Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 53
Filter
1.
FEMS Microbiol Lett ; 204(1): 163-7, 2001 Oct 16.
Article in English | MEDLINE | ID: mdl-11682196

ABSTRACT

A conserved domain, called GGDEF (referring to a conserved central sequence pattern), is detected in many procaryotic proteins, often in various combinations with putative sensory-regulatory components. Most sequenced bacterial genomes contain several different GGDEF proteins. The function of this domain has so far not been experimentally shown. Through genetic complementation using genes from three different bacteria encoding proteins with GGDEF domains as the only element in common, we present genetic data indicating (a) that the GGDEF domain is responsible for the diguanylate cyclase activity of these proteins, and (b) that the activity of cellulose synthase in Rhizobium leguminosarum bv. trifolii and Agrobacterium tumefaciens is regulated by cyclic di-GMP as in Acetobacter xylinum.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Cyclic GMP/analogs & derivatives , Phosphorus-Oxygen Lyases/chemistry , Phosphorus-Oxygen Lyases/metabolism , Amino Acid Motifs , Bacterial Proteins/metabolism , Cellulose/metabolism , Cyclic GMP/metabolism , Escherichia coli Proteins , Gene Expression Regulation, Bacterial , Phosphorus-Oxygen Lyases/genetics , Plasmids/genetics , Protein Structure, Tertiary , Repressor Proteins/chemistry , Repressor Proteins/genetics , Repressor Proteins/metabolism , Rhizobium/enzymology , Rhizobium/genetics
2.
Biochemistry ; 40(12): 3420-6, 2001 Mar 27.
Article in English | MEDLINE | ID: mdl-11297407

ABSTRACT

The phosphodiesterase A1 protein of Acetobacter xylinum, AxPDEA1, is a key regulator of bacterial cellulose synthesis. This phosphodiesterase linearizes cyclic bis(3'-->5')diguanylic acid, an allosteric activator of the bacterial cellulose synthase, to the ineffectual pGpG. Here we show that AxPDEA1 contains heme and is regulated by reversible binding of O(2) to the heme. Apo-AxPDEA1 has less than 2% of the phosphodiesterase activity of holo-AxPDEA1, and reconstitution with hemin restores full activity. O(2) regulation is due to deoxyheme being a better activator than oxyheme. AxPDEA1 is homologous to the Escherichia coli direct oxygen sensor protein, EcDos, over its entire length and is homologous to the FixL histidine kinases over only a heme-binding PAS domain. The properties of the heme-binding domain of AxPDEA1 are significantly different from those of other O(2)-responsive heme-based sensors. The rate of AxPDEA1 autoxidation (half-life > 12 h) is the slowest observed so far for this type of heme protein fold. The O(2) affinity of AxPDEA1 (K(d) approximately 10 microM) is comparable to that of EcDos, but the rate constants for O(2) association (k(on) = 6.6 microM(-)(1) s(-)(1)) and dissociation (k(off) = 77 s(-)(1)) are 2000 times higher. Our results illustrate the versatility of signal transduction mechanisms for the heme-PAS class of O(2) sensors and provide the first example of O(2) regulation of a second messenger.


Subject(s)
Cellulose/biosynthesis , Escherichia coli Proteins , Gluconacetobacter xylinus/enzymology , Heme/chemistry , Oxygen/chemistry , Phospholipases A/chemistry , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Binding Sites , Carbon Monoxide/metabolism , Gluconacetobacter xylinus/metabolism , Heme/metabolism , Molecular Sequence Data , Oxygen/metabolism , Phospholipases A/metabolism , Protein Structure, Tertiary , Spectrophotometry , Type III Secretion Systems
3.
J Bacteriol ; 180(17): 4416-25, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9721278

