Synthesis of cyclic beta-(1,2)-glucans by Rhizobium leguminosarum biovar trifolii TA-1: factors influencing excretion.

The synthesis of cyclic beta-(1,2)-glucans from UDP-[14C]glucose by a crude membrane preparation and whole cells of Rhizobium leguminosarum bv. trifolii TA-1 was investigated. The crude membrane system needed Mn2+, ATP, and NAD+ for optimal activity. Hardly any difference in biosynthetic activity between membrane fractions of TA-1 cells grown in the presence (200 mM) or absence of NaCl was observed. Whole TA-1 cells grown in the presence of NaCl excreted labeled, neutral cyclic beta-(1,2)-glucan during incubation with added UDP-[14C]glucose. With NaCl-free cultured TA-1 cells, no excretion was observed; however, after these cells were alternately frozen and thawed eight times, they excreted glucans. Glucan formation in vitro and glucan excretion by whole cells were strongly inhibited in the presence of 50 mg of cyclic glucan per ml (about 15 mM), indicating that biosynthesis of cyclic beta-(1,2)-glucans in strain TA-1 is controlled by end-product inhibition. These observations indicate that TA-1 cells become more permeable to cyclic glucans at high NaCl concentrations. The constant loss of glucans from cells grown in the presence of 200 mM NaCl prevented end-product inhibition and resulted in glucan accumulation of up to 1,600 mg/liter in the medium.     

Cell Wall Glucans of the Yeast and Mycelial Forms of Paracoccidioides brasiliensis

Glucans were isolated from the cell wall of the yeast (Y) and mycelial (M) forms of Paracoccidioides brasiliensis. The alkali-soluble glucan of the Y form had properties of alpha-1,3-glucan. The alkali-insoluble glucan of the M form was identified as a beta-glucan which contains a beta-(1 --> 3)-glycosidic linkage by infrared absorption spectrum, by effect of beta-1,3-glucanase, and by partial acid hydrolysis. The alkali-soluble glucans of the M form were a mixture of alpha- and beta-glucans and the ratio of alpha- to beta-glucan was variable, depending on the preparations.     

Effects of Ionic and Osmotic Strength on the Glucosyltransferase of Rhizobium meliloti Responsible for Cyclic beta-(1,2)-Glucan Biosynthesis

The cyclic beta-(1,2)-glucans of Rhizobium meliloti and Agrobacterium tumefaciens play an important role during hypoosmotic adaptation, and the synthesis of these compounds is osmoregulated. Glucosyltransferase, the enzyme responsible for cyclic beta-(1,2)-glucan biosynthesis, is present constitutively, suggesting that osmotic regulation of the biosynthesis of these glucans occurs through modulation of enzyme activity. In this study, we examined regulation of cyclic glucan biosynthesis in vitro with membrane preparations from R. meliloti. The results show that ionic solutes inhibit glucan synthesis, even when they are present at low concentrations (e.g., 10 mM). In contrast, neutral solutes (glucose, sucrose, and the compatible solutes glycine betaine and trehalose) were found to stimulate glucan synthesis in vitro when they were present at high concentrations (e.g., 1 M). Furthermore, high concentrations of these neutral solutes were shown to compensate for the inhibition of glucosyltransferase activity by ionic solutes. Consistent with their ionic character, the compatible solute potassium glutamate and the osmoprotectant choline chloride inhibited glucosyltransferase activity in vitro. The results suggest that intracellular ion concentrations, intracellular osmolarity, and intracellular concentrations of nonionic compatible solutes all act as important determinants of glucosyltransferase activity in vivo. Additional experiments were performed with an ndvA mutant defective for transport of cyclic glucans and an ndvB mutant that produces a C-terminal truncated glucosyltransferase. Cyclic beta-(1,2)-glucan biosynthesis, although reduced, was found to be osmoregulated in both mutants. These results reveal that NdvA and the C terminus of NdvB are not required for osmotic regulation of cyclic beta-(1,2)-glucan biosynthesis.     

