Arthrobacter GJM-1 Research Articles

Structure of cell wall mannan of Candida kefyr IFO 0586

We conducted a structural analysis of the antigenic cell wall mannoprotein (mannan) isolated from Candida kefyr (formerly Candida pseudotropicalis) IFO 0586. The result of two-dimensional homonuclear Hartmann-Hahn analysis of this mannan indicates that the molecule is constructed from alpha-1,2- and alpha-1,6-linked mannopyranose residues. Upon alkali treatment (beta-elimination reaction), this mannan released two alpha-1,2-linked mannooligosaccharides, biose and triose. The structure of the alkali-stable mannan (outer chain) moiety was investigated by acetolysis. The structures of the resultant oligosaccharides, biose and triose, from the outer chain moiety were found to be the same as those of the alkali-released ones. Further, the treatment of the parent mannan with an Arthrobacter GJM-1 exo-alpha-mannosidase gave a linear mannan consisting solely of alpha-1,6-linked mannopyranose residues. These results indicate that the mannan forms the long backbone of the alpha-1,6 linkage, with a large number of short alpha-1,2-linked oligomannosyl side chains forming a comblike structure. Moreover, we investigated the serological properties of this mannan by performing an inhibition assay of a slide agglutination reaction with mannooligosaccharides and polyclonal factor sera (Candida Check; Iatron). The result indicates that the factor 1 serum preferentially recognizes the alpha-1,2-linked oligomannosyl side chains in this mannan. On the other hand, the fact that the mannan does not contain an antigenic determinant(s) corresponding to factor 8 suggests that the epitope(s) of this factor resides in other molecules on the cell surface of this strain.

Structure of the d-mannan of the pathogenic yeast, Candida stellatoidea ATCC 20408 (Type II) strain, in comparison with that of C. stellatoidea ATCC 36232 (Type I) strain

Acid treatment of the cell-wall d-mannans of Candida stellatoidea strains ATCC 36232 (Type I, A3 strain) and ATCC 20408 (Type II, A2 strain) gave (1 → 2)-linked β- d-manno-oligosaccharides (dp 2–5), whereas treatment with alkali gave the (1 → 2)-linked α- d-mannobiose. Conventional acetolysis of the acid- and alkali-treated d-mannan of the A3 strain gave oligosaccharides consisting of (1 → 2)- and (1 → 3)-linked α- d-mannopyranose residues, similar to those of Candida albicans serotype B strain. Mild acetolysis of the acid- and alkali-treated d-mannan of the A2 strain gave higher oligosaccharides that were digested by the Arthrobacter GJM-1 strain exo-α- d-mannosidase. The results of 1H- and 13C-NMR analyses indicated this d-mannan to contain branches with the following structures; β- d-Man p-(1 → 2)-α- d-Man p-(1 → 2)-α- d-Man p-Man p-(1 → 2)- d-Man. β- d-(1 → 2)-β- d-Man p-(1 → 2)-α- d-Man p-(1 → 2)-α- d-Man p-(1 → 2)-α- d-Mann p-(1 → 2)- d-Man, and β- d-Man p-(1 → 2)-β- d-Man p-(1 → 2)-β- d-Man p-(1 → 2)-α- d-Man p-(1 → 2)-α- d-Man p-(1 → 2)-α- d-Man p-(1 → 2)- d-Man, in common with the d-mannans of C. albicans serotype A strains.

Structural study of cell wall mannan of a Candida albicans (serotype A) strain

The structure of the mannan of Candida albicans NIH A-207 strain (serotype A) was investigated by adopting mild acetolysis followed by enzymolysis with an Arthrobacter GJM-1 exo-α-mannosidase. The resultant oligosaccharides, from pentaose to octaose (where man p= d-mannopyranose), were identified as man pβ(1→2)man pα(1→2)man pα(1→2)man pα(1→2)man p, man pβ(1→2)man pβ(1→2)man pα(1→2)man pα(1→2)man pα(1→2)man p, man pβ(1→2)man pβ(1→2)man pβ(1→2)man pα(1→2)man pα(1→2)man pα(1→2)man p and man pβ(1→2)man pβ(1→2)man pβ(1→2)man pβ(1→2)man pα(1→2)man pα(1→2)man pα(1→2)man p, respectively. Analyses of α-linked oligosaccharides obtained by acetolysis under conventional conditions gave the same oligosaccharides, from biose to heptaose, as those obtained from the mannans of C. albicans NIH B-792 (serotype B) and J-1012 (serotype A, formerly serotype C).