ABSTRACT

Cyclic di-GMP (c-di-GMP) is the specific nucleotide regulator of beta-1,4-glucan (cellulose) synthase in Acetobacter xylinum. The enzymes controlling turnover of c-di-GMP are diguanylate cyclase (DGC), which catalyzes its formation, and phosphodiesterase A (PDEA), which catalyzes its degradation. Following biochemical purification of DGC and PDEA, genes encoding isoforms of these enzymes have been isolated and found to be located on three distinct yet highly homologous operons for cyclic diguanylate, cdg1, cdg2, and cdg3. Within each cdg operon, a pdeA gene lies upstream of a dgc gene. cdg1 contains two additional flanking genes, cdg1a and cdg1d. cdg1a encodes a putative transcriptional activator, similar to AadR of Rhodopseudomonas palustris and FixK proteins of rhizobia. The deduced DGC and PDEA proteins have an identical motif structure of two lengthy domains in their C-terminal regions. These domains are also present in numerous bacterial proteins of undefined function. The N termini of the DGC and PDEA deduced proteins contain putative oxygen-sensing domains, based on similarity to domains on bacterial NifL and FixL proteins, respectively. Genetic disruption analyses demonstrated a physiological hierarchy among the cdg operons, such that cdg1 contributes 80% of cellular DGC and PDEA activities and cdg2 and cdg3 contribute 15 and 5%, respectively. Disruption of dgc genes markedly reduced in vivo cellulose production, demonstrating that c-di-GMP controls this process.


Subject(s)
Cyclic GMP/analogs & derivatives , Cyclic GMP/metabolism , Gluconacetobacter xylinus/metabolism , Isoenzymes/genetics , Operon , Amino Acid Sequence , Base Sequence , Cyclic GMP/genetics , DNA Primers , Isoenzymes/metabolism , Molecular Sequence Data , Open Reading Frames , Oxygen/metabolism , Phosphoric Diester Hydrolases/genetics , Recombination, Genetic , Sequence Homology, Amino Acid
4.
Plant Cell Physiol ; 39(2): 144-52, 1998 Feb.
Article in English | MEDLINE | ID: mdl-9559560

ABSTRACT

A specific and highly potent inhibitor of diguanylate cyclase, the key regulatory enzyme of the cellulose synthesizing apparatus in the bacterium Acetobacter xylinum, was isolated from extracts of etiolated pea shoots (Pisum sativum). The inhibitor has been purified by a multistep procedure, and sufficient amounts of highly purified compound (3-8 mg) for spectral analysis were obtained. The structure of this compound was established as 3-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-galactopyranosyl-(1--> 2)-beta-D-glucuronopyranosyl soyasapogenol B 22-O-alpha-D-glucopyranoside. The structure was elucidated on the basis of susceptibility to various enzymes, chemical and spectral methods, such as GC-MS, FAB-MS, and the following types of 2D-NMR: COSY, ROESY, TOCSEY, HMQC, HMBC analyses. An identical or a very similar compound with identical biological activity was also isolated from A. xylinum, strongly suggesting that at least certain aspects of cellulose synthesis in the bacteria and in higher plants may be regulated in a similar manner. The content of this saponin in etiolated plants was about 0.04 mumol (g fresh tissue)-1.


Subject(s)
Enzyme Inhibitors/chemistry , Gluconacetobacter xylinus/enzymology , Phosphorus-Oxygen Lyases/antagonists & inhibitors , Pisum sativum/chemistry , Saponins/chemistry , Triterpenes/chemistry , Carbohydrate Conformation , Carbohydrate Sequence , Darkness , Enzyme Inhibitors/isolation & purification , Enzyme Inhibitors/pharmacology , Escherichia coli Proteins , Gas Chromatography-Mass Spectrometry , Molecular Sequence Data , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Saponins/isolation & purification , Saponins/pharmacology , Spectrometry, Mass, Fast Atom Bombardment , Triterpenes/isolation & purification , Triterpenes/pharmacology
5.
FEBS Lett ; 416(2): 207-11, 1997 Oct 20.
Article in English | MEDLINE | ID: mdl-9369216

ABSTRACT

A protein which specifically binds cyclic diguanylic acid (c-di-GMP), the reversible allosteric activator of the membrane-bound cellulose synthase system of Acetobacter xylinum, has been identified in membrane preparations of this organism. c-di-GMP binding is of high affinity (KD 20 nM), saturable and reversible. The equilibrium of the reaction is markedly and specifically shifted towards the binding direction by K+. The c-di-GMP binding protein, structurally associated with the cellulose synthase, appears to play a major role in modulating the intracellular concentration of free c-di-GMP and thus may constitute an essential factor in regulating cellulose synthesis in vivo.


