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1.
The biosynthesis of membrane-derived oligosaccharides. A membrane-bound phosphoglycerol transferase 总被引:16,自引:0,他引:16
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. 相似文献
2.
Regulation of the synthesis of membrane-derived oligosaccharides in Escherichia coli. Assay of phosphoglycerol transferase I in vivo 总被引:8,自引:0,他引:8
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. 相似文献
3.
Isolation and characterization of Escherichia coli mutants blocked in production of membrane-derived oligosaccharides.
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We report a new procedure for the facile selection of mutants of Escherichia coli that are blocked in the production of membrane-derived oligosaccharides. Four phenotypic classes were identified, including two with a novel array of characteristics. The mutations mapped to two genetic loci. Mutations in the mdoA region near 23 min are in two distinct genes, only one of which is needed for the membrane-localized glucosyltransferase that catalyzes the synthesis of the beta-1,2-glucan backbone of membrane-derived oligosaccharides. Another set of mutations mapped near 27 min closely linked to osmZ; these appear to be in the galU gene. 相似文献
4.
Membrane-derived oligosaccharides (MDO) consist of branched substituted beta-glucan chains and are present in the periplasmic space of Escherichia coli and other gram-negative bacteria. A procedure for the isolation of mutants defective in MDO synthesis is described. Their phenotype was compared with a mdoA mutant previously identified, and they are mapped in the mdoA region. Mutants lacking MDO showed imparied chemotaxis on tryptone swarm plates, a reduced number of flagella, and an enhanced expression of the OmpC porin. Revertants able to form swarm rings again had regained the ability to synthesize MDO and showed the wild-type porin pattern. A second group of chemotactic revertants were mutated in the ompB gene region involved in osmoregulation, and they were still devoid of MDO. These findings provide evidence for a link between MDO biosynthesis and other functions of E. coli related to its adaptation to the environment. 相似文献
5.
Characterization of an Escherichia coli mdoB mutant strain unable to transfer sn-1-phosphoglycerol to membrane-derived oligosaccharides 总被引:4,自引:0,他引:4
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. 相似文献
6.
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. 相似文献
7.
8.
Osmotic regulation of biosynthesis of membrane-derived oligosaccharides in Escherichia coli. 总被引:4,自引:1,他引:3
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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. 相似文献
9.
J E Schneider V Reinhold M K Rumley E P Kennedy 《The Journal of biological chemistry》1979,254(20):10135-10138
Membrane-derived oligosaccharides are a novel class of glucose-containing oligosaccharides found in the cell envelope of Escherichia coli and other Gram-negative organisms (Schulman, H., AND Kennedy, E.P. (1979) J. Bacteriol. 137, 686-688). Previous work has shown that these oligosaccharides contain sn-1-glycero-P and smaller amounts of phosphoethanolamine, derived from membrane phospholipids, attached to position 6 of certain of the glucose residues. The structure of the parent oligosaccharides (obtained by reduction with borohydride followed by alkaline hydrolysis) has now been studied. The oligosaccharide was permethylated, followed by hydrolysis and conversion of the products to methylated glucitol acetates, which were then analyzed and identified by gas-liquid chromatography and mass spectrometry. The membrane oligosaccharides contain 10 to 12 D-glucopyranoside residues/mol, linked solely by 1 yields 2 and 1 yields 6 bonds. They are highly branched structures, with four nonreducing termini per mol. Glucose units at the branch points are doubly substituted at positions 2 and 6. The low specific rotation of the oligosaccharide (+8.3 degrees) indicates that the glycosidic bonds are predominantly or entirely beta. 相似文献
10.
