Biosynthesis Of Membrane-derived Oligosaccharides Research Articles

Inactivation of mdoH leads to increased expression of colanic acid capsular polysaccharide in Escherichia coli.

Capsule gene (cps) expression, which normally occurs at low levels in Escherichia coli lon+ cells, increased 38-fold in lon+ cells carrying a Tn10::delta kan insertion mapping to 24 min on the E. coli chromosome. Null mutations in rcsA, rcsB, or rcsC abolished the effect of the Tn10::delta kan insertion. Sequencing of both sides of the Tn10::delta kan insertion localized the insertion to the previously reported mdoH gene, which encodes a protein involved in biosynthesis of membrane-derived oligosaccharides (MDOs). A model suggesting that the periplasmic levels of MDOs act to signal RcsC to activate cps expression is proposed.

Domains of Escherichia coli acyl carrier protein important for membrane-derived-oligosaccharide biosynthesis.

Acyl carrier protein participates in a number of biosynthetic pathways in Escherichia coli: fatty acid biosynthesis, phospholipid biosynthesis, lipopolysaccharide biosynthesis, activation of prohemolysin, and membrane-derived oligosaccharide biosynthesis. The first four pathways require the protein's prosthetic group, phosphopantetheine, to assemble an acyl chain or to transfer an acyl group from the thioester linkage to a specific substrate. By contrast, the phosphopantetheine prosthetic group is not required for membrane-derived oligosaccharide biosynthesis, and the function of acyl carrier protein in this biosynthetic scheme is currently unknown. We have combined biochemical and molecular biological approaches to investigate domains of acyl carrier protein that are important for membrane-derived oligosaccharide biosynthesis. Proteolytic removal of the first 6 amino acids from acyl carrier protein or chemical synthesis of a partial peptide encompassing residues 26 to 50 resulted in losses of secondary and tertiary structure and consequent loss of activity in the membrane glucosyltransferase reaction of membrane-derived oligosaccharide biosynthesis. These peptide fragments, however, inhibited the action of intact acyl carrier protein in the enzymatic reaction. This suggests a role for the loop regions of the E. coli acyl carrier protein and the need for at least two regions of the protein for participation in the glucosyltransferase reaction. We have purified acyl carrier protein from eight species of Proteobacteria (including representatives from all four subgroups) and characterized the proteins as active or inhibitory in the membrane glucosyltransferase reaction. The complete or partial amino acid sequences of these acyl carrier proteins were determined. The results of site-directed mutagenesis to change amino acids conserved in active, and altered in inactive, acyl carrier proteins suggest the importance of residues Glu-4, Gln-14, Glu-21, and Asp-51. The first 3 of these residues define a face of acyl carrier protein that includes the beginning of the loop region, residues 16 to 36. Additionally, screening for membrane glucosyltransferase activity in membranes from bacterial species that had acyl carrier proteins that were active with E. coli membranes revealed the presence of glucosyltransferase activity only in the species most closely related to E. coli. Thus, it seems likely that only bacteria from the Proteobacteria subgroup gamma-3 have periplasmic glucans synthesized by the mechanism found in E. coli.

In vivo nuclear magnetic resonance study of the osmoregulation of phosphocholine-substituted beta-1,3;1,6 cyclic glucan and its associated carbon metabolism in Bradyrhizobium japonicum USDA 110.

