Influence of the culture medium on the synthesis of alpha-D-glucans by Streptococcus cricetus AHT.

Three different alpha-D-glucosyltransferases (GTFs) were separated from culture filtrates of Streptococcus cricetus strain AHT grown in a complex, standard medium in batch culture or under defined conditions of growth in the chemostat. Two of the enzymes (GTF-S1 and GTF-S2) converted sucrose into branched, soluble dextrans, and the third (GTF-I) produced a relatively linear, water-insoluble, predominantly (1----3)-linked alpha-D-glucan. When the organism was grown in complex medium modified by the removal of the fraction of high molecular weight, only GTF-S1 and GTF-S2 were released, and no GTF-I was detected. The water-insoluble glucan fraction obtained by incubating the cell-free filtrate with sucrose contained from 17 to 25% of (1----3)-glucosidic linkages, and accounted for up to 78 and 4% of the total glucans derived from growth in standard and modified medium, respectively. The soluble glucans produced in the same reaction were fractionated with ethanol to give, from both media, two distinct dextrans comprising (1) a highly branched dextran similar to the S1-dextran product of GTF-S1 and (2) a dextran containing fewer branch linkages and up to 86% of (1----6)-alpha-D-glucosidic linkages. A GTF responsible for the synthesis of the latter dextran was not separated. The structures of the glucan fractions and the products of the separated GTF were examined by enzymic degradation and methylation analysis.

Specificity of acceptor binding to Leuconostoc mesenteroides B-512F dextransucrase: binding and acceptor-product structure of alpha-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.

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.

Isolation and characterization of a soybean lectin having 4-O-methylglucuronic acid specificity.

A new lectin from soybeans having specificity toward the extracellular 4-O-methyl-D-glucurono-L-rhamnans produced by certain strains of Rhizobium japonicum has been purified and characterized. Isolation was accomplished initially by isoelectric precipitation of contaminating globulins and subsequently by affinity chromatography on partially hydrolyzed glucuronorhamnan covalently coupled to amino-hexylagarose. Residual globulins were removed by adsorption of the lectin on concanavalin A-agarose and elution with methyl alpha-mannoside. The lectin is a glycoprotein (3-5% carbohydrate) with a molecular weight of approximately 175 000. It is a tetramer with subunit molecular weights of 45 000 when dissociated with sodium dodecyl sulfate. Reverse-phase high-pressure liquid chromatography analysis indicates the presence of two types of subunits, both having equivalent molecular weights. According to amino acid analyses, the lectin is rich in acidic but low in sulfur-containing amino acids. The carbohydrate portion of the lectin contains mannose; no hexosamines could be detected. Chemical modification of the lectin indicated that neither sulfhydryl groups nor amino groups participate in binding. Quantitative binding studies of the lectin with various carbohydrate haptens showed that specificity was directed toward 4-O-methyl-D-glucuronic acid, D-glucuronic acid, and their methyl glycosides with 4-O-methyl-D-glucuronic acid 3-4-fold more effective. In each instance, the methyl glycoside is a more effective hapten.

A soybean lectin having 4-O-methyl-D-glucuronic acid specificity.

Evidence is presented for the presence of a new lectin activity in soybean seeds [Glycine max (L.) Merrill] that has specificity towards the 4-O-methyl-D-glucurono-L-rhamnan exopolysaccharide produced by certain strains of Rhizobium japonicum. Bacterial agglutination and precipitin reactions revealed the lectin activity in phosphate-buffered saline extracts of seeds of all cultivars tested, including the "lectinless" varieties. Reaction of such extracts with carbohydrate haptens demonstrated that the specificity of the binding was towards 4-O-methyl-D-glucuronic acid, D-glucuronic acid and their methyl glycosides.

Growth-related substituent changes in exopolysaccharides of fast-growing rhizobia.

Pyruvic acid and O-acetyl groups are the major noncarbohydrate substituents in exopolysaccharides (EPS) produced by fast-growing species of Rhizobium. EPS substituent variations were observed among strains of the same species. The amounts of these substituents also varied with culture age; pyruvic acid increased in the EPS of all four species, whereas O-acetyl increased in Rhizobium trifolii and R. leguminosarum EPS, decreased in R. meliloti EPS, and remained constant in R. phaseoli EPS. The use of glycerol as a substrate for R. meliloti significantly increased EPS yields, whereas mannitol increased those of the other three Rhizobium species.

Biodegradation of Xanthan Gum by Bacillus sp.

Strains tentatively identified as Bacillus sp. were isolated from sewage sludge and soil and shown to elaborate extracellular enzymes that degrade the extracellular polysaccharide (xanthan gum, polysaccharide B-1459) of Xanthomonas campestris NRRL B-1459. Enzyme production by one strain was greatly enhanced when the strain was incubated in a mixed culture. Products of degradation were identified as d-glucuronic acid, d-mannose, pyruvylated mannose, 6-O-acetyl d-mannose, and a (1-->4)-linked glucan. These products correlate with the known structure of the gum. The complexity of the product mixture indicated that the xanthanase was a mixture of carbohydrases. The xanthanase complexes were similar to one another in temperature stability, pH and temperature optima, degree of substrate degradation, and enzymolysis products. Differences in pH stability, salt tolerance, recoverability, and yields of enzyme were observed.