F Baymann F Zito R Kuras L Minai W Nitschke F A Wollman 《The Journal of biological chemistry》1999,274(33):22957-22967
We have altered the N terminus of cytochrome f by site-directed mutagenesis of the chloroplast petA gene in Chlamydomonas reinhardtii. We have replaced the tyrosine residue, Tyr(32), located immediately downstream of the processing site Ala(29)-Gln(30)-Ala(31) by a proline. Tyr(32) is the N terminus of the mature protein and serves as the sixth axial ligand to the heme iron. This mutant, F32P, accumulated different forms of holocytochrome f and assembled them into the cytochrome b(6)f complex. The strain was able to grow phototrophically. Our results therefore contradict a previous report (Zhou, J., Fernandez-Velasco, J. G., and Malkin, R. (1996) J. Biol. Chem. 271, 1-8) that a mutation, considered to be identical to the mutation described here, prevented cytochrome b(6)f assembly. A comparative functional characterization of F32P with F29L-31L, a site-directed processing mutant in which we had replaced the processing site by a Leu(29)-Gln(30)-Leu(31) sequence (2), revealed that both mutants accumulate high spin cytochrome f, with an unusual orientation of the heme and low spin cytochrome f with an alpha-band peak at 552 nm. Both hemes have significantly lower redox potentials than wild type cytochrome f. We attribute the high spin form to uncleaved pre-holocytochrome f and the low spin form to misprocessed forms of cytochrome f that were cleaved at a position different from the regular Ala(29)-Gln-Ala(31) motif. In contrast to F29L-31L, F32P displayed a small population of functional cytochrome f, presumably cleaved at Ala(29), with characteristics close to those of wild type cytochrome f. The latter form would account for cytochrome b(6)f turnover and photosynthetic electron transfer that sustain phototrophic growth of F32P. 相似文献
11.
In order to purify the glycosyltransferases involved in the assembly of lipid-linked oligosaccharides and to be able to study the acceptor substrate specificity of these enzymes, methods were developed to prepare and purify a variety of lipid-linked oligosaccharides, differing in the structure of the oligosaccharide moiety. Thus, Man9 (GlcNAc)2-pyrophosphoryl-dolichol was prepared by isolation and enzymatic synthesis using porcine pancreatic microsomes, while Glc3Man9(GlcNAc)2-PP-dolichol was isolated from Madin-Darby canine kidney cells. Treatment of these oligosaccharide lipids with a series of selected glycosidases led to the preparation of Man alpha 1,2Man alpha 1,2Man alpha 1,3[Man alpha 1,6(Man alpha 1,3)Man alpha 1,6]Man beta 1,4GlcNAc beta 1,4GlcNAc-PP-dolichol; Man alpha 1,2Man alpha 1,2Man alpha 1,3[Man alpha 1,6]Man beta 1,4GlcNAc beta 1, 4GlcNac-PP-dolichol; and Man alpha 1,6(Man alpha 1,3)Man alpha 1, 6[Man alpha 1,3]Man beta 1,4GlcNAc-beta 1,4GlcNAc-PP-dolichol. The preparation, isolation, and characterization of each of these lipid-linked oligosaccharide substrates are described. 相似文献
12.
Nitrosoguanidine-induced mutants ofAcinetobacter sp. defective in exopolysaccharide biosynthesis did not differ from the parent strain in distinguishing physiological and
biochemical properties, such as requirements for growth factors, utilization of mono- and disaccharides, and resistance to
antibiotics. The genetic relation of parent and mutant strains was shown by 16S rRNA PCR analysis. The comparative study of
parent and mutant strains with respect to resistance to unfavorable environmental factors confirmed our hypothesis thatAcinetobacter sp. exopolysaccharides perform protective functions. Hybridization experiments revealed the conjugal transfer of plasmid
R68.45 fromPseudomonas putida BS228 (R68.45) to mutant but not to the parentAcinetobacter sp. strains. The role of theAcinetobacter sp. exopolysaccharides in providing the genetic stability of this bacterium is discussed. 相似文献
13.
14.
《Experimental mycology》1990,14(3):227-233
Most of the fucosyl transferase activity fromMucor rouxii was detected in a crude membrane fraction. The enzyme transferredl-fucose from GDP-fucose to endogenous and exogenous acceptors. When crude membrane fractions were treated with neutral detergents such as Trition X-100 or Brij 36 T enzyme activity became dependent on exogenous acceptors such as mucoric acid or mucoran. Brij-treated membrane fractions showed maximum fucosyl transferase activity at pH 6.5, and at a temperature between 22 and 28°C. The cations Mn2+, Mg2+, Co2+, Zn2+, Fe2+, and Ca2+ activated the enzyme about twofold. The former was slightly more stimulatory at 4 mM. Km for GDP-fucose was 10 μM. Evidence was obtained that mucoric acid serves as acceptor for fucosyl moieties. Acid hydrolysis of the product synthesized from GDP-fuc by Brij-treated membrane fractions revealed fucose as the major radioactive sugar. 相似文献
15.
Isolation and characterization of Saccharomyces cerevisiae mutants defective in glycerol catabolism.
