The biosynthesis of membrane-derived oligosaccharides. A membrane-bound phosphoglycerol transferase

Membrane-derived oligosaccharides, found in the Escherichia coli periplasmic space (Schulman, H., and Kennedy, E. P. (1979) J. Bacteriol. 137, 686-688), are composed of 8-10 units of glucose, the sole sugar, in beta 1 leads to 2 and beta 1 leads to 6 linkages (Schneider, J. E., Reinhold, V., Rumley, M. K., and Kennedy, E. P. (1979) J. Biol. Chem. 254, 10135-10138). Oligosaccharides in this family are variously substituted with succinyl ester residues, as well as with sn-1-phosphoglycerol and phosphoethanolamine, both derived from membrane phospholipids. These negatively charged oligosaccharides may function in cellular osmoregulation since their synthesis is under osmotic control (Kennedy, E. P. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 1092-1095). We now report initial characterization of an enzyme catalyzing transfer of phosphoglycerol residues from phosphatidylglycerol to membrane-derived oligosaccharides or to synthetic beta-glucoside acceptors. The products are sn-1,2-diglyceride and beta-glucoside-6-phosphoglycerol. Localized in the inner membrane, the transferase has a requirement for divalent cations, of which manganese is most effective, and a pH optimum of 8.9 in vitro.     

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.     

Regulation of the synthesis of membrane-derived oligosaccharides in Escherichia coli. Assay of phosphoglycerol transferase I in vivo

Membrane-derived oligosaccharides are periplasmic constituents of Escherchia coli and other Gram-negative bacteria. Oligosaccharides in this family may be variously substituted with O-succinyl ester residues, and with sn-1-phosphoglycerol and phosphoethanolamine residues derived from membrane phospholipids. Membrane-derived oligosaccharides appear to be important in osmoregulation, because their synthesis is under strict control (Kennedy, E.P. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 1092-1095). Maximum rate of synthesis is at very low osmolarity of the medium. Phosphoglycerol residues are transferred from phosphatidylglycerol to membrane-derived oligosaccharides, or to certain beta-glucoside acceptors, in a reaction catalyzed by phosphoglycerol transferase I, an enzyme of the inner membrane (Jackson, B. J., and Kennedy, E.P. (1983) J. Biol. Chem. 258, 2394-2398). We now report that this enzyme catalyzes the transfer of phosphoglycerol residues to arbutin (p-hydroxyphenyl-beta-D-glucoside) added to the medium with Km similar to that observed with the cell-free enzyme. The active site of the enzyme must therefore be on the periplasmic face of the inner membrane. We assayed phosphoglycerol transferase I in vivo and found that it is present and completely active even in cells growing in medium of very high osmolarity, in which the synthesis of membrane-derived oligosaccharides is severely reduced. We conclude that osmotic regulation must occur at the stage of the synthesis of oligosaccharide chains. A study of the kinetics of transfer of phosphoglycerol residues to membrane-derived oligosaccharides in vivo revealed that synthesis of the polyglucose chains must stop abruptly upon transfer of cells from medium of low to high osmolarity, inconsistent with a model postulating simple dilution of some rate-limiting enzyme during growth at the higher osmolarity.     

Transfer of phosphoethanolamine residues from phosphatidylethanolamine to the membrane-derived oligosaccharides of Escherichia coli.

The membrane-derived oligosaccharides (MDO) of Escherichia coli are periplasmic constituents composed of glucose residues linked by beta-1,2 and beta-1,6 glycosidic bonds. MDO are substituted with phosphoglycerol, phosphoethanolamine, and succinic acid moieties. The phosphoglycerol residues present on MDO are derived from phosphatidylglycerol (B. J. Jackson and E. P. Kennedy, J. Biol. Chem. 258:2394-2398, 1983), but evidence as to the source of the phosphoethanolamine residues has been lacking. We now report that phosphatidylethanolamine, exogenously added to intact cells of E. coli, provides a source of phosphoethanolamine residues that are transferred to MDO. The biosynthesis of phosphoethanolamine-labeled MDO is osmotically regulated, with maximum synthesis occurring during growth in medium of low osmolarity.     