Cell-associated glucans of Burkholderia solanacearum and Xanthomonas campestris pv. citri: a new family of periplasmic glucans.

The cell-associated glucans produced by Burkholderia solanacearum and Xanthomonas campestris pv. citri were isolated by trichloroacetic acid treatment and gel permeation chromatography. The compounds obtained were characterized by compositional analysis, matrix-assisted laser desorption ionization mass spectrometry, and high-performance anion-exchange chromatography. B. solanacearum synthesizes only a neutral cyclic glucan containing 13 glucose residues, and X. campestris pv. citri synthesizes a neutral cyclic glucan containing 16 glucose residues. The two glucans were further purified by high-performance anion-exchange chromatography. Methylation analysis revealed that these glucans are linked by 1,2-glycosidic bonds and one 1,6-glycosidic bond. Our 600-MHz homonuclear and 1H-13C heteronuclear nuclear magnetic resonance experiments revealed the presence of a single alpha-1,6-glycosidic linkage, whereas all other glucose residues are beta-1,2 linked. The presence of this single alpha-1,6 linkage, however, induces such structural constraints in these cyclic glucans that all individual glucose residues could be distinguished. The different anomeric proton signals allowed complete sequence-specific assignment of both glucans. The structural characteristics of these glucans contrast with those of the previously described osmoregulated periplasmic glucans.     

Biosynthesis and excretion of cyclic glucans by Rhizobium meliloti 1021.

Cyclic beta-1,2-glucans produced by Agrobacterium and Rhizobium species play an important role in the interaction of these bacteria with plant hosts. In this study, we show that (i) the neutral cyclic glucans are the biosynthetic precursors of anionic cyclic glucans; (ii) the conversion of neutral to anionic glucans is much more rapid and more extensive in exponentially growing cultures than in cultures in the stationary phase, although the latter synthesize large amounts of glucan; and (iii) the excretion of glucan, as well as the total amount synthesized, is strongly influenced by the medium.     

The Brucella abortus cyclic beta-1,2-glucan virulence factor is substituted with O-ester-linked succinyl residues

Brucella periplasmic cyclic beta-1,2-glucan plays an important role during bacterium-host interaction. Nuclear magnetic resonance spectrometry analysis, thin-layer chromatography, and DEAE-Sephadex chromatography were used to characterize Brucella abortus cyclic glucan. In the present study, we report that a fraction of B. abortus cyclic beta-1,2-glucan is substituted with succinyl residues, which confer anionic character on the cyclic beta-1,2-glucan. The oligosaccharide backbone is substituted at C-6 positions with an average of two succinyl residues per glucan molecule. This O-ester-linked succinyl residue is the only substituent of Brucella cyclic glucan. A B. abortus open reading frame (BAB1_1718) homologous to Rhodobacter sphaeroides glucan succinyltransferase (OpgC) was identified as the gene encoding the enzyme responsible for cyclic glucan modification. This gene was named cgm for cyclic glucan modifier and is highly conserved in Brucella melitensis and Brucella suis. Nucleotide sequencing revealed that B. abortus cgm consists of a 1,182-bp open reading frame coding for a predicted membrane protein of 393 amino acid residues (42.7 kDa) 39% identical to Rhodobacter sphaeroides succinyltransferase. cgm null mutants in B. abortus strains 2308 and S19 produced neutral glucans without succinyl residues, confirming the identity of this protein as the cyclic-glucan succinyltransferase enzyme. In this study, we demonstrate that succinyl substituents of cyclic beta-1,2-glucan of B. abortus are necessary for hypo-osmotic adaptation. On the other hand, intracellular multiplication and mouse spleen colonization are not affected in cgm mutants, indicating that cyclic-beta-1,2-glucan succinylation is not required for virulence and suggesting that no low-osmotic stress conditions must be overcome during infection.     

