Hyphal Wall Compositions of Marine and Terrestrial Fungi of the Genus Leptosphaeria

Hyphal wall compositions of six Leptosphaeria species were compared to assess whether gross changes have occurred in the hyphal wall chemistry of closely related fungi which have become ecologically restricted to marine or terrestrial habitats. Unfractionated, lipid-extracted hyphal walls of each Leptosphaeria species had qualitatively identical compositions consisting of glucose, mannose, galactose, glucosamine, amino acids, and traces of galactosamine. Quantitative analyses showed that the hyphal wall components varied from species to species. Qualitative compositions of alkali-soluble wall fractions from each species were identical and contained the same sugars found in the unfractionated walls. The alkali-insoluble residues contained glucose, glucosamine, and amino acids. The alkali-soluble fractions were composed predominantly of glucose, galactose, and mannose. The alkali-insoluble fractions contained high concentrations of glucose and glucosamine and relatively low concentrations of amino acids.     

Carbohydrate composition of lymphocyte plasma membrane from pig mesenteric lymph node.

Pig lymphocyte plasma membrane isolated from mesenteric lymph node contained 69 mug of carbohydrate/mg dry wt., which was made up of neutral sugar, amino sugar and sialic acid in the molar proportions 5:1.7:1. The neutral sugar comprised fucose, ribose, mannose, glucose, galactose and inositol (molar proportions 2:9:11:15:26:1), and the amino sugar glucosamine and galactosamine (molar ratio 2:1). The ribose was most probably derived from RNA. All of the fucose and mannose and almost all of the glucosamine were associated with the membrane protein whereas the membrane lipid contained all of the inositol. The remaining sugars were distributed in various ratios between the protein and lipid fractions.     

Studies on the Autolysis of Aspergillus oryzae

The conditions of autolysis of washed mycelia of Aspergillus oryzae were systematically examined as for temperature, pH, aeration, energy supply, and chemicals which stimulate autolysis. Below 45°C, the higher the temperature the faster was the rate of autolysis. Optimum pH of autolysis with special reference to the excretion of nucleic acid components and amino acids was 5. With the optimum conditions of autolysis settled by us, 90 to 100% of nucleic acids, 75% of protein, and 20% of sugars in the mycelia were excreted into the medium within three days.

In the presence of lipophilic compounds such as toluene and sodium salts of fatty acids, autolysis occurred much faster than in distilled water. Autolysis was inhibited by the addition of glucose and aeration.

Mycelia of Aspergillus oryzae were autolyzed in distilled water, in toluene-saturated water, or in acetate buffer, pH 5.4, at 30°C. The cytoplasmic materials disappeared from cells during autolysis, but the cell wall retained its shape even after autolysis. The disappearance of the cytoplasmic materials started from the inner part under an aerobic condition and from the outer part under an anaerobic condition. During the autolysis, 15% of the cellular proteins was excreted as free amino acids (60%) and peptides (15%). Glucose, ribose, glucosamine, and three unidentified sugars were found in autolyzate. After eighteen hours of autolysis stimulated by toluene, 81% of the cellular nucleic acids was excreted as uridine (28%), xanthine (24%), hypoxanthine (17%), and two other nucleosides or bases.     

Cell wall composition and deoxyribonucleic acid similarities among the anaerobic coryneforms, classical propionibacteria, and strains of Arachnia propionica

