5.0. These data suggest that calcium-dependent electrostatic interactions play a role in binding. By immunoblotting was demonstrated that blood group antigens and Lewis antigens were present on agglutinin. Synthetic blood group antigens and Lewis antigens covalently coupled to polyacrylamide were tested for binding to S.mutans. Only Le^a(Gal1,3(Fuc1,4)GlcNAc) bound to S.mutans, whereas the blood group antigens Le^b, Le^x, Le^y, H1, H2, A, B and sialylated Le^a did not. Le^a without galactose (Fuc1,4GlcNAc) still bound to S. mutans, but Le^a without fucose (Gal1,3GlcNAc) did not. Binding of agglutinin to S. mutans was not inhibited by Le^a. In conclusion, S. mutans can bind to Le^a carbohydrate epitopes in which the fucose is an essential residue. Le^a carbohydrate epitopes are present on salivary agglutinin but play no major role in binding.     

Composition of the cell wall of Chlorella fusca

Isolated cell walls of mature Chlorella fusca consisted of about 80% carbohydrate, 7% protein, and 13% unidentified material. Mannose and glucose were present in a ratio of about 2.7:1 and accounted for most of the carbohydrate. Minor components were glucuronic acid, rhamnose, and traces of other sugars; galactose was absent. After treatment with 2 M trifluoroacetic acid or with 80% acetic acid/HNO₃ (10/1, v/v), a residue with a mannose/glucose ratio of 0.3:1 was obtained, probably representing a structural polysaccharide. An X-ray diffraction diagram of the walls showed one diffuse reflection at 0.44 nm and no reflections characteristic of cellulose. Walls from young cells contained about 51% carbohydrate, 12% protein, and 37% unidentified material. Mannose and glucose were also the main sugars; their absolute amounts per wall increased 6–7 fold during cell growth. Walls isolated with omission of a dodecylsulphate/mercaptoethanol/urea extraction step had a higher protein content and, with young walls, a significantly higher glucose and fucose content. These data and other published cell wall analyses show a wide variability in cell wall composition of the members of the genus Chlorella.Abbreviations GLC gas liquid chromatography - TFA trifluoroacetic acid     

Immunochemical characteristics of Streptococcus mutans serotype h carbohydrate antigen

Serotype h carbohydrate antigen was prepared from cell walls of Streptococcus mutans strain MFe28 of monkey origin. The h antigen was extracted from the cell walls with 5% trichloracetic acid at 4 C, and purified by DEAE-Sephadex A-25 ion exchange chromatography followed by Sephacryl S-300 gel filtration. The purified antigen was composed of galactose (75%), glucose (16%), and rhamnose (3%). Although the antiserum against whole cells of S. mutans MFe28 gave a strong cross reaction with serotype d S. mutans, serotype h-specific antiserum could be obtained by adequate adsorption. The precipitin reactions and hapten inhibition test using serotype h-specific antiserum showed that galactose, glucose, and their derivative sugars were markedly potent inhibitors. It was concluded that the serotype h antigen is immunologically distinguishable from the known serotypes of S. mutans, although it is closely related to serotype d antigen of S. mutans.     

Enzymic analysis of cell wall structure in apple fruit cortical tissue

Galactanase from Phytophthora infestans and an arabinosidase isoenzyme from Sclerotinia fructigena attacked the cortical cell walls of apple fruits liberating galactose and arabinose residues, respectively. Other arabinosidase isoenzymes from S. fructigena attacked cell walls very slowly. A S. fructigena polygalacturonase isoenzyme liberated half of the uronic acid residues with few associated neutral residues, while a second polygalacturonase isoenzyme released more uronic acid with a substantial proportion of arabinose and galactose and lesser amounts of xylose, rhamnose and glucose; reaction products of this enzyme could be further degraded by the first isoenzyme to give high MW fragments, rich in arabinose with most of the xylose, rhamnose and glucose, and low MW fragments rich in galactose and uronic acid. Endoglucanase from Trichoderma viride released a small proportion of the glucose residues from cell walls together with uronic acid, arabinose, xylose and galactose; more extensive degradation occurred if walls were pre-treated with the second polygalacturonase isoenzyme. Endoglucanase reaction products were separated into a high MW fraction, rich in arabinose, and lower MW fractions rich in galactose and glucose residues. The high MW polygalacturonase and endoglucanase products could be degraded with an arabinosidase isoenzyme to release about 75% of their arabinose. Cell walls from ripe fruit showed similar susceptibility to arabinosidase and galactanase to those from unripe apples. Cell walls from fruit, ripened detached from the tree were more susceptible to degradation by polygalacturonase than walls from unripe fruit or fruit ripened on the tree. Endoglucanase released less carbohydrate from ripe fruit cell walls than from unripe fruit cell walls.     