Subject(s)
Bacterial Proteins/metabolism , Carrier Proteins/metabolism , Cellulose/biosynthesis , Cyclic GMP/analogs & derivatives , Gluconacetobacter xylinus/metabolism , Allosteric Regulation , Bacterial Proteins/isolation & purification , Carrier Proteins/isolation & purification , Chromatography, Gel , Cyclic GMP/metabolism , Energy Metabolism/drug effects , Enzyme Activation , Ethanolamines/pharmacology , Glucosyltransferases/metabolism , Kinetics , Potassium/pharmacology
6.
Plant Physiol ; 103(2): 307-308, 1993 Oct.
Article in English | MEDLINE | ID: mdl-12231937
7.
Plant Physiol ; 101(1): 187-191, 1993 Jan.
Article in English | MEDLINE | ID: mdl-12231676

ABSTRACT

[beta]-Furfuryl-[beta]-glucoside (FG) has been shown to be a specific endogenous activator of higher plant callose synthase (P. Ohana, D.P. Delmer, G. Volman, J.C. Steffens, D.E. Matthews, M. Benziman [1992] Plant Physiol 98: 708-715). Because glycosides such as FG are usually sequestered in vacuoles, we have proposed that activation of callose synthesis in vivo may involve a change in the compartmentation of FG and Ca2+, resulting in a synergistic activation of callose synthase. The use of suspension-cultured barley (Hordeum bulbosum L.) cells provides evidence that FG is largely sequestered in the vacuole. Furthermore, conditions that lead to induction of callose synthesis in vivo correspondingly lead to elevation of the cytoplasmic concentration of FG. These conditions include the lowering of cytoplasmic pH or elevation of cytoplasmic Ca2+. Oligogalacturonide elicitors have also been reported to cause similar changes in cytoplasmic pH and Ca2+ concentration (Y. Mathieu, A. Kurkdjian, H. Xia, J. Guern, A. Koller, M.D. Spiro, M. O'Neill, P. Albersheim, A. Darvill [1991] The Plant Journal 1: 333-343), and such an elicitor also causes an elevation in cytoplasmic FG coupled with stimulation of callose synthesis. These results support the concept that a relative redistribution of FG between cytoplasm and vacuole may be one of the components of the signal transduction pathway for elicitation of callose synthase in vivo.

8.
Plant Physiol ; 98(2): 708-15, 1992 Feb.
Article in English | MEDLINE | ID: mdl-16668699

ABSTRACT

In a recent paper (P Ohana, DP Delmer, JC Steffens, DE Matthews, R Mayer, M Benziman [1991] J Biol Chem 266: 13472-13475), we described the purification and structural characterization of beta-furfuryl-beta-glucoside (FG), an endogenous activator of plant UDP-glucose:(1-->3)-beta-glucan (callose) synthase. In the present report, we provide evidence that FG specifically stimulates callose synthase. The effects of FG on the kinetic properties of callose synthase were studied, and we ascertained that FG, or at least a very similar compound, is present in other plant systems. Chemically synthesized alpha-furfuryl-beta-glucoside also stimulates callose synthase, exhibiting a slightly higher K(a) of 80 micromolar, compared with 50 micromolar for FG. In addition, we have identified and partially characterized an enzyme that catalyzes the synthesis of FG using beta-furfuryl alcohol and UDP-glucose as substrates. A model for the regulation of callose synthesis in vivo, involving changes in intracellular compartmentation of FG and Ca(2+), is proposed.