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Mutants of the yeast Saccharomyces cerevisiae that are defective in the catabolism of glycerol were isolated, and two types of mutants were obtained. One type was deficient in glycerol kinase activity, whereas the other type was deficient in sn-glycerol 3-phosphate dehydrogenase activity. Genetic analysis indicated that each mutant strain owed its phenotype to a single nuclear mutation, and that the two mutations were complementary. The mutations were not linked to each other or to any of 10 loci tested. In addition, neither mutation was centromere linked. Possible mechanisms for the regulation of these enzymes were tested by growing the parental strain in the presence of various carbon sources. 相似文献
16.
17.
The yeastSaccharomyces cerevisiae X2180-1A (wild) and its mutants X2180-1A-4 (mnn 1) and X2180-1A-5 (mnn 2) defective in mannan biosynthesis were used as enzyme sources to catalyzein vitro mannosyl transfer from GDP-[14C-U]-mannose to endogenous glycoproteins as well as to exogenous, low-molecular weight acceptors. While the enzyme preparation
from the wild strain exhibited all mannosyl transferase activities involved in mannan biosynthesis by catalyzing the synthesis
of characteristic mannoprotein, the enzyme frommnn 1 mutant failed to catalyze the synthesis of α(1→3) mannoside linkages both with endogenous as well as with exogenous acceptors.
The enzyme preparation from themnn 2 mutant catalyzed the formation of mannoprotein very similar to that obtained with the enzyme from the wild strain. The
most important difference was the formation of a higher number of unsubstituted mannosyl units in the α(1→ 6) linked mannan
backbone. The observed results support the hypothesis that in themnn 1 the mutation has altered the structural gene involved in biosynthesis of an α(1→3) mannosyl transferase catalyzing the addition
of α(1→3) linked mannosyl units to α(1→2) linked mannotrioses in the polysaccharide side chains and in the oligosaccharides
attached to serine and/or threonine in the protein part of mannan molecule. Themnn 2 mutant represents most probably a kind of regulatory mutation where the activity of an α(1→2) mannosyl transferase adding
the mannosyl units directly to α(1→6) linked backbone in the outer region of polysacoharide part of yeast mannan is repressedin vivo but becomes significantin vitro. 相似文献
18.
《Journal of Fermentation and Bioengineering》1995,79(1):6-10
Two mutants defective in salt tolerance were identified among hygromycin B (HygB)-resistant mutants of Zygosaccharomyces rouxii. These mutants showed different phenotypes in terms of sensitivity towards high concentrations of glucose and KCl. Recovery of salt tolerance by the addition of KCl and CaCl2 or by lowering pH (pH 4.0) was different for the two mutants. Moreover, both mutants showed lowered plasma membrane (PM-) ATPase activity and proton pumpout activity. They exhibited neither growth nor proton pumpout activity in a medium containing 5% NaCl. The proton pumpout activity was inhibited by vanadate, an inhibitor of PM-ATPase, only when cells were incubated in the presence of more than 1% NaCl. Damage of the proton pumpout activity seems to be the reason for the salt sensitivity of both mutants. We showed that it was essential for Z. rouxii cells to pump out protons under a high salt environment using mutants defective in this ability. 相似文献
19.
Salmonella typhimurium mutants defective in acetohydroxy acid synthases I and II. 总被引:16,自引:11,他引:5
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An analysis of transposon-induced mutants shows that Salmonella typhimurium possesses two major isozymes of acetohydroxy acid synthase, the enzymes which mediate the first common step in isoleucine and valine biosynthesis. A third (minor) acetohydroxy acid synthase is present, but its significance in isoleucine and valine synthesis may be negligible. Mutants defective in acetohydroxy acid synthase II (ilvG::Tn10) require isoleucine, alpha-ketobutyrate, or threonine for growth, a mutant defective in acetohydroxy acid synthase I (ilvB::Tn5) is a prototroph, and a double mutant (ilvG::Tn10 ilvB::Tn5) requires isoleucine plus valine for growth. 相似文献
20.
Guanylate cyclase activity in Escherichia coli mutants defective in adenylate cyclase. 总被引:3,自引:2,他引:1
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Guanylate cyclase, which catalyzes the synthesis of guanosine 3',5'-monophosphate, has been assayed in several strains of Escherichia coli. They include wild-type cells and mutants defective in adenylate cyclase, which is responsible for the synthesis of adenosine 3',5'-phosphate. Our results demonstrate that adenylate cyclase and guanylate cyclase are two different enzymes in E. coli and suggest that the gene that encodes adenylate cyclase also plays a regulatory role in the synthesis of guanylate cyclase. 相似文献