Immunochemistry of the Cell Walls of Listeria monocytogenes

The antigenic specificity of Listeria monocytogenes types I, II, III, IVa, and IVb was studied by immunochemical techniques. Immunologically active carbohydrates of the various types were extracted from cell walls and were chemically analyzed. Types I and II contained predominantly glucosamine and rhamnose; type III, galactose, rhamnose, and glucosamine; and types IVa and IVb, glucose and galactose. Quantitative precipitin inhibition tests with purified monosaccharides indicated that the major antigenic determinant of types I and II is rhamnose. Precipitin reactions could not be detected with type III carbohydrate and homologous or heterologous antisera. The major determinants of types IVa and IVb were found to be galactose and glucose, respectively. As much as 87% inhibition of the quantitative precipitin test for types I and II was obtained with rhamnose, 72% for type IVa with galactose, and 72% for type IVb with glucose. The immunochemical basis for the antigenic specificity of L. monocytogenes types I, II, IVa, and IVb was further confirmed by using agar gel diffusion. Cross-reactions among the various type-specific carbohydrates and heterologous antisera were also studied. Type II carbohydrate was found to contain galactose and react with type IVa antisera. This reaction could be blocked by galactose. Type I carbohydrate did not contain galactose nor did it react with antiserum prepared from type IVa cells. Therefore, the somatic antigens of type I and type II L. monocytogenes, previously thought to be identical, appeared to differ. The dominant immuno-specific group in the cross-reaction between type IVb carbohydrate and type IVa antisera was found to be galactose. Type IVa absorbed antisera did not produce a significant cross-reaction with type IVb carbohydrate. The results obtained from this investigation indicate a lesser degree of antigenic relationship between type IVa and type IVb L. monocytogenes than was previously believed to exist.     

Influence of fructose and glucose on the production of exopolysaccharides and the activities of enzymes involved in the sugar metabolism and the synthesis of sugar nucleotides in Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772

 The effect of fructose and glucose on the growth, production of exopolysaccharides and the activities of enzymes involved in the synthesis of sugar nucleotides in Lactobacillus delbrueckii subsp. bulgaricus grown in continuous culture was investigated. When grown on fructose, the strain produced 25 mg l^-1 exopolysaccharide composed of glucose and galactose in the ratio 1:2.4. When the carbohydrate source was switched to a mixture of fructose and glucose, the exopolysaccharide production increased to 80 mg l^-1, while the sugar composition of the exopolysaccharide changed to glucose, galactose and rhamnose in a ratio of 1:7.0:0.8. A switch to glucose as the sole carbohydrate source had no further effect. Analysis of the enzymes involved in the synthesis of sugar nucleotides indicates that in cell-free extracts of glucose-grown cells the activity of UDP-glucose pyrophosphorylase was higher than that in cell-free extracts of fructose-grown cells. The activities of dTDP-glucose pyrophosphorylase and the rhamnose synthetic enzyme system were very low in glucose-grown cultures but could not be detected in fructose-grown cultures. Cells grown on a mixture of fructose and glucose showed similar enzyme activities as cells grown on glucose. Analysis of the intracellular level of sugar nucleotides in glucose-grown cultures of L. delbrueckii subsp. bulgaricus showed the presence of UDP-glucose and UDP-galactose in a ratio of 3.3:1, respectively, a similar ratio and slightly lower concentrations were found in fructose-grown cultures. The lower production of exopolysaccharides in cultures grown on fructose may be caused by the more complex pathway involved in the synthesis of sugar nucleotides. The absence of activities of enzymes leading to the synthesis of rhamnose nucleotides in fructose-grown cultures appeared to result in the absence of rhamnose monomer in the exopolysaccharides produced on fructose. Received: 1 February 1996/Received revision: 31 May 1996/Accepted: 2 June 1996     