Osmoregulated periplasmic glucans of the free-living photosynthetic bacterium Rhodobacter sphaeroides.

The osmoregulated periplasmic glucans (OPGs) produced by Rhodobacter sphaeroides, a free-living organism, were isolated by trichloracetic acid treatment and gel permeation chromatography. Compounds obtained were characterized by compositional analysis, matrix-assisted laser desorption ionization mass spectrometry and nuclear magnetic resonance. R. sphaeroides predominantly synthesizes a cyclic glucan containing 18 glucose residues that can be substituted by one to seven succinyl esters residues at the C6 position of some of the glucose residues, and by one or two acetyl residues. The glucans were subjected to a mild alkaline treatment in order to remove the succinyl and acetyl substituents, analyzed by MALDI mass spectrometry and purified by high-performance anion-exchange chromatography. Methylation analysis revealed that this glucan is linked by 17 1,2 glycosidic bonds and one 1,6 glycosidic bond. Homonuclear and (1)H/(13)C heteronuclear NMR experiments revealed the presence of a single alpha-1,6 glycosidic linkage, whereas all other glucose residues are beta-1,2 linked. The different anomeric proton signals allowed a complete sequence-specific assignment of the glucan. The structural characteristics of this glucan are very similar to the previously described OPGs of Ralstonia solanacearum and Xanthomonas campestris, except for its different size and the presence of substituents. Therefore, similar OPGs are synthesized by phytopathogenic as well as free-living bacteria, suggesting these compounds are intrinsic components of the Gram-negative bacterial envelope.     

Cellulose synthesis by extracts of Acanthamoeba castellanii during encystment. Stimulation of the incorporation of radioactivity from UDP-(14C)glucose into alkali-soluble and insoluble beta-glucans by glucose 6-phosphate and related compounds.

1. The activity of a particulate enzyme prepared from encysting cells of Acanthamoeba castellanii (Neff), previously shown to catalyze the incorporation of glucose from UDP-[14C]glucose into both alkali-soluble and alkali-insoluble beta-(1 leads to 4) glucans, was stimulated several fold by glucose-6-phosphate and several related compounds. 2. Incorporation was observed when [14C]glucose-6-P was incubated with the particles in the presence of UDP-glucose. The results of product analysis by partial acid hydrolysis indicated that glucose-6-P stimulates the formation of both alkali-soluble and alkali-insoluble beta-(1 leads to 4) glucans from UDP-[14C]glucose and was itself incorporated into an alkali-insoluble beta-(1 leads to 4)glucan. 3. When particles incubated with UDP-[14C]glucose and glucose-6-P were reisolated and then reincubated with unlabeled UDP-glucose and glucose-6-P, a loss of counts from the alkali-soluble fraction was detected along with a corresponding rise in the radioactivity of the alkali-insoluble fraction. This suggests that the alkali-soluble beta-glucan was converted to an alkali-insoluble product and possibly may be an intermediate stage in cellulose synthesis.     

Mechanism of action of cyclic beta-1,2-glucan synthetase from Agrobacterium tumefaciens: competition between cyclization and elongation reactions.

We have examined some aspects of the mechanism of cyclic beta-1,2-glucan synthetase from Agrobacterium tumefaciens (235-kDa protein, gene product of the chvB region). The enzyme produces cyclic beta-1,2-glucans containing 17 to 23 glucose residues from UDP-glucose. In the presence of added cyclic beta-1,2-glucans (> 0.5 mg/ml) (containing 17 to 23 glucose residues), the enzyme instead synthesizes larger cyclic beta-1,2-glucans containing 24 to 30 glucose residues. This is achieved by de novo synthesis and not by disproportion reactions with the added product. This is interpreted as inhibition of the specific cyclization reaction for the synthesis of cyclic beta-1,2-glucans containing 17 to 23 glucose residues but with no concomitant effect on the elongation (polymerization) reaction. Temperature and detergents both affect the distribution of sizes of cyclic beta-1,2-glucans, but glucans containing 24 to 30 glucose residues are not produced. We suggest that the size distribution of cyclic beta-1,2-glucan products depends on competing elongation and cyclization reactions.     