9.
Plant Cell ; 3(9): 989-95, 1991 Sep.
Article in English | MEDLINE | ID: mdl-1668373

ABSTRACT

Because numerous attempts to detect an activity for a cellulose synthase in plants have failed, we have taken a different approach toward detecting polypeptides involved in this process. The uniqueness of the structure and function of cyclic diguanylic acid (c-di-GMP) as an activator of the cellulose synthase of the bacterium Acetobacter xylinum makes it an attractive probe to use in a search for a c-di-GMP receptor that might be involved in the process in plants. Direct photolabeling with 32P-c-di-GMP has been used, therefore, to identify in plants two membrane polypeptides of 83 and 48 kD derived from cotton fibers that possess properties consistent with their being components of a c-di-GMP-dependent cellulose synthase. Based upon several criteria, the 48-kD species is proposed to be derived by proteolytic cleavage of the 83-kD polypeptide. Both polypeptides bind c-di-GMP with high affinity and specificity and show antigenic relatedness to the bacterial cellulose synthase, and the N-terminal sequence of the 48-kD polypeptide also indicates relatedness to the bacterial synthase. Ability to detect both cotton fiber polypeptides by photolabeling increases markedly in extracts derived from fibers entering the active phase of secondary wall cellulose synthesis. These results provide a basis for future work aimed at identifying and characterizing genes involved in cellulose synthesis in plants.


Subject(s)
Glucosyltransferases/analysis , Gossypium/enzymology , Membrane Proteins/analysis , Plant Proteins/analysis , Acetobacter/enzymology , Amino Acid Sequence , Cloning, Molecular , Cyclic GMP/analogs & derivatives , Cyclic GMP/metabolism , Glucosyltransferases/genetics , Gossypium/genetics , Gossypium/growth & development , Membrane Proteins/genetics , Membrane Proteins/immunology , Membrane Proteins/metabolism , Plant Proteins/genetics , Plant Proteins/immunology , Plant Proteins/metabolism , Sequence Homology, Nucleic Acid , Substrate Specificity
10.
J Biol Chem ; 266(21): 13742-5, 1991 Jul 25.
Article in English | MEDLINE | ID: mdl-1830307

ABSTRACT

We have recently established the existence of endogenous activators of higher plant UDP-glucose: (1----3)-beta-glucan synthase (Callaghan, T., Ross, P., Weinberger-Ohana, P., and Benziman, M. (1988) Plant Physiol. 86, 1099-1103). Here we report the purification and chemical analysis of the most abundant and specific compound, termed Activator I, isolated from Vigna radiata. This compound was extensively purified by a multistep procedure which yielded 0.1 mg of purified activator/g of fresh tissue. Enzyme digestion, neutral sugar analysis, GC/MS of permethylated derivatives, and NMR analysis of native Activator I indicated that the compound contains a single beta-linked glucosyl residue. High resolution FAB-MS indicated an elemental composition of C11H16O7 (Mr = 260), with a calculated Mr of 98 for the aglycone. 13C, DEPT, and COSY NMR spectra showed that the aglycone molecule is an oxygen heterocycle of 5 carbons, consistent with a structure of beta-furfuryl alcohol. Comparison of IR and GC/EI-MS spectra of authentic beta-furfuryl alcohol with native aglycone confirmed the conclusion that Activator I is beta-furfuryl-beta-glucoside. Chemically synthesized beta-furfuryl-beta-glucoside has identical chemical properties and biological activity when compared with the purified endogenous activator (Ka = 50 microM).