Synthesis of (±)-Xanthoxins

The enzymatic properties of P2-2 enzyme were determined by using cells of M. radiodurans. The enzyme was: most active at 60°C incubation temperature, stable at 40°C in neutral buffer, and inactivated by heating at 80°C for 15min. Maximal lytic activity occurred at pH 8.5 in Tris-HCl buffer. The range of enzyme stability was between pH 5.5 and 8. Bivalent metal ions, p-chloromercuribenzoate and monoiodo acetate inhibited lytic activity. The molecular weight was estimated to be 16,000 daltons by gel filtration on Sephadex G-75. The enzymatic digestion of peptidoglycans from the cell walls of M. radiodurans and M. lysodeikticus liberated free amino groups, but neither reducing groups nor N-acetylhexosamine, indicating that the enzyme was an endopeptidase. From analysis of the N-terminal amino acids of the digests, it is suggested that the P2-2 enzyme cleaves the peptide bond at the carboxyl group of D-alanine in peptidoglycan.     

Effective Immunity to Dental Caries: Protection of Gnotobiotic Rats by Local,Immunization with a Ribosomal Preparation from Streptococcus mutans

A ribosomal preparation from Streptococcus mutans 6715 was characterized for its ability to induce an immune response in gnotobiotic rats which was protective against S. mutans-induced dental caries. Animals injected in the salivary gland region with the S. mutans ribosomal vaccine developed significantly higher (P < 0.01) salivary IgA and serum IgG antibody activities against whole S. mutans cells and ribosomal preparations than nonimmunized rats. Vaccinated animals had significantly lower (67%; P < 0.01) levels of S. mutans adherent to their molar surfaces than the control rats after infection with the homologous, cariogenic S. mutans. The immunized animals had significantly fewer (P < 0.01) carious lesions on their buccal, sulcal, and proximal molar surfaces than the nonimmunized rats following challenge with the virulent organism. Animals injected with the ribosomal preparation developed salivary IgA and serum IgG antibodies with specificities to various cell surface-associated antigens such as lipoteichoic acid and glucosyltransferase, suggesting that the observed protection may be due to antibodies against cell surface contaminants of the ribosomal vaccine. These results are the first demonstration that a ribosomal preparation from S. mutans protected rats from caries formation after challenge with the homologous, virulent S. mutans.     

Epidemiological Survey of Streptococcus mutans among Japanese Children

An epidemiological investigation was carried out to identify and determine the serotypes of Streptococcus mutans from carious lesions of young Japanese children. For this purpose, a direct fluorescent antibody technique was mainly used. Fluorescein isothiocyanate-conjugated antibodies were prepared for the five known serotypes of S. mutans. Cross reactions and nonspecific reactions were eliminated by adsorption, counterstaining, or DEAE-cellulose column chromatography. Agar-gel immunodiffusion was used to distinguish between serotypes a and d. The epidemiological survey suggested that serotype c strains were most prevalent in dental plaques of Japanese children. The d and e serotypes were rare and serotypes a and b were not detected. It was also noted that more than one serotype of S. mutans could be found in the same locus of a carious lesion and that there might be no relationship between the degree of caries and the causative serotype(s) of S. mutans.     

Isolation of a polysaccharide from the inner cell wall, intine, of pollen of Cryptomeria japonica

Isolation and solubilization of the intine of pollen grainsof Cryptomeria japonica were attempted. The intine was separatedeffectively from other cell fragments by passing it througha column of glass beads and dissolving it in an EDTA solution.The extract was fractionated by column chromatography on DEAE-celluloseand gelfiltration through Sephadex G-200, and a polysaccharidecomponent was obtained. This was found to consist of galactose,rhamnose, arabinose, xylose and uronic acid. (Received October 13, 1976; )     

Composition of ethanol-insoluble polysaccharides in water extracts of ripening tomatoes