Eighty strains of anaerobic coryneforms were compared with 29 strains of classical propionibacteria and 8 strains of Arachnia propionica by cell wall analysis, deoxyribonucleic acid (DNA) base compositions, and nucleotide sequence similarities. The anaerobic coryneforms have DNA base compositions in the range of 58 to 64% guanine + cytosine (GC) and show at least three homology groups. The largest group corresponds to organisms identified as Propionibacterium acnes and shows about 50% homology to strains in the P. avidum homology group. The third group, P. granulosum, shows low levels of similarities to the other two. All strains of anaerobic coryneforms have some combination of galactose, glucose, or mannose as cell wall sugars, and most have alanine (ala), glutamic acid (glu), glycine (gly), and l-alpha-epsilon-diaminopimelic acid (l-DAP) as amino acids of peptidoglycan. However, a few strains in the P. acnes and P. avidum homology groups have meso-DAP and minimal amounts of glycine. Two serological types, based on cell wall antigens, were found in the P. acnes homology group. One type had galactose, glucose, and mannose as cell wall sugars, the other glucose and mannose only. The classical propionibacteria have DNA base compositions in the range of 65 to 68% GC and show four homology groups which correspond closely to van Niel's classification as given in the 7th edition of Bergey's Manual. The P. jensenii group showed about 50% homology to the P. thoenii group and about 30 to 35% to the P. acidi-propionici group. The P. freudenreichii strains showed a rather lower level of similarity (8 to 25%) to the other homology groups. Most of the strains of classical propionibacteria also have some combination of galactose, glucose, or mannose as cell wall sugars and ala, glu, gly, and l-DAP as peptidoglycan amino acids, but P. shermanii and P. freudenreichii strains, which form a single homology group, have galactose, mannose, and rhamnose as cell wall sugars and ala, glu, and meso-DAP in their peptidoglycan. There is a rather low level of DNA homology (10 to 20%) between the anaerobic coryneforms and classical propionibacteria. However, the strains of A. propionica which have a GC content of 64 to 65% and form a single homology group, show no homology to either of the other two major groups.     

Further Studies of Pollen Wall Glycoproteins in Cucurbita pepo L.

Samples of pollen wall protein of Cucurbita pepo were prepared as reported in previous paper. Gas chromoatographic analyses snowed that the carbohydrate fraction of the pollen wall glycoprotcin contained 20.4% rhamnose, 15.3% fucose, 11% mannose, 11% galactose, 31% glucose, 4% arabinose and traces of xylose. The glycoproteins were further purified by Con. A affinity chromatography, Isoelectric focussing electrophoresis of the purified sample showed 3 PAS-positive bands, with respective PI 5.2, 6.0 and 6.3. The glycoprotein samples were subjected to hydrolysis with 6N HC1. After hydrolysis, the mixture was analyzed for amino acid composition with Backman 121-MB automatic amino acid analyzer, Results show the amino acid composition of the 3 glycoprotein was very similar, They all have glycine, glutamic acid and serine as their major component (these three amino acids constitute 50–60% of the total amino acids); and they all contain very small amount of methionine, phenylalanine, isoleucine and tyrosine. The lysine content of each glycoprotein is consistent with its respective PI, the glycoprotein which contains more lysine has higher PI.     

Autolytic enzymes in hyphae of Aspergillus nidulans: their action on old and newly formed walls.

Walls, purified from hyphae of the ascomycete Aspergillus nidulans, autolyzed on incubation and liberated glucose, mannose, galactose, N-acetylglucosamine, and soluble oligosaccharides. Digestion proceeded at linear rates until approximately 3% of the wall polymers had been hydrolyzed and then slowed markedly. The change in the rate of autolysis was not due to loss of enzyme activity but was caused by the disappearance of a fraction of the wall which was highly susceptible to digestion. Radioactive labeling showed that this fraction was the newly formed wall. The new wall was highly susceptible to enzyme action both when it was deposited at the apex in growing hyphae or when deposited laterally in hyphae treated with cycloheximide. The relations between wall modification and apical growth are discussed.     

Cell wall composition in the ascomycete sphaerostilbe repens at different developmental stages

Changes in the biochemical composition of isolated cell walls were analysed during the differentiation of coremia and rhizomorphs in Sphaerostilbe repens.Differentiation was accompanied by exclusively quantitative variations of the wall components: the content in carbohydrates, chitin and free amino sugars increased; on the contrary, amino acids, uronic acids, lipids and mineral substances decreased.Carbohydrates were composed of glucose, galactose and mannose; glucosamine was the main component of amino sugars. The predominant amino acid in the walls was cysteine the amount of which increased during hyphal aggregation, while quantities of the sixteen other determined amino acids decreased.Mineral matter was present in large quantities in the walls of the fungus, especially in vegetative mycelium. Iron, phosphorus and calcium were the most abundant elements.Possible relations between the variations in chemical composition of the wall and the capability of hyphae to aggregate are discussed.     