Subject(s)
Furans/pharmacology , Glucosides/pharmacology , Glucosyltransferases/metabolism , Membrane Proteins , Schizosaccharomyces pombe Proteins , Enzyme Activation , Fabaceae/enzymology , Furans/chemical synthesis , Furans/chemistry , Furans/isolation & purification , Glucosides/chemical synthesis , Glucosides/chemistry , Glucosides/isolation & purification , Magnetic Resonance Spectroscopy , Plants, Medicinal
11.
Proc Natl Acad Sci U S A ; 88(12): 5472-6, 1991 Jun 15.
Article in English | MEDLINE | ID: mdl-1647035

ABSTRACT

To comprehend the catalytic and regulatory mechanism of the cyclic diguanylic acid (c-di-GMP)-dependent cellulose synthase of Acetobacter xylinum and its relatedness to similar enzymes in other organisms, the structure of this enzyme was analyzed at the polypeptide level. The enzyme, purified 350-fold by enzyme-product entrapment, contains three major peptides (90, 67, and 54 kDa), which, based on direct photoaffinity and immunochemical labeling and amino acid sequence analysis, are constituents of the native cellulose synthase. Labeling of purified synthase with either [32P]c-di-GMP or [alpha-32P]UDP-glucose indicates that activator- and substrate-specific binding sites are most closely associated with the 67- and 54-kDa peptides, respectively, whereas marginal photolabeling is detected in the 90-kDa peptide. However, antibodies raised against a protein derived from the cellulose synthase structural gene (bcsB) specifically label all three peptides. Further, the N-terminal amino acid sequences determined for the 90- and 67-kDa peptides share a high degree of homology with the amino acid sequence deduced from the gene. We suggest that the structurally related 67- and 54-kDa peptides are fragments proteolytically derived from the 90-kDa peptide encoded by bcsB. The anti-cellulose synthase antibodies crossreact with a similar set of peptides derived from other cellulose-producing microorganisms and plants such as Agrobacterium tumefaciens, Rhizobium leguminosarum, mung bean, peas, barley, and cotton. The occurrence of such cellulose synthase-like structures in plant species suggests that a common enzymatic mechanism for cellulose biogenesis is employed throughout nature.


Subject(s)
Arabidopsis Proteins , Cyclic GMP/analogs & derivatives , Glucosyltransferases/metabolism , Peptides/analysis , Plants/metabolism , Affinity Labels , Amino Acid Sequence , Bacteria/enzymology , Blotting, Western , Cross Reactions , Cyclic GMP/metabolism , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Molecular Sequence Data , Substrate Specificity
12.
Microbiol Rev ; 55(1): 35-58, 1991 Mar.
Article in English | MEDLINE | ID: mdl-2030672

ABSTRACT

The current model of cellulose biogenesis in plants, as well as bacteria, holds that the membranous cellulose synthase complex polymerizes glucose moieties from UDP-Glc into beta-1,4-glucan chains which give rise to rigid crystalline fibrils upon extrusion at the outer surface of the cell. The distinct arrangement and degree of association of the polymerizing enzyme units presumably govern extracellular chain assembly in addition to the pattern and width of cellulose fibril deposition. Most evident for Acetobacter xylinum, polymerization and assembly appear to be tightly coupled. To date, only bacteria have been effectively studied at the biochemical and genetic levels. In A. xylinum, the cellulose synthase, composed of at least two structurally similar but functionally distinct subunits, is subject to a multicomponent regulatory system. Regulation is based on the novel nucleotide cyclic diguanylic acid, a positive allosteric effector, and the regulatory enzymes maintaining its intracellular turnover: diguanylate cyclase and Ca2(+)-sensitive bis-(3',5')-cyclic diguanylic acid (c-di-GMP) phosphodiesterase. Four genes have been isolated from A. xylinum which constitute the operon for cellulose synthesis. The second gene encodes the catalytic subunit of cellulose synthase; the functions of the other three gene products are still unknown. Exclusively an extracellular product, bacterial cellulose appears to fulfill diverse biological roles within the natural habitat, conferring mechanical, chemical, and physiological protection in A. xylinum and Sarcina ventriculi or facilitating cell adhesion during symbiotic or infectious interactions in Rhizobium and Agrobacterium species. A. xylinum is proving to be most amenable for industrial purposes, allowing the unique features of bacterial cellulose to be exploited for novel product applications.