Characterization of an Escherichia coli mdoB mutant strain unable to transfer sn-1-phosphoglycerol to membrane-derived oligosaccharides

A procedure for the isolation of mutants affected in components containing glycerol derived from phospholipids yielded two mutant strains that contain membrane-derived oligosaccharides (MDO) devoid of glycerol (Rotering, H., Fiedler, W., Rollinger, W., and Braun, V. (1984) FEMS Microbiol. Lett. 22, 61-68). MDO are found in the periplasmic space of Escherichia coli and other Gram-negative bacteria, and they may comprise up to 7% of the cells dry weight. The biosynthesis of MDO is osmoregulated (Kennedy, E. P. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 1092-1095) and linked to the metabolism of phospholipids (van Golde, L. M. G., Schulman, H., and Kennedy, E. P. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 1368-1372). This leads to substitution of MDO with sn-1-phosphoglycerol and phosphoethanolamine (Kennedy, E. P., Rumley, M. K., Schulman, P., and van Golde, L. M. G. (1976) J. Biol. Chem. 251, 4208-4213). MDO also contain succinate in O-ester linkage. We now report that one mutant strain lacks phosphoglycerol transferase I activity and thus is unable to transfer sn-1-phosphoglycerol residues from phosphatidylglycerol to MDO. The mdoB gene affected in this mutant has been located at 99.2 min on the E. coli chromosome. The ethanolamine content of MDO isolated from the mutant strain is elevated, whereas the number of succinate residues is not affected. The only phenotype of mdoB mutants we found is a dramatic reduction of the diglyceride content observed in dgk mdoB double mutants when the beta-glucoside arbutin is present in the growth medium.     

Biosynthesis of membrane-derived oligosaccharides: characterization of mdoB mutants defective in phosphoglycerol transferase I activity.

Phosphoglycerol transferase I, an enzyme of the inner, cytoplasmic membrane of Escherichia coli, catalyzes the in vitro transfer of phosphoglycerol residues from phosphatidylglycerol to membrane-derived oligosaccharides or to the model substrate arbutin (p-hydroxyphenyl-beta-D-glucoside). The products are a phosphoglycerol diester derivative of membrane-derived oligosaccharides or arbutin, respectively, and sn-1,2-diglyceride (B. J. Jackson and E. P. Kennedy, J. Biol. Chem. 258:2394-2398, 1983). Because this enzyme has its active site on the outer aspect of the inner membrane, it also catalyzes the transfer of phosphoglycerol residues to arbutin added to the medium (J.-P. Bohin and E. P. Kennedy, J. Biol. Chem. 259:8388-8393, 1984). When strains bearing the dgk mutation, which are defective in the enzyme diglyceride kinase, are grown in medium containing arbutin, they accumulate large amounts of sn-1,2-diglyceride, a product of the phosphoglycerol transferase I reaction. Growth is inhibited under these conditions. A further mutation in such a dgk strain, leading to the loss of phosphoglycerol transferase I activity, should result in the phenotype of arbutin resistance. We have exploited this fact to obtain strains with such mutations, designated mdoB, that map near min 99. Such mutants lack detectable phosphoglycerol transferase I activity, cannot transfer phosphoglycerol residues to arbutin in vivo, and synthesize membrane-derived oligosaccharides devoid of phosphoglycerol residues. These findings offer strong genetic support for the function of phosphoglycerol transferase I in membrane-derived oligosaccharide biosynthesis.     

Phosphoglycerol substituents present on the cyclic beta-1,2-glucans of Rhizobium meliloti 1021 are derived from phosphatidylglycerol.

The synthesis of periplasmic cyclic beta-1,2-glucans is a property unique to species of the family Rhizobiaceae. For this reason, it is generally believed that these molecules may play an important role in the plant infection process. In the present study, we determined that the cyclic beta-1,2-glucans produced by Rhizobium meliloti 1021 were predominantly anionic in character and contained both phosphoglycerol and succinic acid substituents. In addition, we demonstrated that phosphatidylglycerol was the source of the phosphoglycerol substituents present on these oligosaccharides and that greater than 60% of the total phospholipid turnover in this organism involved this substitution reaction.     

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.     

Molecular cloning and expression of a locus (mdoA) implicated in the biosynthesis of membrane-derived oligosaccharides in Escherichia coli

Mutants of Escherichia coli defective in the mdoA locus are blocked at an early stage in the biosynthesis of membrane-derived oligosaccharides. The mdoA locus has now been cloned into multicopy plasmids. A 5 kb DNA fragment is necessary to complement mdoA mutations. Cells harbouring the mdoA+ plasmid produced three to four times more MDO than wild-type cells. MDO overproduction did not affect the degree of MDO substitution with sn-1-phosphoglycerol residues. The biosynthesis of MDO remained under osmotic control in overproducing strains.     