Structural determination of d-mannans of pathogenic yeasts Candida stellatoidea type I strains: TIMM 0310 and ATCC 11006 compared to IFO 1397

The structures of the cell-wall d-mannans of pathogenic yeasts of Candida stellatoidea Type I strains, IFO 1397, TIMM 0310, and ATCC 11006, were investigated by mild acid and, alkaline hydrolysis, by digestion with the Arthrobacter GJM-1 strain exo-α- d-mannosidase, and by acetolysis. The modified d-mannans and their degradation products were studied by 1H- and 13C-n.m.r. analysis. d-Manno-oligosaccharides released by acid treatment from the parent d-mannans were identified as the homologous β-(1 → 2)-linked d-manno-oligosaccharides from biose to hexaose, whereas those obtained by alkaline degradation were the homologous α-(1 → 2)-linked d-mannobiose and d-mannotriose. The acid- and alkali-modified d-mannans lacking 1H-n.m.r. signals above 4.900 p.p.m. [corresponding to β-(1 → 2)-linked d-mannopyranose units] were acetolyzed with 10:10:1 (v/v) Ac 2O-AcOH-H 2SO 4, and the resultant d-manno-oligosaccharides were also analyzed. It was found that the longest branches of the d-mannans, corresponding to hexaosyl residues, had the following structures: α- d-Man p-(1 → 3)-α- d-Man p-(1 → 2)-α- d-Man p-(1 → 2)-α- d-Man p-(1 → 2)- d-Man and α- d-Man p-(1 → 2)-α- d-Man p-(1 → 3)-α- d-Man p-(1 → 2)-α- d-Man p-(1 → 2)-α- d-Man p-(1 → 2)- d-Man. These results indicate that the d-mannans of C. stellatoidea Type I strains possess structures in common with the d-mannans of Candida albicans serotype B strain (see ref. 4) containing phosphate-bound β-(1 → 2)-linked oligo- d-mannosyl residues.

Structural study of cell wall phosphomannan of Candida albicans NIH B-792 (serotype B) strain, with special reference to 1H and 13C NMR analyses of acid-labile oligomannosyl residues

Chemical structures of manno-oligosaccharides, from biose to heptaose, released from the phosphomannan of Candida albicans NIH B-792 strain (serotype B) by mild acid hydrolysis were investigated. The results of 1H NMR, 13C NMR, and fast atom bombardment mass spectrometry analyses confirmed that these manno-oli gosaccharides belong to a homologous β-1,2-linked series. Although chemical shifts of 1H NMR patterns of these oligosaccharides were considerably too complicated to be assigned, their 13C NMR patterns were sufficiently simple to be interpreted, exhibiting a regular increase of downfield shift of ppm values of the C-1 atom from each mannopyranose residue in proportion to their molecular weights. In order to determine the whole chemical structure of the parent phosphomannan, the acid-stable domain was subjected to acetolysis and then enzymolysis with the Arthrobacter GJM-1 α-mannosidase and the resultant manno-oligosaccharides were investigated for their chemical structures by 1H NMR spectroscopy. The results of a precipitin-inhibition test using the β-1,2-linked manno-oligosaccharides, from biose to hexaose, in comparison with the corresponding isomers containing α-1,2 linkage with small amounts of α-1,3 linkage, indicated that the haptens possessing the former linkage exhibited much higher inhibitory effects than the corresponding isomers containing the latter linkages did. Based on the present findings, a chemical structure of the phosphomannan of this C. albicans strain was proposed.

Structural study of phosphomannan of yeast-form cells of Candida albicans J-1012 strain with special reference to application of mild acetolysis

Structural analysis of the phosphomannan isolated from yeast-form cells of a pathogenic yeast, Candida albicans J-1012 strain, was conducted. Treatment of this phosphomannan (Fr. J) with 10 m m HCl at 100 °C for 60 min gave a mixture of β-1,2-linked manno-oligosaccharides, from tetraose to biose plus mannose, and an acid-stable mannan moiety (Fr. J-a), which was then acetolyzed by means of an acetolysis medium, 100:100: 1 ( v v ) mixture of (CH 3CO) 2O, CH 3COOH, and H 2SO 4, at 40 °C for 36 h in order to avoid cleavage of the β-1,2 linkage. The resultant manno-oligosaccharide mixture was fractionated on a column of Bio-Gel P-2 to yield insufficiently resolved manno-oligosaccharide fractions higher than pentaose and lower manno-oligosaccharides ranging from tetraose to biose plus mannose. The higher manno-oligosaccharide fraction was then digested with the Arthrobacter GJM-1 α-mannosidase in order to cleave the enzymesusceptible α-1,2 and α-1,3 linkages, leaving manno-oligosaccharides containing the β-1, 2 linkage at their nonreducing terminal sites, Man pβ1 → 2Man pα1 → 2Man pα1 → 2Man pα1 → 2Man, Man pβ1 → 2Man pβ1 → 2Man pα1 → 2Man pα1 → 2Man pα1 → 2Man, and Man pβ1 → 2Man pβ1 → 2Man pβ1 → 2Man pα1 → 2Man pα1 → 2Man pα1 → 2Man. However, the result of acetolysis of Fr. J-a by means of a 10:10:1 ( v v ) mixture of (CH 3CO) 3O, CH 3COOH, and H 2SO 4 at 40 °C for 13 h was significantly different from that obtained by the mild acetolysis method; i.e., the amount of mannose was apparently larger than that formed by the mild acetolysis method. In summary, a chemical structure for Fr. J as a highly branched mannan containing 14 different branching moieties was proposed.