Cyclic glucans produced by Agrobacterium tumefaciens are substituted with sn-1-phosphoglycerol residues

In a previous study (Miller, K.J., Kennedy, E.P. and Reinhold, V.N. (1986) Science 231, 48-51) it was reported that the biosynthesis of periplasmic cyclic beta-1,2-glucans by Agrobacterium tumefaciens is strictly osmoregulated in a pattern closely similar to that found for the membrane-derived oligosaccharides of Escherichia coli (Kennedy, E.P. (1982) Proc. Natl. Acad. Sci. USA 79, 1092-1095). In addition to the well-characterized neutral cyclic glucan, the periplasmic glucans were found to contain an anionic component not previously reported. Biosynthesis of the anionic component is osmotically regulated in a manner indistinguishable from that of the neutral cyclic beta-1,2-glucan. We now find that the anionic component consists of cyclic beta-1,2-glucans substituted with one or more sn-1-phosphoglycerol residues. The presence of sn-1-phosphoglycerol residues represents an additional, striking similarity to the membrane-derived oligosaccharides of E. coli.     

Bacterial cyclic beta-(1,2)-glucan acts in systemic suppression of plant immune responses

Although cyclic glucans have been shown to be important for a number of symbiotic and pathogenic bacterium-plant interactions, their precise roles are unclear. Here, we examined the role of cyclic beta-(1,2)-glucan in the virulence of the black rot pathogen Xanthomonas campestris pv campestris (Xcc). Disruption of the Xcc nodule development B (ndvB) gene, which encodes a glycosyltransferase required for cyclic glucan synthesis, generated a mutant that failed to synthesize extracellular cyclic beta-(1,2)-glucan and was compromised in virulence in the model plants Arabidopsis thaliana and Nicotiana benthamiana. Infection of the mutant bacterium in N. benthamiana was associated with enhanced callose deposition and earlier expression of the PATHOGENESIS-RELATED1 (PR-1) gene. Application of purified cyclic beta-(1,2)-glucan prior to inoculation of the ndvB mutant suppressed the accumulation of callose deposition and the expression of PR-1 in N. benthamiana and restored virulence in both N. benthamiana and Arabidopsis plants. These effects were seen when cyclic glucan and bacteria were applied either to the same or to different leaves. Cyclic beta-(1,2)-glucan-induced systemic suppression was associated with the transport of the molecule throughout the plant. Systemic suppression is a novel counterdefensive strategy that may facilitate pathogen spread in plants and may have important implications for the understanding of plant-pathogen coevolution and for the development of phytoprotection measures.     

Chemical analysis of the hyphal wall of Schizophyllum commune.

1. Purified hyphal wall fragments of Schizophyllum commune are analysed and shown to consist of glucose (67.6%), mannose (3.4%), xylose (0.2%), (N-acetyl)glucosamine (12.5%), amino acids (6.4%) and some lipid material (3.0%). 2. The previously proposed structures of two glucans located at the hyphal wall surface (Wessels et al. (1972) Biochim. Biophys. Acta 273, 346-358) were essentially confirmed using methylation analysis. The mucilaginous glucan consists of 1,3-linked beta-glucan chains with branches of single glucose units attached by beta-1,6 linkages on every third unit, on average, along the chain. The alkali soluble S-glucan is an exclusively 1,3-linked alpha-glucan. 3. The alkali-insoluble R-glucan, occurring in close association with chitin, in the inner wall layer, has been characterised by methylation analysis, X-ray diffraction, enzymatic hydrolysis with purified exo-beta-1,3-glucanase and Smith degradation. It appears to be a highly branched beta-1,3,beta-1,6-glucan and a model of this glucan is proposed. Certain parts of this highly insoluble R-glucan bear a close structural similarity to the mucilaginous glucan present at the outer wall surface and in the medium.     