Chemical Studies on the Cell Walls of Leptospira biflexa Strain Urawa and Treponema pallidum Strain Reiter

The preparation and chemical poperties of the cell walls of Leptospira biflexa Urawa and Treponema pallidum Reiter are described. Both cell walls are composed mainly of polysaccharides and peptidoglycans. The data of chemical analysis indicate that the cell wall of L. biflexa Urawa contains rhamnose, arabinose, xylose, mannose, galactose, glucose and unidentified sugars as neutral sugars, and alanine, glutamic acid, α,ε-diaminopimelic acid, glucosamine and muramic acid as major amino acids and amino sugars. As major chemical constituents of the cell wall of T. pallidum Reiter, rhamnose, arabinose, xylose, mannose, galactose, glucose, alanine, glutamic acid, ornithine, glycine, glucosamine and muramic acid have been detected. The chemical properties of protein and polysaccharide fractions prepared from the cells of T. pallidum Reiter were also partially examined.     

The quantitative determination of metabolites of 6-mercaptopurine in biological materials. VI. Evidence for posttranscriptional modification of 6-thioguanosine residues in RNA from L5178Y cells treated with 6-mercaptopurine

Cell surface glycoconjugates of epimastigotes of Trypanosoma cruzi have been isolated and analyzed to give their amino acid and carbohydrate compositions. Those which have been investigated are a complex of three closely associated glycoproteins, GP24, GP31, GP37, and a lipopeptidophosphoglycan. The GP24-GP31-GP37 complex has an unusual amino acid composition with very low levels of hydrophobic amino acids, it contains 56% (w/w) carbohydrate, with mannose, galactose and glucosamine (presumably N-acetyl) being present in approximately equal quantities. The lipopeptidophosphoglycan also has low levels of hydrophobic amino acids and contains equal levels of mannose and galactose together with lesser amounts of (N-acetyl) glucosamine. The glycoconjugates are contrasted and compared with two other previously characterised cell surface glycoproteins (GP25 and GP72) from T. cruzi.     

Purification and characterisation of lipoglycan macroamphiphiles from Propionibacterium acnes

Lipidated macroamphiphiles such as the lipoteichoic acids and mycobacterial lipoarabinomannans are cell envelope components of Gram-positive bacteria that have been extensively associated with the pathogenesis of disease. In order to study such associations, purification of these macroamphiphiles is essential for resolving their structures and diverse biological effects. We describe herein a method for purification of lipoglycan components from Propionibacterium acnes. This method uses the existing phenol-water extraction, followed by hydrophobic interaction chromatography and an additional purification step that utilises preparative electrophoresis for the separation of two lipoglycan components. Analysis of these lipoglycans revealed evidence for a lipid anchor based on fatty acids whilst the polysaccharide moiety contained significant amounts of mannose, glucose and galactose, together with an amino sugar suspected of being a diaminohexuronic acid. These latter components have been previously identified as components of the P. acnes cell wall polysaccharide. Consequently, it is proposed that there may be a relationship between the structures of these distinctive cell envelope polymers.     

Chemical composition and serological analysis of the cell wall of Peptostreptococcus

Bahn, Arthur N. (Northwestern University, Chicago, Ill.), Patrick C. Y. Kung, and James A. Hayashi. Chemical composition and serological analysis of the cell wall of Peptostreptococcus. J. Bacteriol. 91:1672-1676. 1966.-Chemical and serological analyses were made of the cell wall of Peptostreptococcus to characterize taxonomically this genus of anaerobic streptococci. Cell wall hydrolysates of P. putridus strains 06 and 85, P. intermedius strains 11 and 87, and P. elsdenii strain B-159 were prepared, and the cell wall sugars were measured quantitatively by paper chromatography. Strain 85 contained only glucose, whereas strain 06 contained 93% glucose and 7% mannose. Strain 87 contained only rhamnose, and strain 11 contained approximately equal amounts of glucose and rhamnose. Strain B-159 differed from all the other strains in having a low (3.1%) content of total carbohydrate, consisting of rhamnose, galactose, and glucose. Quantitative amino acid analyses showed that the major amino compounds present in the cell wall were glutamic and aspartic acids, alanine, lysine, muramic acid, glucosamine, and galactosamine. Strains 06 and 85 possessed this complement of amino compounds, but strains 11 and 87 had relatively little aspartic acid. Strain B-159 was markedly different in having a high content of glycine and diaminopimelic acid, with only traces of lysine; it was the only strain in which teichoic acid was found. Serological analyses were made with the use of cell wall extracts as antigenic material and with homologous antisera, as well as streptococcal group antisera for groups A through S. The only strong agglutination was obtained between strain 87 antigen and group C antisera; weak agglutination was obtained with 87 against N, O, and K, and between strain 11 and groups E and F. All other antisera gave negative reactions. It is concluded that strain B-159 does not belong to the genus Peptostreptococcus, that strains 06 and 85 are members of P. putridus, and that strains 11 and 87 may be members of two different genera.     