The carbohydrate composition of the 80% ethanol-insoluble polysaccharides (EIP) from water extracts of ‘Rutgers,’ rin (ripening inhibitor) and nor (non-ripening) tomatoes has been determined. The amount of EIP extracted from ‘Rutgers’ fruit increased from 0.34 to 0.61 mg/g fr. wt during ripening little change occurred in rin or nor fruit. The carbohydrate composition (μg/g fr. wt) of EIP from mature green fruit was: galacturonic acid (48); rhamnose (3); arabinose (20); xylose (48); mannose (31); glucose (139); galactose (51). The most obvious changes that accompanied ripening were a 7.4-fold and 4-fold increase in galacturonic acid and rhamnose content, respectively. These changes were attenuated in the ripening mutants. EIP was fractionated into three major peaks by using DEAE-cellulose ion exchange chromatography. The first peak, which was not retained by the column, contained predominantly glucose and mannose, with lower amounts of galacturonic acid and galactose. The two retained peaks which eluted at 0.1 and 0.2 M sodium chloride contained primarily galacturonic acid, xylose, galactose and arabinose. The galacturonic acid content of these two fractions increased substantially during ripening, whereas the other components decreased. No changes were evident in the ripening mutants. No increase in water-soluble polysaccharides high in galactose content was observed during ripening.     

The capsule and slime polysaccharides of the wild type and a phage resistant mutant ofRhodopseudomonas capsulata St. Louis

A rhamnose, galactose and pyruvic acid containing polysaccharide (capsule) together with the peptidoglycan was isolated fromRhodopseudomonas capsulata St. Louis as the insoluble sediment after sodium dodecyl sulfate extraction of cell envelope fractions. Treatment with pronase E separated the soluble polysaccharide from the insoluble peptidoglycan. After lysozyme-digestion, both the capsule polysaccharide and peptidoglycan were soluble.The capsule was also accumulated in the combined interphase/phenol-phase of hot phenol-water extracts of whole cells. Again, the capsule and peptidoglycan were sedimented together as long as no pronase E-treatment was performed. With the phage-resistant mutant (R. capsulata St. Louis RC1^-), no capsule polysaccharide was obtained in the combined interphase/phenol phase.An acidic polysaccharide (slime) different from the capsule in composition and serology was obtained by Cetavlon fractionation of hot phenol/water extracts of cells of both the wild-type and the mutant cells. It was shown to consist mainly of rhamnose, glucosamine and galacturonic acid.The use of O/K-antisera and of capsule polysaccharideantisera allowed a separate visualization of the capsule and slime layers.This paper is dedicated to Professor Hans G. Schoegel on the occasion of his 60th birthday     

Purification and Properties of a Lytic Enzyme from Streptomyces griseus H-402

A lytic enzyme which was capable of lysing cells of Streptococcus mutans was purified from the culture filtrate of Streplomyces griseus H–402 by Amberlite CG–50 treatment, CM-cellulose and hydroxylapatite column chromatographies, and Sephadex G–150 gelfiltration. The lytic enzyme was obtained in a crystalline form which was homogeneous in polyacrylamide gel electrophoresis. The molecular weight was estimated to be 2×10⁴ by the thin-layer gel-filtration method on Sephadex G–75, and 2.3 × 10⁴ by the method of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme was found to be a N-acetylmuramidase whose activity was lost by N-bromosuccinimide as an inhibitor.     

Streptococcal dTDP‐L‐rhamnose biosynthesis enzymes: functional characterization and lead compound identification