Intracellular mannitol, a product of glucose metabolism in staphylococci.

Mannitol (Mtl), not previously reported as an intracellular component of bacteria, although it has been found as an extracellular end product of anaerobic carbohydrate metabolism, accumulated within strains of all 10 staphylococcal species tested after aerobic incubation of washed cell suspensions in phosphate-buffered 1% glucose for 2 h. Phenol extracts of the cells, before and after incubation, were analyzed for Mtl content by periodate utilization and paper chromatography and for Mtl 1-phosphate content, with Mtl 1-phosphate dehydrogenase. In Staphylococcus aureus Towler, the content of Mtl increased from a 0-h value of less than 2.4 to 16 mumol/g (dry weight) after incubation, and the level of Mtl 1-phosphate increased from a 0-h value of 1 to 8 mumol/g. The identification of Mtl was confirmed as the per-O-acetyl ester by gas-liquid chromatography and as the per-O-methyl ether by mass spectrometry. Also tested were 5 additional S. aureus strains and 32 coagulase-negative staphylococcal strains. All strains accumulated Mtl, even those strains that could not utilize exogenous Mtl during aerobic growth, usually in the range 4 to 25 mumol/g. Furthermore, three strains accumulated very high Mtl levels. Bacteria from several other genera were tested, and some were found to accumulate low to moderate levels of Mtl under similar incubation conditions. The metabolic conversion of glucose to intracellular Mtl, probably via Mtl 1-phosphate, is a common feature of staphylococci and also occurs in some other bacteria.

Effect of Ultraviolet-B (280 to 320 nm) Radiation on Blue-Green Algae (Cyanobacteria), Possible Biological Indicators of Stratospheric Ozone Depletion.

The effect of ultraviolet-B (280 to 320 nm) irradiation on physiological activities of Anabaena flos-aquae and the water fern Azolla caroliniana has been studied under conditions where lethal effects of irradiation are absent. Nitrogenase activity, measured as acetylene reduction, specifically declined during irradiation with low levels of ultraviolet-B, whereas other physiological activities were unaffected. These findings indicate that measurement of acetylene reduction by cyanobacteria may serve as a specific, sensitive biochemical assay to assess environmental ultraviolet-B effects due to depletion of stratospheric ozone.

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.

Phosphorus-31 nuclear magnetic resonance spectroscopy of extracellular, yeast O-phosphonohexoglycans.

P nuclear magnetic resonance spectra of a number of purified yeast O-phosphonohexoglycans were recorded. The data therefrom were correlated with established chemicals aspects of individual and collective polymer structures, permitting (a) conclusions to be drawn regarding the chemical environment of the phosphate groups of these polymers, and (b) assignment of anormeric configurations to the hexosyl phosphate residues.

Growth of Hansenula holstii on cadavers.

Growth of a yeast was observed on prosected cadavers used for demonstration purposes in a medical school. An asporogenous yeast was isolated and identified as an atypical form of Hansenula holstii by analysis of the extracellular polysaccharide. The isolate showed resistance to embalming fluid but was eventually eradicated by addition of picloxidine digluconate to the fluid.

Variation in composition of yeast phosphohexosans.

Omitting of KH(2)PO(4) from culture media leads to the production of altered phosphohexosans or neutral extracellular mannans by yeasts that otherwise elaborate phosphogalactans and phosphomannans.

Predicted and unpredicted cross-reactions of an acetylphosphogalactan of Sporobolomyces yeast.

The teichoic acid of streptococcal Group N, with end groups of galactose phosphate, had been shown to cross-react with antipneumococcal sera of types VI, XIV, XVI, and XXVII. End groups of D-galactose-1-phosphate in the phosphogalactans of Sporobolomyces yeasts made it predictable that these galactans would precipitate the same antipneumococcal sera and also antisera to streptococcal Group N. The predictions were verified, and other unpredicted reactions were found. Precipitation of much of the antibody in an antipneumoccal type XVIII horse serum was shown to be due to O-acetyl-D-galactose residues in the phosphogalactan, in accord with earlier information that an O-acetyl sugar was a principal determinant of S XVIII. The new results identify this sugar as D-galactose. Since it is linked 1,3- in S XVIII, the O-acetyl group in the Sporobolomyces galactan is probably also on a 1,3-linked residue. Another major cross-reaction in anti-S. paratyphi A serum characterizes the galactose residues in the "O" polysaccharide of the bacillus as members of the D-series probably linked in tandem 1,6-, 1,6-; 1,6-, 1,3-; 1,3-, 1,6-; or 1,3-, 1,3-. Reactions of periodate-oxidized-reduced products confirm the conclusions stated above. Quantitative data are given.