Structural analysis of phospho- d-mannan-protein complexes isolated from yeast and mold form cells of Candida albicans NIH A-207 serotype a strain

The immunochemical properties between phospho- d-mannan-protein complexes of yeast (Y) and mycelial (M) forms of Candida albicans NIH A-207 (serotype A) strain were compared. Hydrolysis of the Y-form complex gave a mixture of β-(1→2)-linked d-mannooligosaccharides consisting mainly of tri- and tetra-ose, whereas the M-form complex gave preponderantly d-mannose. The antiserum against Y-form cells exhibited a lower reactivity with the M-form than with the Y-form complex, whereas the antiserum to M-form cells could not distinguish significantly between both complexes. Moreover, these acid-modified complexes showed lower antibody-precipitating effect than each corresponding intact complex against antisera of Y- and M-form cells. Digestion of the acid-modified Y- and M-form complexes with the Arthrobacter GJM-1 strain α- d-mannosidase yielded 35- and 40-% degradation products, respectively. Acetolysis of each modified complex under mild conditions gave the same d-mannohexaose, β- d-Man p-(1→2)-β- d-Man p-(1→2)-α- d-Man p-(1→2)-α- d-Man p-(1→2)-α- d-Man p-(1→2)- d-Man. Because the complexes of Y- and M-form cells of C. albicans NIH B-792 (serotype B) strain did not give any hexaose fraction containing β-(1→2) linkages, the presence of this hexaose can be regarded as one of the dominant characteristics of the serotype-A specificity of C. albicans spp.

A glucomannan as an extracellular product of Candida utilis. II Structure of a glucomannan. Characterization of oligosaccharides obtained by partial hydrolysis.

The structure of an extracellular glucomannan produced by Candida utilis was studied. Up to 15% hydrolysis of the glucomannan with the α-mannosidase isolated from Arthrobacter GJM-1 did not alter its viscosity. Sequential treatment of the glucomannan with periodate, borohydride and 0.1 N HCl gave a nondialyzable periodate resistant portion which amounted to 15% of the original glucomannan. The carbohydrate residue of the periodate resistant portion was found to consist only of mannose. Methylation analysis indicated that all the glucose residues in the molecule were doubly branched at the C 3 and C 4 positions, and the non-reducing ends were composed of only mannose. Partial hydrolysis of the glucomannan yielded di-(PHP 1, 2), tri-(PHP 3), tetra-(PHP 4) and pentasaccharides (PHP 5) which were isolated by TLC. They all contained one mole of glucose residue per mole of oligosaccharide. Arthrobacter α-mannosidase completely hydrolyzed PHP 1 to glucose and mannose, but did not hydrolyze PHP 2. In addition, the enzyme degraded PHP 3 and 4 into PHP 2 and mannose, and PHP 5 into mannose and a tetrasaccharide which did not correspond to any of the series PHP 1-5. The structures of these oligosaccharides were studied, by a joint analyses using methylation and α-mannosidase digestion.

Chitin structures of the cell walls of synchronously grown virgin cells ofSaccharomyces cerevisiae

The ability of a lytic beta-glucanase of Arthrobacter GJM-1 to dissolve cell walls of Saccharomyces cerevisiae with exception of the chitin-containing fraction was employed for the isolation of chitin-rich residues of the cell walls of synchronously growing populations of virgin cells. Electron microscopical examination of such wall residues isolated from cells at various stages of the budding cycle showed that the first phase of chitin deposition in the wall corresponds to the formation of an annular structure found as a part of the bud scar after cell division. The annular chitin-rich structure could not be isolated at cell cycle stages preceding the bud emergence and at earliest stages of bud development. The observations confirmed that the annular structure (chitin ring) formed during bud growth represents a major part of total chitin present in the bud scar after septum closure.

