Morey E. Slodki

Structure and antigenic activity of the capsular polysaccharide of Cryptococcus neoformans serotype A

Abstract The glucuronoxylomannan (GXM) of C. neoformans serotype A contained molar ratios of xylose: mannose: glucuronic acid: O -acetyl of 2:5:1:2. The xylose and glucuronic acid occurred as single pyranosyl residues and were oxidized by periodate; the Smith-degraded product was an insoluble mannan. Mannose was linked (1→3) with three out of five mannose residues (1→2)-substituted with one glucuronosyl and two single xylosyl end groups. A repeating octasaccharide was proposed as the structure. GXM was separated into species-specific and serotype-specific components by chromatography on a serotype-D-antibody agarose column, but these showed no obvious differences in molar ratios of the monosaccharides. Decreased serological activity was observed when GXM was de- O -acetylated or carboxy-reduced, but not when carboxy-esterified. The Smith-degraded mannan was inactive. Autohydrolysis caused a gradual release of xylose and some mannose, and a decline in serological activity. Xylose and glucuronic acid were implicated in a single antigenic group since selective inactivation of either residue led to loss of more than half of the activity. Extensive crossreactions were observed with the GXMs from C. laurentii and Tremella mesenterica , but not with types II or XIV pneumococcal polysaccharides.

Gas-liquid chromatography-mass spectrometry of methylated and deuteriomethylated per-O-acetyl-aldononitriles from D-mannose

Abstract Peracetylated aldononitriles of the tetra-, tri-, and di-methyl ethers of D-mannopyranose were separated by gas-liquid chromatography, and analyzed by mass spectrometry. Through introduction of deuteriomethyl ether groups, various fragmentions constituting the mass spectra were identified and related to the parent methylated sugar structures. Also identified were several characteristic series of fragment ions that are common to two or more methylated D-mannopyranosides. As expected, mass spectra of the D-mannose derivatives were identical to those previously observed for D-glucose methylated in the same positions. Distinctive mass spectra were also recorded for all additional di- O -methyl-D-mannose derivatives. This information permits use of peracetylated aldononitrite derivatives in methylation-fragmentation analysis of aldohexans.

Laboratory production of a phosphorylated mannan by Hansenula holstii

Abstract Conditions for the production of a phosphorylated mannan by strains of the yeast Hansenula holstii were investigated. Polysaccharide was produced from glucose in media containing organic nitrogen and inorganic salts. The ratio of mannose to phosphate in the polymer was not altered by changes in medium constituents. Yields of polymer in the range of 50–55% based on glucose were realized in shaken-flask experiments.

Methylation analyses of NRRL dextrans by capillary gas-liquid chromatography

Abstract Methylation analyses were conducted on 38 NRRL dextrans with the aid of capillary gas-liquid chromatography (g.l.c.)-mass spectrometry. The superior chromatographic resolution of capillary g.l.c. permitted better quantitation of linkage types and detection of trace components than had been obtained with packed columns. Only a few of the dextrans analyzed contain a single type of non-(1→6) linkage; seven contain at least traces of all three possible secondary links at points of branching. The results amend some earlier methylation analyses conducted by packed-column g.l.c., and extend the utility of the NRRL dextran collection by providing more-precise, quantitative data on the types of α- d -glucopyranosyl residues present.

Tryptophan catabolism to indolepyruvic and indoleacetic acids byRhizobium japonicum L-259 mutants

Tan-colored (tan) mutants, which retain the capacity to reduce acetylene, were selected and isolated fromRhizobium japonicum L-259 after growth in a glutamate-limited medium supplemented with tryptophan (trp). The mutants catabolized trp (500 mg per liter) to produce organe-colored fermentation broths containing extracellular indolepyruvic (IPA) and indoleacetic (IAA) acids. In contrast, parental strain L-259 produced a colorless broth containing only IAA when grown on the trp-supplemented medium. Trp alone was determined to be an incomplete nitrogen source for mutant growth and did not stimulate acetylenereduction activity.

Methylation and acetolysis of extracellular D-mannans from yeast

Methylation-fragmentation analyses were conducted on a series of extra-cellular, yeast alpha-D-linked mannans representing six different structural types. D-Mannans of low degree of branching were produced by Hansenula capsulata strains and by species related to H. holstii, The former consisted primarily of (1 leads to 2)- and (1 leads to 6)-linked D-mannosyl residues; the latter, of (1 leads to 2)- and (1 leads to 3)-linked D-mannosyl residues. Although the remaining structural types were highly branched, each gave distinct methylation-patterns indicative of (1 leads to 6)-linked backbones to which are appended non-(1 leads to 6)-linked side-chains. Acetolysis studies were correlated with the methylation analyses, and the correlation demonstrated that each branched polymer possesses side chains of heterogeneous length.