Subject(s)
Bacteria/metabolism , Cellulose/biosynthesis , Bacteria/genetics , Cellulose/chemistry
13.
J Biol Chem ; 265(31): 18933-43, 1990 Nov 05.
Article in English | MEDLINE | ID: mdl-2172238

ABSTRACT

An unusual compound, cyclic-bis(3'----5') diguanylic acid (c-di-GMP or cGpGp), is involved in the regulation of cellulose synthesis in the bacterium Acetobacter xylinum. This cyclic dinucleotide acts as an allosteric, positive effector of cellulose synthase activity in vitro (Ka = 0.31 microM) and is inactivated via degradation by a Ca2(+)-sensitive phosphodiesterase, PDE-A (Km = 0.25 microM). A series of 13 analogs cyclic dimer and trimer nucleotides were synthesized, employing a phosphotriester approach, and tested for the ability to mimick c-di-GMP as activators of cellulose synthase and as substrates for PDE-A. Seven of the synthetic compounds stimulate cellulose synthase activity and all of these activators undergo the Ca2(+)-inhibited degradation reaction. The order of affinities for synthase activators is cGpGp approximately cdGpGp approximately cGp(S)Gp (S-diastereomer) greater than cIpGp greater than cdGpdGp greater than cXpGp greater than cIpIp greater than cGp(S)Gp (R-diastereomer). Three cyclic dinucleotides of negligible affinity for either enzyme are cApAp, cUpUp, and cCpCp. This same order of affinities essentially pertains to the analogs as inhibitors of PDE-A activity, but at least one cyclic dinucleotide, cXpXp, which does not bind to cellulose synthase, is also a substrate for the degradation reaction, demonstrating that although the two enzymes share a similar, high degree of specificity for c-di-GMP, their cyclic dinucleotide binding sites are not identical. Phosphodiester bonds of activators in which an exocyclic oxygen is replaced with an atom of sulfur (cGp(S)Gp isomers) resist the action of PDE-A, and such derivatives may be prototypes for synthetic non-hydrolyzable c-di-GMP analogs.


Subject(s)
Arabidopsis Proteins , Cellulose/biosynthesis , Cyclic GMP/analogs & derivatives , Gluconacetobacter xylinus/metabolism , Allosteric Regulation , Calcium/pharmacology , Cyclic GMP/chemical synthesis , Cyclic GMP/pharmacology , Glucosyltransferases/metabolism , Indicators and Reagents , Structure-Activity Relationship , Uridine Diphosphate Glucose/metabolism
14.
Proc Natl Acad Sci U S A ; 87(20): 8130-4, 1990 Oct.
Article in English | MEDLINE | ID: mdl-2146681

ABSTRACT

An operon encoding four proteins required for bacterial cellulose biosynthesis (bcs) in Acetobacter xylinum was isolated via genetic complementation with strains lacking cellulose synthase activity. Nucleotide sequence analysis indicated that the cellulose synthase operon is 9217 base pairs long and consists of four genes. The four genes--bcsA, bcsB, bcsC, and bcsD--appear to be translationally coupled and transcribed as a polycistronic mRNA with an initiation site 97 bases upstream of the coding region of the first gene (bcsA) in the operon. Results from genetic complementation tests and gene disruption analyses demonstrate that all four genes in the operon are required for maximal bacterial cellulose synthesis in A. xylinum. The calculated molecular masses of the proteins encoded by bcsA, bcsB, bcsC, and bcsD are 84.4, 85.3, 141.0, and 17.3 kDa, respectively. The second gene in the operon (bcsB) encodes the catalytic subunit of cellulose synthase. The functions of the bcsA, bcsC, and bcsD gene products are unknown. Bacterial strains mutated in the bcsA locus were found to be deficient in cellulose synthesis due to the lack of cellulose synthase and diguanylate cyclase activities. Mutants in the bcsC and bcsD genes were impaired in cellulose production in vivo, even though they had the capacity to make all the necessary metabolic precursors and cyclic diguanylic acid, the activator of cellulose synthase, and exhibit cellulose synthase activity in vitro. When the entire operon was present on a multicopy plasmid in the bacterial cell, both cellulose synthase activity and cellulose biosynthesis increased. When the promoter of the cellulose synthase operon was replaced on the chromosome by E. coli tac or lac promoters, cellulose production was reduced in parallel with decreased cellulose synthase activity. These observations suggest that the expression of the bcs operon is rate-limiting for cellulose synthesis in A. xylinum.