Enzymes of the yeast lytic system produced byArthrobacter GJM-1 bacterium and their role in the lysis of yeast cell walls

Yeast lytic system produced by Arthrobacter GJM-1 bacterium during growth on baker's yeast cell walls contains a complete set of enzymes which can hydrolyze all structural components of cell walls of Saccharomyces cerevisiae. Chromatographic fractionation of the lytic system showed the presence of two types of endo-beta-1,3-glucanase. Rapid lysis of isolated cell walls of yeast was induced only by endo-beta-1,3-glucanase exhibiting high affinity to insoluble beta-1,3-glucans and releasing laminaripentaose as the main product of hydrolysis of beta-1,3-glucans. This enzyme was able to lyse intact cells of S. cerevisiae only in the presence of an additional factor present in the Arthrobacter GJM-1 lytic system, which was identified as an alkaline protease. This enzyme possesses the lowest molecular weight among other identified enzyme components present in the lytic system. Its role in the solubilization of yeast cell walls from the outer surface by endo-beta-1,3-glucanase could be substituted by preincubation of cells with Pronase or by allowing the glucanase to act on cells in the presence of thiol reagents. The mechanism of lysis of intact cells and isolated cell walls by the enzymes of Arthrobacter GJM-1 is discussed in the light of the present conception of yeast cell wall structure.

Lysis of intact yeast cells and isolated cell walls by an inducible enzyme system of Arthrobacter GJM-1.

Bacterium Arthrobacter GJM-1 known in the literature as a good producer of alpha-mannanase was found to accumulate in the culture fluid lytic activities against viable yeast cells during growth on isolated cell walls or beta-glucan fractions of yeast. The accumulation of the lytic activities showed an inducible character. The lytic system produced in the medium containing baker's yeast cell walls was capable of complete solubiliaztion of cell wals in vitro. The system lysed viable cells of a number of yeast species and induced their conversion to protoplasts in an osmotically stabilized medium. The lytic system showed different pH and temperature optima when viable cells or isolated cell walls were used as substrates. The pH optimum of the lysis of isolated cell walls was identical with pH optimum of beta-glucanase activities in the crude system. The results pointed out that in the lysis of intact cells, in addition to beta-glucanases, some other factor is involved. Substantial differences in the nature of the outer and the inner surface of cell walls of Saccharomuces cerevisiae were confirmed in this paper based on the different susceptibility to lysis of the cell walls in vivo and in vitro.

Metabolism of 2-deoxy- d-glucose by baker's yeast. VI. A study on cell wall mannan

The incorporation of 2-deoxy- d-glucose into cell wall mannan of growing Saccharomyces cerevisiae proceeded continuously during culture growth and followed the cell multiplication. About 10% of mannan labelled with deoxyglucose was concurrently released into the medium. The distribution of deoxyglucose between the side-chains and the main chain of mannan has been established. Approximately 90% of deoxyglucose present in the polysaccharide was bound in the side-chains and only 10% was located in the (1 å 6)-linked main chain. This result suggested that deoxyglucose metabolites serving as glycosyl donors in mannan biosynthesis were much worse substrates for the enzyme(s) responsible for the formation of the main chain of the polysaccharide than for the mannosyl transferases involved in the formation of the mannan side-chains. Degradation of deoxyglucose-containing mannan by α-mannosidase of Arthrobacter GJM-1 stopped at the deoxyglucosyl residues.

Enzymic degradation of yeast cell-wall mannans and Galactomannans to polymeric fragments containing α-(1→6)-linked D-mannopyranose residues

The cell-wall D-mannans of Candida parapsilosis, Endomycopsis fibuliger, Saccharomyces rouxii, Torulopsis apicola (Hajsig strain), and Torulopsis bombi were degraded with an exo α- D-mannosidase from Arthrobacter GJM-1 to their α-(1→6)-linked D-mannopyranose main-chains, as demonstrated by p.m.r. spectroscopy. D-galacto- D-mannans from Candida lipolytica, Torulopsis gropengiesseri, Torulopsis lactis-condensi, Torulopsis magnoliae, and Trichosporon fermentans could be degraded to polysaccharides containing mainly 6- O-linked α- D-mannopyranosyl residues following preferential removal of their enzyme-resistant, D-galactopyranosyl non-reducing end-units with acid. The D-mannans of Saccharomyces lodderi, Citeromyces matritensis, and Pichia pastoris could also be enzymically degraded to polysaccharides containing predominantly α-(1→6)-linked D-mannopyranosyl residues after hydrolysis of most of the β- D-linked residues in their side chains with acid. The exo α- D-mannosidase, as would be expected, produced β- D-mannose on splitting of an α-(1→2)-linked D-mannopyranose tetramer. It is, however, very selective in its action since it did not cleave α- D-Man p-(1→2)-β- D-Man p-(1→2)-β- D-Man p-(1→2)-β- D-Man p-(1→2)- D-Man. Apparently a D-mannopyranose non-reducing end-unit and two consecutive α- D-mannopyranose residues are required by the enzyme for cleavage of a substrate to take place.