A phosphocholine-substituted beta-1,3;1,6 cyclic glucan (PCCG), an unusual cyclic oligosaccharide, has been isolated from Bradyrhizobium japonicum USDA 110 (D. B. Rolin, P. E. Pfeffer, S. F. Osman, B. S. Swergold, F. Kappler, and A. J. Benesi, Biochim. Biophys. Acta 1116:215-225, 1992). Data presented here suggest that PCCG synthesis is dependent on the carbon metabolism and that osmotic regulation of its biosynthesis parallels regulation of membrane-derived oligosaccharide biosynthesis observed in Escherichia coli (E. P. Kennedy, M. K. Rumley, H. Schulman, and L. M. G. van Golde, J. Biol. Chem. 251:4208-4213, 1976) and Agrobacterium tumefaciens (G. A. Cangelosi, G. Martinetti, and E. W. Nester, J. Bacteriol. 172:2172-2174, 1990). Growth of B. japonicum USDA 110 cells in the reference medium at relatively low osmotic pressures (LO) (65 mosmol/kg of H2O) caused a large accumulation of PCCG and unsubstituted beta-1,3;1,6 cyclic glucans (CG). Sucrose and polyethylene glycol, nonionic osmotica, reduce all growth rates and inhibit almost completely the production of PCCG at high osmotic pressures (HO) above 650 and 400 mosmol/kg of H2O), respectively. We used in vivo 13C nuclear magnetic resonance spectroscopy to identify the active osmolytes implicated in the osmoregulation process. The level of alpha,alpha-trehalose in B. japonicum cells grown in autoclaved or filter-sterilized solutions remained constant in HO (0.3 M sucrose or 250 g of polyethylene glycol 6000 per liter) medium. Significant amounts of glycogen and extracellular polysaccharides were produced only when glucose was present in the autoclaved HO 0.3 M sucrose media. The results of hypo- and hyperosmotic shocking of B. japonicum USDA 110 cells were monitored by using in vivo 31P and 13C nuclear magnetic resonance spectroscopy. The first observed osmoregulatory response of glycogen-containing cells undergoing hypoosmotic shock was release of P(i) into the medium. Within 7 h, reabsorption of P(i) was complete and production of PCCG was initiated. After 12 h, the PCCG content had increased by a factor of 7. Following the same treatment, cells containing little or no glycogen released trehalose and failed to produce PCCG. Thus the production of PCCG/CG in response to hypoosmotic shocking of stationary-phase cells was found to be directly linked to the interconversion of stored glycogen. Hyperosmotic shocking of LO-grown stationary-phase cells with sucrose had no effect on the content of previously synthesized CG/PCCG. The PCCG/CG content and its osmotically induced biosynthesis are discussed in terms of carbon metabolism and a possible role in hypoosmotic adaptation in B. japonicum USDA 110.

Mechanisms of regulation of the biosynthesis of membrane-derived oligosaccharides in Escherichia coli.

The periplasmic glucans of Gram-negative bacteria, including the membrane-derived oligosaccharides (MDO) of Escherichia coli and the cyclic glucans of the Rhizobiaceae, have important but poorly understood functions in osmotic adaptation and, in the case of the Rhizobiaceae, in the complex cell-signaling of these bacteria with specific plant hosts. Experiments on the mechanisms of osmotic regulation of the biosynthesis of MDO in E. coli reported here support a model in which osmotic regulation occurs principally at the level of modulation of enzyme activity rather than at the level of gene expression. 1) Activity of the membrane-bound glucosyltransferase thought to catalyze the first and rate-making step in the biosynthesis of MDO is not altered by the osmolarity of the medium in which cells are grown. 2) Upon dilution of cells growing at high osmolarity into a medium of low osmolarity, the increased synthesis of MDO begins at maximum rate without detectable lag. 3) The activity of the membrane glucosyltransferase in vitro is strongly inhibited by high levels of salts, consistent with the view that synthesis in vivo is regulated chiefly by this mechanism, rather than by regulation of the synthesis of biosynthetic enzymes. We also find that the biosynthesis of MDO is regulated not only osmotically but also by strong feedback inhibition in response to the levels of MDO in the periplasm.