Subject(s)
Arabidopsis Proteins , Gluconacetobacter xylinus/genetics , Glucosyltransferases/genetics , Operon , Base Sequence , Cloning, Molecular , Escherichia coli/genetics , Genes, Bacterial , Gluconacetobacter xylinus/enzymology , Glucosyltransferases/isolation & purification , Macromolecular Substances , Molecular Sequence Data , Mutagenesis, Site-Directed , Oligonucleotide Probes , Plasmids , Promoter Regions, Genetic , Restriction Mapping
15.
J Bacteriol ; 171(12): 6649-55, 1989 Dec.
Article in English | MEDLINE | ID: mdl-2556370

ABSTRACT

The occurrence of the novel regulatory nucleotide bis(3',5')-cyclic diguanylic acid (c-di-GMP) and its relation to cellulose biogenesis in the plant pathogen Agrobacterium tumefaciens was studied. c-di-GMP was detected in acid extracts of 32P-labeled cells grown in various media, and an enzyme responsible for its formation from GTP was found to be present in cell-free preparations. Cellulose synthesis in vivo was quantitatively assessed with [14C]glucose as a tracer. The organism produced cellulose during growth in the absence of plant cells, and this capacity was retained in resting cells. Synthesis of a cellulosic product from UDP-glucose in vitro with membrane preparations was markedly stimulated by c-di-GMP and its precursor GTP and was further enhanced by Ca2+. The calcium effect was attributed to inhibition of a c-di-GMP-degrading enzyme shown to be present in the cellulose synthase-containing membranes.


Subject(s)
Arabidopsis Proteins , Cellulose/biosynthesis , Cyclic GMP/analogs & derivatives , Rhizobium/metabolism , Cyclic GMP/metabolism , Glucosyltransferases/metabolism , Guanosine Triphosphate/metabolism , Kinetics , Phosphorus Radioisotopes , Radioisotope Dilution Technique , Rhizobium/growth & development
16.
Mol Gen Genet ; 217(1): 26-30, 1989 May.
Article in English | MEDLINE | ID: mdl-2549367

ABSTRACT

Three cellulose-negative (Cel-) mutants of Acetobacter xylinum strain ATCC 23768 were complemented by a cloned 2.8 kb DNA fragment from the wild type. Biochemical analysis of the mutants showed that they were deficient in the enzyme uridine 5'-diphosphoglucose (UDPG) pyrophosphorylase. The analysis also showed that the mutants could synthesize beta(1-4)-glucan in vitro from UDPG, but not in vivo from glucose. This result was expected, since UDPG is known to be the precursor for cellulose synthesis in A. xylinum. In order to analyze the function of the cloned gene in more detail, its biological activity in Escherichia coli was studied. These experiments showed that the cloned fragment could be used to complement an E. coli mutant deficient in the structural gene for UDPG pyrophosphorylase. It is therefore clear that the cloned fragment must contain this gene from A. xylinum. This is to our knowledge the first example of the cloning of a gene with a known function in cellulose biosynthesis from any organism, and we suggest the gene be designated celA.


Subject(s)
Cloning, Molecular , Genes , Gluconacetobacter xylinus/genetics , Nucleotidyltransferases/genetics , Phosphorus-Oxygen Lyases , UTP-Glucose-1-Phosphate Uridylyltransferase/genetics , Cellulose/biosynthesis , Cellulose/genetics , Conjugation, Genetic , DNA, Bacterial/genetics , Electrophoresis, Agar Gel , Escherichia coli Proteins , Genetic Complementation Test , Gluconacetobacter xylinus/enzymology , Lyases/metabolism , Mutation , Phosphoglucomutase/metabolism , Phosphoric Diester Hydrolases/metabolism , Plasmids
17.
Plant Physiol ; 86(4): 1099-103, 1988 Apr.
Article in English | MEDLINE | ID: mdl-16666038