Periplasmic glucans of Pseudomonas syringae pv. syringae.

We report the initial characterization of glucans present in the periplasmic space of Pseudomonas syringae pv. syringae (strain R32). These compounds were found to be neutral, unsubstituted, and composed solely of glucose. Their size ranges from 6 to 13 glucose units/mol. Linkage studies and nuclear magnetic resonance analyses demonstrated that the glucans are linked by beta-1,2 and beta-1,6 glycosidic bonds. In contrast to the periplasmic glucans found in other plant pathogenic bacteria, the glucans of P. syringae pv. syringae are not cyclic but are highly branched structures. Acetolysis studies demonstrated that the backbone consists of beta-1,2-linked glucose units to which the branches are attached by beta-1,6 linkages. These periplasmic glucans were more abundant when the osmolarity of the growth medium was lower. Thus, P. syringae pv. syringae appears to synthesize periplasmic glucans in response to the osmolarity of the medium. The structural characteristics of these glucans are very similar to the membrane-derived oligosaccharides of Escherichia coli, apart from the neutral character, which contrasts with the highly anionic E. coli membrane-derived oligosaccharides.     

Cyclic beta-glucans of members of the family Rhizobiaceae.

Cyclic beta-glucans are low-molecular-weight cell surface carbohydrates that are found almost exclusively in bacteria of the Rhizobiaceae family. These glucans are major cellular constituents, and under certain culture conditions their levels may reach up to 20% of the total cellular dry weight. In Agrobacterium and Rhizobium species, these molecules contain between 17 and 40 glucose residues linked solely by beta-(1,2) glycosidic bonds. In Bradyrhizobium species, the cyclic beta-glucans are smaller (10 to 13 glucose residues) and contain glucose linked by both beta-(1,6) and beta-(1,3) glycosidic bonds. In some rhizobial strains, the cyclic beta-glucans are unsubstituted, whereas in other rhizobia these molecules may become highly substituted with moieties such as sn-1-phosphoglycerol. To date, two genetic loci specifically associated with cyclic beta-glucan biosynthesis have been identified in Rhizobium (ndvA and ndvB) and Agrobacterium (chvA and chvB) species. Mutants with mutations at these loci have been shown to be impaired in their ability to grow in hypoosmotic media, have numerous alterations in their cell surface properties, and are also impaired in their ability to infect plants. The present review will examine the structure and occurrence of the cyclic beta-glucans in a variety of species of the Rhizobiaceae. The possible functions of these unique molecules in the free-living bacteria as well as during plant infection will be discussed.     

Molecular dynamics simulations of a cyclic-beta-(1-->2) glucan containing an alpha-(1-->6) linkage as a 'molecular alleviator' for the macrocyclic conformational strain

The conformational preferences of a cyclic osmoregulated periplasmic glucan of Ralstonia solanacearum (OPGR), which is composed of 13 glucose units and linked entirely via beta-(1-->2) linkages excluding one alpha-(1-->6) linkage, were characterized by molecular dynamics simulations. Of the three force fields modified for carbohydrates that were applied to select a suitable one for the cyclic glucan, the carbohydrate solution force field (CSFF) was found to most accurately simulate the cyclic molecule. To determine the conformational characteristics of OPGR, we investigated the glycosidic dihedral angle distribution, fluctuation, and the potential energy of the glucan and constructed hypothetical cyclic (CYS13) and linear (LINEAR) glucans. All beta-(1-->2)-glycosidic linkages of OPGR adopted stable conformations, and the dihedral angles fluctuated in this energy region with some flexibility. However, despite the inherent flexibility of the alpha-(1-->6) linkage, the dihedral angles have no transition and are more rigid than that in a linear glucan. CYS13, which consists of only beta-(1-->2) linkages, is somewhat less flexible than other glycans, and one of its linkages adopts a higher energy conformation. In addition, the root-mean-square fluctuation of this linkage is lower than that of other linkages. Furthermore, the potential energy of glucans increases in the order of LINEAR, OPGR, and CYS13. These results provide evidence of the existence of conformational constraints in the cyclic glucan. The alpha-(1-->6)-glycosidic linkage can relieve this constraint more efficiently than the beta-(1-->2) linkage. The conformation of OPGR can reconcile the tendency for individual glycosidic bonds to adopt energetically favorable conformations with the requirement for closure of the macrocyclic ring by losing the inherent flexibility of the alpha-(1-->6)-glycosidic linkage.     