17] Diacylglycerol kinase from escherichia coli

Publisher Summary Diacylglycerol kinases (DG kinases) catalyze the ATP-dependent phosphorylation of sn -l,2-diacylglycerols. Escherichia coli DG kinase functions to recycle diacylglycerol (DAG), which is generated largely as a by-product of membrane-derived oligosaccharide biosynthesis. Recent observations with mutants defective in the regulation of DG kinase expression suggest that DAG may play a regulatory role in E. coli analogous to its role in eukaryotic signal transduction. Diacylglycerol kinase from E. coli has found use in a radiochemical assay of DAG levels in crude lipid extracts of eukaryotic cells. The structural gene for E. coli DG kinase has been cloned and its DNA sequence determined. Bacterial strains expressing high levels of enzyme activity have been constructed and employed for purifying the enzyme to homogeneity. Sequence analysis of the purified kinase has allowed alignment of the protein with the structural gene. Kinetic analyses indicate that in addition to MgATP and diacylglycerol, E. coli DG kinase requires a second divalent metal cation and a lipid cofactor. Several lines of kinetic evidence suggest that the lipid cofactor induces a conformational change in the enzyme prior to its interaction with DAG, MgATP, and the divalent metal activator.

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.

Lack of inhibition by colicin M suggests bactoprenol independence of MDO biosynthesis

Biosynthesis of membrane-derived oligosaccharides (MDO), located in the periplasmic space of Escherichia coli, was not inhibited by colicin M, an inhibitor of bactoprenyl phosphate regeneration. This result suggests that bactoprenol does not serve as a lipid carrier of MDO oligosaccharides across the cytoplasmic membrane.

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.

Membrane-binding sites for acyl carrier protein in Escherichia coli

We report that membrane vesicles of Escherichia coli contain protein-binding sites for acyl carrier protein. Scatchard analysis of the binding indicates a dissociation constant around 0.35 μM and a maximum number of protein-binding sites around 50 pmol per mg of membrane protein. Binding is on the inner membrane while the outer membrane is devoid of binding sites. These results are consistent with the fact that some acyl carrier protein-dependent enzymes implicated in phospholipid- and membrane-derived oligosaccharide biosynthesis are localized in the cytoplasmic membrane.

Osmotic regulation of biosynthesis of membrane-derived oligosaccharides in Escherichia coli

The osmotic regulation of the biosynthesis of membrane-derived oligosaccharides (MDO) in strains UB1005 and DC2 of Escherichia coli K-12 was examined; this regulation was previously reported by Clark (J. Bacteriol. 161:1049-1053, 1985) to be different from that observed by Kennedy for other strains of E. coli (Proc. Natl. Acad. Sci. USA 79:1092-1095, 1982). Osmotic regulation of the synthesis of MDO in UB1005 and DC2 is in fact indistinguishable from that previously reported for other strains of E. coli, with maximum production of MDO occurring in the medium of lowest osmolarity. The report of Clark to the contrary was apparently based on the inadequate methods for the measurement of MDO employed in that study. MDO are localized in the periplasm of wild-type E. coli cells. However, strain DC2, selected for hypersensitivity to a range of antibiotics, released most of its MDO into the medium, apparently as a result of greater outer membrane permeability.

An essential function for acyl carrier protein in the biosynthesis of membrane-derived oligosaccharides of Escherichia coli.

Membrane-derived oligosaccharides are branched, substituted beta-glucans localized in the periplasmic space of Escherichia coli and other Gram-negative bacteria. The biosynthesis of membrane-derived oligosaccharides and of analogous periplasmic oligosaccharides found in plant bacteria is of particular interest because it is subject to strict osmotic regulation [Miller, K.J., Kennedy, E.P., and Reinhold, V.N. (1986) Science 231, 48-51]. An enzyme system catalyzing the synthesis of the (beta 1-2)-linked glucan backbone of E. coli membrane-derived oligosaccharides from UDP-glucose requires both a membrane component and a cytosolic protein termed transglucosylation factor. The factor has now been purified to apparent homogeneity and has been found to be identical to acyl carrier protein (ACP), the phosphopantetheine-containing protein of low molecular weight that has long been known to be essential for fatty acid synthesis in E. coli and other organisms. Both are small, heat-stable, highly anionic proteins with identical chromatographic and electrophoretic behavior. ACP of the highest purity has an activity in the transglucosylation system indistinguishable from that of the protein independently purified as transglucosylation factor. Antibody raised against pure ACP completely inhibits transglucosylation activity; this inhibition is overcome by titration of the antibody with either ACP or transglucosylation factor. These findings provide evidence for an essential function of ACP unrelated to the biosynthesis of lipid.