ABSTRACT

Heat-stable activators of membranous beta-glucan synthase have been isolated from the supernatant fraction of crude mung bean (Vigna radiata) extracts by DEAE-cellulose and silica-gel chromatography. One of the activators has been partially purified and characterized on the basis of susceptibility to various enzymes and by analysis of the products formed upon total acid hydrolysis, alkaline-methanolysis, and beta-glucosidase digestion. This activator has the characteristics of a 1,2-dioleoyl diglyceride containing beta-linked glucose residue(s) at the C-3 position. When expressed per mole of glucosyl residues, the maximal K(a) value of the activator is estimated to be 25 micromolar. Both the intact glucosyl and fatty acid moiety are essential to the stimulatory effect of the activator.

18.
Plant Physiol ; 86(4): 1104-7, 1988 Apr.
Article in English | MEDLINE | ID: mdl-16666039

ABSTRACT

n-Alkyl (C(6)-C(12)) beta-d-monoglucopyranosides have been found to be highly potent activators of mung bean beta-glucan synthase in vitro, increasing the V(max) of the enzyme as much as 60-fold and with K(a) values as low as 10 micromolar. Activation is highly specific for the beta-linked terminal glucose residue; other alkyl glycosides such as, octyl-alpha-glucoside, dodecyl beta-maltoside, 6-lauryl sucrose, 6-lauryl glucose, which lack this structure, are ineffective as activators. Based on the similarities in their structure and effects on beta-glucan synthesis under a variety of conditions, it is proposed that the alkyl beta-glucosides are structural analogs of the native glucolipid activator of beta-glucan synthase isolated from mung bean extracts.

19.
Nature ; 325(6101): 279-81, 1987.
Article in English | MEDLINE | ID: mdl-18990795

ABSTRACT

Cellulose is the most abundant renewable carbon resource on earth and is an indispensable raw material for the wood, paper, and textile industries. A model system to study the mechanism of cellulose biogenesis is the bacterium Acetobacter xylinum which produces pure cellulose as an extracellular product. It was from this organism that in vitro preparations which possessed high levels of cellulose synthase activity were first obtained in both membranous and soluble forms. We recently demonstrated that this activity is subject to a complex multi-component regulatory system, in which the synthase is directly affected by an unusual cyclic nucleotide activator enzymatically formed from GTP, and indirectly by a Ca (2+) -sensitive phosphodiesterase which degrades the activator. The cellulose synthase activator (CSA) has now been identified as bis-(3' 5')-cyclic diguanylic acid (5'G3'p5'G3'p) on the basis of mass spectroscopic data, nuclear magnetic resonance analysis and comparison with chemically synthesized material. We also report here on intermediary steps in the synthesis and degradation of this novel circular dinucleotide, which have been integrated into a model for the regulation of cellulose synthesis.


Subject(s)
Gluconacetobacter xylinus , Glucosyltransferases , Bacteria/metabolism , Cellulose/metabolism , Glucosyltransferases/metabolism
20.
FEBS Lett ; 186(2): 191-6, 1985 Jul 08.
Article in English | MEDLINE | ID: mdl-19160595

ABSTRACT

The mechanism of GTP-specific activation of the membrane-bound cellulose synthase system of Acetobacter xylinum has been further elucidated. The supernatant fraction derived from washed membranes of this organism contains an enzyme which reacts with GTP to form a low molecular mass, heat-stable compound,tentatively characterized as a cyclic oligonuleotide composed of GMP residues, which is the immediate activator of the cellulose synthase. This activation is reversed by a membrane-bound enzyme that degrades the activator; the latter enzyme is inhibited by Ca (2+). It is suggested that the interaction between these enzymes and nucleotide derivatives, mediated by Ca (2+), may regulate cellulose synthesis in VIVO.

SELECTION OF CITATIONS
SEARCH DETAIL
...