基于pH调控的环β-1,2-葡聚糖合成及结构鉴定

目的:研究pH调控对发酵法生产环β-1,2-葡聚糖的影响,并对pH调控条件下所产环葡聚糖的结构进行解析。方法:以根瘤菌ATCC 1333为研究对象,进行pH调控与不调控的发酵过程分析,并结合乙醇分级沉淀对pH调控后的发酵液中多糖进行分离提纯,经Superose 12层析纯化,利用基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization time of flight mass spectrometry,MALDI-MS)、单糖组成分析、电喷雾串联质谱(electrospray ionization mass spectrometry,ESI-MS),傅里叶红外光谱(fourier transform infrared spectoscopy,FTIR),核磁共振nuclear magnetic resonance,NMR)手段对葡聚糖进行结构鉴定。结果:提出两阶段pH调控发酵策略,生长期pH控制为7.0,产糖期pH控制为5.5,与自然发酵模式相比,环葡聚糖产量增加了52%,细胞浓度增加了102%,并且发酵颜色不再发生褐化现象,更有利于后续环葡聚糖的分离纯化。并确定pH调控对根瘤菌ATCC 1333发酵生产的葡聚糖的结构并无影响,合成的环葡聚糖是以葡萄糖为单体,通过β-1,2糖苷键连接的环葡聚糖,聚合度从17~22,以19为主的环状葡聚糖,无支链结构。结论:pH调控对于发酵生产环β-1,2-葡聚糖的结构无影响,为环β-1,2-葡聚糖的发酵工艺研究提供理论基础,也为研究β-1,2-葡聚糖提供可靠的糖源。     

Effects of Ionic and Osmotic Strength on the Glucosyltransferase of Rhizobium meliloti Responsible for Cyclic β-(1,2)-Glucan Biosynthesis

The cyclic β-(1,2)-glucans of Rhizobium meliloti and Agrobacterium tumefaciens play an important role during hypoosmotic adaptation, and the synthesis of these compounds is osmoregulated. Glucosyltransferase, the enzyme responsible for cyclic β-(1,2)-glucan biosynthesis, is present constitutively, suggesting that osmotic regulation of the biosynthesis of these glucans occurs through modulation of enzyme activity. In this study, we examined regulation of cyclic glucan biosynthesis in vitro with membrane preparations from R. meliloti. The results show that ionic solutes inhibit glucan synthesis, even when they are present at low concentrations (e.g., 10 mM). In contrast, neutral solutes (glucose, sucrose, and the compatible solutes glycine betaine and trehalose) were found to stimulate glucan synthesis in vitro when they were present at high concentrations (e.g., 1 M). Furthermore, high concentrations of these neutral solutes were shown to compensate for the inhibition of glucosyltransferase activity by ionic solutes. Consistent with their ionic character, the compatible solute potassium glutamate and the osmoprotectant choline chloride inhibited glucosyltransferase activity in vitro. The results suggest that intracellular ion concentrations, intracellular osmolarity, and intracellular concentrations of nonionic compatible solutes all act as important determinants of glucosyltransferase activity in vivo. Additional experiments were performed with an ndvA mutant defective for transport of cyclic glucans and an ndvB mutant that produces a C-terminal truncated glucosyltransferase. Cyclic β-(1,2)-glucan biosynthesis, although reduced, was found to be osmoregulated in both mutants. These results reveal that NdvA and the C terminus of NdvB are not required for osmotic regulation of cyclic β-(1,2)-glucan biosynthesis.     