Cell Wall Composition of the Yeastlike and Mycelial Forms of Blastomyces dermatitidis

The thermally induced changes in the cell wall polysaccharides of Blastomyces dermatitidis strain BD64, which produces a yeastlike form (Y form) at 37 C and a mycelial form (M form) at 20 C, were examined. The cell walls of the Y and M forms contained 36 and 51% of hexoses, respectively. The M-form cell wall contained glucose, galactose, and mannose in a molar ratio of 1:0.1:0.2. The Y-form cell wall contained mainly glucose and a very small amount of galactose and mannose. The glucans of the cell wall of the Y form consisted of about 95% alpha-glucan and 5% beta-glucan, whereas those of the M-form cell wall consisted of about 60% alpha-glucan and 40% beta-glucan.     

Purification and partial characterization of two glycoproteins in bovine peripheral nerve myelin membrane

Two major glycoproteins of bovine peripheral nerve myelin were isolated from the acid-insoluble residue of the myelin by a procedure involving delipidation with chloroform/methanol (2:1, v/v) and chromatography on Sephadex G-200 column with a buffer containing sodium dodecyl sulfate. The separation patterns of the proteins on the gel were affected considerably by the dodecyl sulfate concentration in the elution buffer. At above 2% dodecyl sulfate concentration in the elution buffer, the glycoproteins could be separated clearly on the gel and were purified. The purified proteins, the BR protein (mol. wt. 28 000) and the PAS-II protein (mol. wt. 13 000), were homogeneous on dodecyl sulfate-polyacrylamide gel electrophoresis. The NH₂-terminal amino acids of the BR and the PAS-II proteins were isoleucine and methionine, respectively. The BR protein contained glucosamine, mannose, galactose, fucose and sialic acids and the PAS-II protein contained glucosamine, mannose, galactose, fucose and glucose. Neither the BR protein nor the PAS-II were a glycosylated derivative of a basic protein of bovine peripheral nerve myelin, a deduction based on the results of amino acid analysis. The two major glycoproteins were observed commonly in the peripheral nerve myelin of cows, pigs, rabbits and guinea pigs, using dodecyl sulfate-polyacrylamide gel electrophoresis.     

Cell wall composition in Corynebacterium bovis and some other corynebacteria

The cell wall compositions of two strains of Corynebacterium bovis were found to differ: one contained lysine, rhamnose, mannose, and glucose, the other meso-alpha, epsilon, diaminopimelic acid (DAP), arabinose, galactose, and mannose. The walls of a strain of C. nephridii were characterized by l-DAP and galactose. Those of a strain of C. paurometabolum and of two strains of "lipophilic diphtheroids" contained meso-DAP, arabinose, galactose, and mannose as did walls of a reference strain of C. xerosis. The results are discussed in relation to the taxonomy of the organisms examined.     

Studies on the Cell Wall of Piricularia oryzae†

The chemical constituents of the cell wall of Piricularia oryzae, the pathogenic fungus of rice blast disease, were studied with the aids of chemical analysis, X-ray diffraction, infra-red absorption and enzymatic degradation. The sugar constituents were identified by chromatography as glucose (62%), mannose (4%), galactose (0.5%), and hexosamine (13%). The acidic amino acid rich protein was comprised 4.6% in the cell wall. The cell wall consists of at least three different polysaccharide complexes: a) α-Heteropolysaccharide protein complex containing mannose, glucose and galactose, b) β-1,3-Glucan containing β-1, 6-linked branch, c) Chitin like substance.     

The cell wall of Fusarium oxysporum.