Effect of Phosphate Limitation on Synthesis of Periplasmic Cyclic (beta)-(1,2)-Glucans

Rhizobium meliloti and Agrobacterium tumefaciens synthesize periplasmic cyclic (beta)-(1,2)-glucans during adaptation to hypoosmotic environments. It also appears that these glucans provide important functions during the interactions of these bacteria with plant hosts. A large fraction of these glucans may become modified with anionic substituents such as phosphoglycerol or succinic acid; however, the role(s) of these substituents is unknown. In this study, we show that growth of these bacteria in phosphate-limited media leads to a dramatic reduction in the levels of phosphoglycerol substituents present on the periplasmic cyclic (beta)-(1,2)-glucans. Under these growth conditions, R. meliloti 1021 was found to synthesize anionic cyclic (beta)-(1,2)-glucans containing only succinic acid substituents. Similar results were obtained with R. meliloti 7154 (an exoH mutant which lacks the ability to succinylate its high-molecular-weight exopolysaccharide), revealing that succinylation of the cyclic (beta)-(1,2)-glucans is mediated by an enzyme system distinct from that involved in the succinylation of exopolysaccharide. In contrast, when A. tumefaciens C58 was grown in a phosphate-limited medium, it was found to synthesize only neutral cyclic (beta)-(1,2)-glucans.     

Mapping of a locus (mdoA) that affects the biosynthesis of membrane-derived oligosaccharides in Escherichia coli.

Mutants of Escherichia coli defective in the newly discovered mdoA locus are blocked at an early stage in the biosynthesis of membrane-derived oligosaccharides. The mutation has now been mapped and found to be located near 23 min on the E. coli chromosome between putA and pyrC. The mdoA mutants are defective in the membrane-localized component of the glucosyl transferase system described by Weissborn and Kennedy (A. C. Weissborn and E. P. Kennedy, Fed. Proc. 42:2122, 1983).     

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.     

Biosynthesis of membrane-derived oligosaccharides. Membrane-bound glucosyltransferase system from Escherichia coli requires polyprenyl phosphate.

The periplasmic glucans of Gram-negative bacteria, including the membrane-derived oligosaccharides (MDO) of Escherichia coli and the cyclic glucans of the Rhizobiaceae, are now recognized to be a family of closely related substances with important functions in osmotic adaptation and cell signaling. The synthesis of the beta-1,2-glucan backbone of MDO is catalyzed by a membrane-bound glucosyltransferase system previously shown to require UDP-glucose and (surprisingly) acyl carrier protein (Therisod, H., Weissborn, A. C., and Kennedy, E. P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7236-7240). In the present study, no glucan intermediates bound to acyl carrier protein or to UDP could detected. The enzyme system, however, was found to be strongly inhibited by bacitracin and by amphomycin. Because the two antibiotics function by forming specific complexes with polyprenyl phosphates, their inhibitory effect suggests a prenol requirement for MDO biosynthesis. Furthermore, the activity of the glucosyltransferase was greatly stimulated by the addition of polyprenyl phosphates such as decaprenyl-P and dihydroheptaprenyl-P, but not by farnesyl-P. The same membrane preparations carry out the synthesis of polyprenyl-P-glucose, which is also stimulated by added polyprenyl-P, including farnesyl-P, the most active of those tested. Pulse chase experiments, however, indicate that the endogenous pool of polyprenyl-P-glucose cannot be an obligate intermediate in the MDO glucosyltransferase system.     

Synthesis of glycerophosphorylated cyclic beta-(1,2)-glucans by Rhizobium meliloti ndv mutants.

The periplasmic cyclic beta-(1,2)-glucans of Rhizobium spp. are believed to provide functions during hypoosmotic adaptation and legume nodulation. In Rhizobium meliloti, cyclic beta-(1,2)-glucans are synthesized at highest levels when cells are grown at low osmolarity, and a considerable fraction (> or = 35%) of these glucans may become substituted with phosphoglycerol moieties. Thus far, two chromosomally encoded proteins, NdvA and NdvB, have been shown to function during cyclic beta-(1,2)-glucan biosynthesis; however, the precise roles for these proteins remain unclear. In the present study, we show that R. meliloti mutants lacking up to one-third of the downstream region of ndvB synthesize cyclic beta-(1,2)-glucans similar to those produced by wild-type cells with respect to size and phosphoglycerol substituent profile. In contrast, no phosphoglycerol substituents were detected on the cyclic beta-(1,2)-glucans synthesized by an R. meliloti ndvA mutant.     

Identification of UDP-glucose as an intermediate in the biosynthesis of the membrane-derived oligosaccharides of Escherichia coli.