Productivity of four α-d-glucosyltransferases released by Streptococcus sobrinus under defined conditions in continuous culture

Abstract The distribution and activity of four different α- d -glucosyltransferases (GTF) were determined in culture filtrates of Streptococcus sobrinus grown in the chemostat. The rate of production of each enzyme depended on the growth rate, the pH of the medium, the limiting nutrient and on the presence of Tween 80. Changes in the relative proportion of GTF were reflected in the altered structures and adhesive qualities of the water-insoluble α- d -glucans synthesized from sucrose by the culture filtrates.

High-temperature, salt-tolerant xanthanase

SummaryA new high-temperature, salt-tolerant xanthanase suitable as an enzymic viscosity breaker for xanthanbased hydraulic fracture fluids was obtained by soil enrichment growth on xanthan gum incubated at 45°C in the presence of 3% NaCl. The mixed culture produces exoenzymes functional up to 65°C in the presence of salts. Degradation products include the pyruvic acetal of mannose and branched oligosaccharides derived from cleavage of main-chain β-(1→4)-d-glucosyl linkages. Release of the terminal pyruvic acetal ofd-mannose leads to oligosaccharide products that evidently contain the ene-4,5-unsaturated glucuronic acid residue.

Type-specific polysaccharides of Cryptococcus neoformans. N.m.r.-spectral study of a glucuronomannan chemically derived from a Tremella mesenterica exopolysaccharide

A glucuronomannan (GM) was derived by removal, through Smith degradation, of xylose from the native (3-O-acetylglucurono)xylomannan exopolysaccharide isolated from Tremella mesenterica. 13C-N.m.r. chemical shifts measured at various pD values were compared for p-nitrophenyl beta-D-glucopyranosiduronic acid (1) and two GMs (2 and 3) differing in GlcA content (Man:GlcA; 2, 10:1; and 3, 5:1). Also measured and compared were pKa values for 1 and 2. One-dimensional and two-dimensional (COSY and HETCOR) n.m.r. data allowed unambiguous assignments of pD-sensitive chemical shifts due to 2-O-beta-D-GlcpA substituents attached to a (1----3)-linked alpha-D-Manp backbone. The pKa and n.m.r. data indicated that the CO2H groups in either GM are independent of each other, and are similar in behavior to those of p-nitrophenyl beta-D-glucopyranosiduronic acid molecules. The n.m.r. data confirmed the previous, chemically deduced, structural role of GlcpA in the native polysaccharide from T. mesenterica, and indicated that significant pD-induced changes occur in the stabilities of the glycosidic orientations in the GM. Previous 13C-n.m.r. assignments for 2-O-beta-D-GlcpA in polysaccharides derived from Cryptococcus neoformans serotype A-variant were confirmed, except for the signal due to the anomeric carbon atom. This signal is now known to be pD-sensitive. In acidic solutions, it is coincident with the signal (104.5 p.p.m.) due to the anomeric carbon atoms of the unsubstituted alpha-D-Manp backbone residues. In basic solutions, the 2-O-beta-D-GlcpA anomeric carbon resonance is shifted upfield by approximately 0.2 p.p.m., and is observed as a separate signal.

Specificity of acceptor binding to Leuconostoc mesenteroides B-512F dextransucrase: Binding and acceptor-product structure of α-methyl-d-glucopyranoside analogs modified at C-2, C-3, and C-4 by inversion of the hydroxyl and by replacement of the hydroxyl with hydrogen

The specificity of acceptor binding to the active site of dextransucrase was studied by using alpha-methyl-D-glucopyranoside analogs modified at C-2, C-3, and C-4 positions by (a) inversion of the hydroxyl group and (b) replacement of the hydroxyl group with hydrogen. 2-Deoxy-alpha-methyl-D-glucopyranoside was synthesized from 2-deoxyglucose; 3- and 4-deoxy-alpha-methyl-D-glucopyranosides were synthesized from alpha-methyl-D-glucopyranoside; and alpha-methyl-D-allopyranoside was synthesized from D-glucose. The analogs were incubated with [14C]sucrose and dextransucrase, and the products were separated by thin-layer chromatography and quantitated by liquid scintillation spectrometry. Structures of the acceptor products were determined by methylation analyses and optical rotation. The relative effectiveness of the acceptor analogs in decreasing order were 2-deoxy, 2-inverted, 3-deoxy, 3-inverted, 4-inverted, and 4-deoxy. The enzyme transfers D-glucopyranose to the C-6 hydroxyl of analogs modified at C-2 and C-3, to the C-4 hydroxyl of 4-inverted, and to the C-3 hydroxyl of 4-deoxy analogs of alpha-methyl-D-glucopyranoside. The data indicate that the hydroxyl group at C-2 is not as important for acceptor binding as the hydroxyl groups at C-3 and C-4. The hydroxyl group at C-4 is particularly important as it determines the binding orientation of the alpha-methyl-D-glucopyranoside ring.