Osmotic Adaptation by Gram-Negative Bacteria: Possible Role for Periplasmic Oligosaccharides

The cyclic (1----2)-beta-D-glucans produced by species of Agrobacterium and Rhizobium resemble the membrane-derived oligosaccharides of Escherichia coli in their periplasmic localization, intermediate size, and (1----2)-beta-D-glucan backbones. The regulation of the biosynthesis of cyclic (1----2)-beta-D-glucan by Agrobacterium tumefaciens is now shown to parallel the osmotic regulation of membrane-derived oligosaccharide biosynthesis in Escherichia coli. This result suggests a general role for periplasmic oligosaccharides in the osmotic adaptation of Gram-negative bacteria as ecologically diverse as enteric and soil bacteria.

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.

Biosynthesis of membrane-derived oligosaccharides. Novel glucosyltransferase system from Escherichia coli for the elongation of beta 1—-2-linked polyglucose chains.

Membrane-derived oligosaccharides (MDO) of Escherichia coli are a family of substituted branched oligomers containing 8-12 residues of glucose that are joined by beta 1----2 and beta 1----6 linkages. MDO are localized in the periplasmic space of the cell, and their biosynthesis is regulated by the osmolarity of the medium (Kennedy, E. P. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 1092-1095). We report here the initial characterization of a novel glucosyltransferase system that catalyzes the elongation of beta 1----2-linked polyglucose chains. The system requires: 1) a beta-D-glucoside such as the disaccharide sophorose (2-O-beta-D-glucosyl-glucose) or octyl beta-D-glucoside; 2) a trypsin-sensitive membrane fraction; 3) a heat-stable protein from the soluble fraction; 4) UDP-glucose; and 5) Mg2+ ions. Oligomers containing 6-10 glucose units (about the same size as MDO) that are joined by beta 1----2 linkages are major products of the enzyme system. Mutants in the recently mapped mdoA locus (Bohin, J. -P., and Kennedy, E. P. (1984) J. Bacteriol. 157, 956-957) are blocked in vivo at an early stage of MDO synthesis. It has now been found that mdoA mutants are defective in the membrane component, but not in the heat-stable protein that is required for the in vitro synthesis of beta 1----2-linked glucosyl oligomers. We conclude that the glucosyltransferase system described here has an essential function in the synthesis of MDO in vivo.

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).

Osmotic regulation and the biosynthesis of membrane-derived oligosaccharides in Escherichia coli.

The membrane-derived oligosaccharides (MDO) of Escherichia coli are periplasmic constituents containing 8-10 glucose units in a highly branched structure, linked by beta 1-2 and beta 1-6 bonds [Schneider, J. E. Reinhold, V., Rumley, M. K. & Kennedy, E. P. (1979) J. Biol. Chem. 254, 10135-10138]. The MDO are multiply substituted with sn-1-phosphoglycerol residues (derived from membrane phosphatidylglycerol) and with O-succinyl ester residues and, thus, are high anionic. Experiments in this paper offer evidence that the biosynthesis of MDO is an important aspects of osmoregulation in E. coli. Cells grown in medium of low osmolarity (ca. 50 mosM) synthesize 16 times more MDO than those grown in the same medium with 0.4 M NaCl. In cells grown in medium of low osmolarity, it appears that MDO is the principal source of fixed anion in the periplasmic space and, thus, acts to maintain the high osmotic pressure and Donnan membrane potential of the periplasmic compartment Regulation of MDO synthesis in response to changes in osmolarity of the medium appears to occur at the genetic level because the synthesis of new protein is needed to permit the production of MDO at high rates after shift of cells to medium of low osmolarity.