The membrane-derived oligosaccharides of Escherichia coli constitute a closely related family of oligosaccharides containing approximately 9 glucose units variously substituted with sn-glycero-1-phosphate and phosphoethanolamine residues derived from the head groups of membrane phospholipids, and also with succinate in O-ester linkage (Kennedy, E.P., Rumley, M.K., Schulman, H., and van Golder, L.M.G. (1976) J. Biol. Chem. 251, 4208-4213). Studies with mutant strains defective in the synthesis of various nucleoside diphosphate sugars have now revealed that UDP-glucose is an essential intermediate in the biosynthesis of these oligosaccharides. Mutants unable to synthesize UDP-glucose do not contain significant amounts of the membrane-derived oligosaccharides. In contrast, a strain unable to synthesize ADP-glucose, the glucosyl donor for glycogen synthesis in E. coli, contained normal amounts of the membrane-derived oligosaccharides, although with a somewhat different pattern of distribution of the various subspecies. In confirmation of these genetic studies, pulse-label isotope tracer studies have been carried out with glucose of high specific activity, under conditions in which UDP-glucose comprises a large fraction of the total radioactivity in the low molecular weight pool. Subsequent "chase" experiments clearly revealed the conversion of UDP-glucose to the higher molecular weight membrane-derived oligosaccharides.     

Identification of the diacylglycerol kinase structural gene of Rhizobium meliloti 1021.

The cyclic beta-1,2-glucans of Rhizobium may function during legume nodulation. These molecules may become highly substituted with phosphoglycerol moieties from the head group of phosphatidylglycerol; diglyceride is a by-product of this reaction (K. J. Miller, R. S. Gore, and A. J. Benesi, J. Bacteriol. 170:4569-4575, 1988). We recently reported that R. meliloti 1021 produces a diacylglycerol kinase (EC 2.7.1.107) activity that shares several properties with the diacylglycerol kinase enzyme of Escherichia coli (W. P. Hunt, R. S. Gore, K. J. Miller, Appl. Environ. Microbiol. 57:3645-3647, 1991). A primary function of this rhizobial enzyme is to recycle diglyceride generated during cyclic beta-1,2-glucan biosynthesis. In the present study, we report the cloning and initial characterization of a single-copy gene from R. meliloti 1021 that encodes a diacylglycerol kinase homolog; this homolog can complement a diacylglycerol kinase deficient strain of E. coli. The sequence of the rhizobial diacylglycerol kinase gene was predicted to encode a protein of 137 amino acids; this protein shares 32% identity with the E. coli enzyme. Analysis of hydropathy and the potential to form specific secondary structures indicated a common overall structure for the two enzymes. Because diglyceride metabolism and cyclic beta-1,2-glucan biosynthesis are metabolically linked, future studies with diacylglycerol kinase mutants of R. meliloti 1021 should further elucidate the roles of the cyclic beta-1,2-glucans in the Rhizobium-legume symbiosis.     

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.     

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.     

A novel phosphodiesterase from Aspergillus niger and its application to the study of membrane-derived oligosaccharides and other glycerol-containing biopolymers.

A novel phosphodiesterase has been found in commercially available extracts of Aspergillus niger and has been partially purified by fractionation with acetone and chromatography on carboxymethylcellulose. The enzyme attacks glycerophosphodiester bonds with the liberation of free glycerol only. The synthetic substrate glucose 6-phospho-sn-1'(3')-glycerol is hydrolyzed with production of equivalent amounts of free glycerol and glucose 6-phosphate. Similarly, the enzymic hydrolysis of sn-glycero-3-phosphocholine liberates glycerol and phosphocholine. The hydrophilic head groups of membrane phospholipids of Escherichia coli are continuously transferred to a closely related family of oligosaccharides ("membrane-derived oligosaccharides") containing glucose as the sole sugar (van Golde, L. M. G., Schulman, H., and Kennedy, E. P. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 1368--1372). Oligosaccharide A-2 contains sn-1-glycerophosphate residues (derived from phosphatidylglycerol) in phosphodiester linkage. Treatment of this oligosaccharide with the phosphodiesterase led to the liberation of nearly all of the glycerol as free glycerol. Subsequent partial acid hydrolysis of the enzyme-treated oligosaccharide led to the recovery of glucose 6-phosphate in almost quantitative yield. The sn-1-glycerophosphate residues are therefore linked to position 6 of glucose units of the oligosaccharide. The activity of the enzyme is not restricted to glycerophosphodiesterases since it will hydrolyze phosphodiesters containing other polyols such as the synthetically prepared glucose 6-phospho-DL-1'(2'-hydroxy-3'-ethoxy)propane.