Sugar analysis of isolated cell walls from three formae speciales of Fusarium oxysporum showed that they contained not only glucose and (N-acetyl)-glucosamine, but also mannose, galactose, and uronic acids, presumably originating from cell wall glycoproteins. Cell wall glycoproteins accounted for 50-60% of the total mass of the wall. X-ray diffraction studies showed the presence of alpha-1, 3-glucan in the alkali-soluble cell wall fraction and of beta-1, 3-glucan and chitin in the alkali-insoluble fraction. Electron microscopy and lectin binding studies indicated that glycoproteins form an external layer covering an inner layer composed of chitin and glucan.     

Cell-Wall Autohydrolysis in Isolated Endosperms of Lettuce (Lactuca sativa L.)

Cell walls prepared from the endosperm tissue of hydrated lettuce (Lactuca sativa L.) seeds undergo autohydrolysis. Release of carbohydrates is most rapid (0.4-0.6 [mu]g per endosperm) within the 1st h of incubation in buffer, but substantial autolysis is sustained for at least 10 h. Autolysis is temperature sensitive, and the optimum rate occurs at pH 5. The rate of autolysis increases markedly in the period just prior to radicle emergence. The cell-wall polysaccharide composition in micropylar and lateral endosperm regions differs significantly; the micropylar walls are rich in arabinose and glucose with substantially lower amounts of mannose. Although walls prepared from both micropylar and lateral regions undergo autolysis, micropylar walls release carbohydrates at a higher rate than lateral walls. Autolysis products elute as large polymers when subjected to size-exclusion chromatography, suggesting that endo-enzyme activity is responsible for release of fragments containing arabinose, galactose, mannose, and uronic acids. Arabinose, galactose, mannose, and glucose are also released as monomers. As a function of time, the ratio of polymers to monomers decreases, indicating that exo-enzyme activity is also present. Thermoinhibition or treatment with abscisic acid suppresses germination and reduces the rates of autolysis of walls isolated from the endosperm by about 25%. Treatments that alleviate thermoinhibition (kinetin and gibberellic acid) increase the rates of autolysis by 20 to 30% when compared to thermoinhibited controls.     

CHEMICAL COMPOSITION AND STRUCTURE OF THE CELL WALLS OF THE CONCHOCELIS AND THALLUS PHASES OF PORPHYRA TENERA (RHODOPHYCEAE)1,2

Purified cell walls were prepared from both the conchocelis and thallus phases of Porphyra tenera (Kjellm.). The nitrogen content of cell walls from the conchocelis was significantly greater than that for the thallus cell walls, being 3.35 ± 0.26% and 2.39± 0.03%, respectively. Amino acid analysis revealed important differences. The conchocelis cell wall hydrolyzates were richer in aspartic acid, glutamic acid, methionine, and basic amino acids. The thallus cell wall hydrolyzates, however, contained much more glycine and alanine than did those of the conchocelis. Hydroxyproline was not detected in cell walls of either phase. The neutral sugar content of cell wall hydrolyzates from the thallus was more than double that from the conchocelis being 83.6% and 34.5%, respectively. The former contained predominantly mannose which accounted for 72.2% of the neutral sugars while the latter was principally galactose (49.9%) and glucose (36.4%). Methylation analysis confirmed the presence of cellulose microfibrils in the conchocelis in contrast to xylan microfibrils in the thallus. The results establish that the conchocelis and thallus phases of P. tenera differ markedly in the structure and composition of the cell walls.     

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.     

Halococcus morrhuae: a sulfated heteropolysaccharide as the structural component of the bacterial cell wall.

The qualitative and quantitative composition of purifed cell wall of Halococcus morrhuae CCM 859 was determined. Glucose, mannose, galactose; glucuronic and galacturonic acids; glucosamine, galactosamine, gulosaminuronic acid; acetate, glycine and sulfate are found as major constituents. The amino sugars are N-acetylated. It was not possible to fractionate the cell wall in chemically different polymers. Evidence is presented that the major cell wall polymer of this strain is a complex heterolgycan which seems, like the peptidoglycan of most bacteria, to be responsible for the rigidity and stability of the cell wall. In addition it could be proved that this heteroglycan is sulfated and therefore differs considerably from previously described bacterial cell wall polymers.