Biosynthesis of the nucleotide sugar precursor dTDP‐L‐rhamnose is critical for the viability and virulence of many human pathogenic bacteria, including Streptococcus pyogenes (Group A Streptococcus; GAS), Streptococcus mutans and Mycobacterium tuberculosis. Streptococcal pathogens require dTDP‐L‐rhamnose for the production of structurally similar rhamnose polysaccharides in their cell wall. Via heterologous expression in S. mutans, we confirmed that GAS RmlB and RmlC are critical for dTDP‐L‐rhamnose biosynthesis through their action as dTDP‐glucose‐4,6‐dehydratase and dTDP‐4‐keto‐6‐deoxyglucose‐3,5‐epimerase enzymes respectively. Complementation with GAS RmlB and RmlC containing specific point mutations corroborated the conservation of previous identified catalytic residues. Bio‐layer interferometry was used to identify and confirm inhibitory lead compounds that bind to GAS dTDP‐rhamnose biosynthesis enzymes RmlB, RmlC and GacA. One of the identified compounds, Ri03, inhibited growth of GAS, other rhamnose‐dependent streptococcal pathogens as well as M. tuberculosis with an IC₅₀ of 120–410 µM. Importantly, we confirmed that Ri03 inhibited dTDP‐L‐rhamnose formation in a concentration‐dependent manner through a biochemical assay with recombinant rhamnose biosynthesis enzymes. We therefore conclude that inhibitors of dTDP‐L‐rhamnose biosynthesis, such as Ri03, affect streptococcal and mycobacterial viability and can serve as lead compounds for the development of a new class of antibiotics that targets dTDP‐rhamnose biosynthesis in pathogenic bacteria.     

Analysis of the exopolysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 grown in continuous culture on glucose and fructose

The exopolysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 grown in defined medium were investigated. At equal cell densities, the strain produced 95 mg l⁻¹ exopolysaccharides with glucose and 30 mg l⁻¹ with fructose as the carbohydrate source. High-performance size-exclusion chromatography of the exopolysaccharides produced on glucose showed the presence of two fractions with relative molecular masses (M _r) of 1.7 × 10⁶ and 4 × 10⁴ in almost equal amounts. The exopolysaccharides produced on fructose contained mainly a fraction of low M _r of 4 × 10⁴. The high-M _r fraction of the purified exopolysaccharides produced on glucose appeared to have a sugar composition of galactose, glucose and rhamnose in the molar ratio of 5:1:1, whereas the low-M _r weight fraction contained galactose, glucose and rhamnose in the molar ratio of approximately 11:1:0.4. The purified exopolysaccharide fractions produced on fructose showed comparable ratios. The high-molecular-mass fractions contained terminally linked galactose, 1,2,3-linked galactose, 1,3,4-linked galactose, 1,3-linked glucose and terminally linked rhamnose. The low-molecular-mass fractions contained mainly 1,3-linked galactose and 1,6-linked galactose and lower amounts of other sugar linkages. The production of the high-M _r fractions appeared to be dependent on the carbohydrate source, whereas the low-M _r fractions were produced more continuously. Received: 30 April 1997 / Received revision: 11 June 1997 / Accepted: 14 June 1997     

Cell wall carbohydrates in Phycomyces blakesleeanus burgeff

The carbohydrate composition of the cell walls from spores, mycelium and sporangiophores of Phycomyces blakesleeanus was analyzed. Spore wall polysaccharides contained over 50% glucose, about 20% uronic acids, 10% mannose and 10% amino-sugars. During the growth of the hyphae amino-sugars became the main carbohydrate (45%); uronic acids contributed some 25%, glucose and fucose 10% and galactose nearly 6%. Sporangiophores contained almost 90% aminosugars and some 6% uronic acids. Traces of rhamnose were found in all wall preparations. A similar picture emerged from studies on the incorporation of [U-¹⁴C]-glucose into wall materials.Furthermore we looked for a GDP-fucose synthesizing system and found an increasing activity during early germination. This rise in activity was inhibited by cycloheximide but not by 5-fluorouracil.     

Binding of Polysaccharide to Peptidoglycan in Cell Wall of Streptomyces roseochromogenes

The polysaccharide-peptidoglycan complex, which was prepared with lysozyme from Streptomyces roseochromogenes IAM53 cell walls, was hydrolyzed with lytic enzyme of Flavo-bacterium to separate polysaccharide. The enzymatically prepared polysaccharide (100 mg) contained 500 μmoles of hexoses, 40 μmoles of hexosamines and 31 μmoles of phosphate. Hexoses consisted of mannose and galactose in a molar ratio of 5 to 1. Hexosamines consisted of equimolar glucosamine and muramic acid, a half of which was identified as muramic acid 6-phosphate. The reducing end of the polysaccharide was muramic acid. The polysaccharide extracted with trichloroacetic acid contained no muramic acid-phosphate. So the polysaccharide moiety of S. roseochromogenes cell walls must be linked covalently to 6-position of muramic acid in peptidoglycan through phosphate,