A novel cyclic beta-1,2-glucan mutant of Rhizobium meliloti.

The periplasmic cyclic beta-1,2-glucans produced by bacteria within the Rhizobiaceae family provide functions during hypo-osmotic adaptation and plant infection. In Rhizobium meliloti, these molecules are highly modified with phosphoglycerol and succinyl substituents, and it is possible that the anionic character of these glucans is important for their functions. In the present study, we have used a thin-layer chromatographic screening method to identify a novel R. meliloti mutant specifically blocked in its ability to transfer phosphoglycerol substituents to the cyclic beta-1,2-glucan backbone. Further analysis revealed that the cyclic glucans produced by this mutant contained elevated levels of succinyl substituents. As a result, the overall anionic charge on the cyclic beta-1,2-glucans was found to be similar to that of wild-type cells. Despite this difference in cyclic beta-1,2-glucan structure, the mutant was shown to effectively nodulate alfalfa and to grow as well as wild-type cells in hypo-osmotic media.     

Isolation and purification of Flavobacterium alpha-1,3-glucanase-hydrolyzing, insoluble, sticky glucan of Streptococcus mutans.

Studies were made on the physical and chemical properties of polysaccharides synthesized by cell-free extracts of Streptococcus mutans, Streptococcus sanguis, and Streptococcus sp. and their susceptibilities to dextranases. Among the polysaccharides examined, insoluble glucans were rather resistant to available dextranase preparations, and the insoluble, sticky glucan produced by S. mutans OMZ 176, which could be important in formation of dental plaques, was the most resistant. By enrichment culture of soil specimens, using OMZ 176 glucans as the sole carbon source, an organism was isolated that produced colonies surrounded by a clear lytic zone on opaque agar plates containing the OMZ 176 glucan. The organism was identified as a strain of Flavobacterium and named the Ek-14 bacterium. EK-14 bacterium was grown in Trypticase soy broth, and an enzyme capable of hydrolyzing the OMZ 176 glucan was concentrated from the culture supernatant and purified by negative adsorption on a diethylaminoethyl-cellulose (DE-32) column and gradient elution chromatography with a carboxymethyl-cellulose (CM-32) column. The enzyme was a basic protein with an isoelectric point of pH 8.5 and molecular weight of 65,000. Its optimum pH was 6.3 and its optimal temperature was 42 C. The purified enzyme released 11% of the total glucose residues of the OMZ 176 glucan as reducing sugars and solubilized about half of the substrate glucan. The products were found to be isomaltose, nigerose, and nigerotriose, with some oligosaccharides. The purified enzyme split the alpha-1,3-glucan endolytically and was inactive toward glucans containing alpha-1,6, alpha-1,4, beta-1,3, beta-1,4, and/or beta-1,6 bonds as the main linkages.     

Cell-associated oligosaccharides of Bradyrhizobium spp.

We report the initial characterization of the cell-associated oligosaccharides produced by four Bradyrhizobium strains: Bradyrhizobium japonicum USDA 110, USDA 94, and ATCC 10324 and Bradyrhizobium sp. strain 32H1. The cell-associated oligosaccharides of these strains were found to be composed solely of glucose and were predominantly smaller than the cyclic beta-1,2-glucans produced by Agrobacterium and Rhizobium species. Linkage studies and nuclear magnetic resonance analyses demonstrated that the bradyrhizobial glucans are linked primarily by beta-1,6 and beta-1,3 glycosidic bonds. Thus, the bradyrhizobia appear to synthesize cell-associated oligosaccharides of structural character substantially different from that of the cyclic beta-1,2-glucans produced by Agrobacterium and Rhizobium species.