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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.

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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.

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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.

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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.

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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.

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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.

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Nervous system dysfunction and hearing loss are part of the clinical picture of hypothyroidism. Several studies on visual evoked potential and two brainstem auditory evoked potential studies have shown abnormalities in this disease that are reversible with treatment. It has been suggested that visual evoked potential and brainstem auditory evoked potential could be useful to evaluate the effects of hypothyroidism on the central nervous system and to monitor the response to treatment. We recorded brainstem auditory evoked potentials in 15 adult hypothyroid patients immediately before treatment. All patients were women, ranging in age from 34 to 82 years. Fourteen also had an audiometric study. In five patients, both tests were repeated 20 to 22 months after treatment. Audiometry showed that hearing loss increased with age, suggesting that hearing loss in these patients could be secondary more to aging than to hypothyroidism. When compared to sex-matched controls of similar ages, our patients showed no statistically significant differences in brainstem auditory evoked potentials before treatment. Brainstem auditory evoked potential values were not modified in the five patients whose tests were repeated after treatment. The normality of these results raises serious objections to the clinical use of brainstem auditory evoked potential for central nervous system evaluation and therapy monitoring in adult hypothyroidism.

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The asparagine-linked oligosaccharide structures of chronic lymphocytic leukemia cells (CLL) were studied by sequential lectin affinity chromatography. Glycopeptides were isolated from a Concanavalin A (ConA)-binding glycoprotein fraction prepared from a soluble CLL extract. This fraction, which contained 1.8% of the proteins present in the soluble extract, greater than 30 polypeptides revealed by SDS-PAGE analysis, and known cell surface antigens such as HLA-DR and p85 glycoprotein, must include a major proportion of the glycoproteins of the CLL cells. Glycopeptides were prepared by digestion with pronase, were separated into four pools (A-D) by Bio-Gel P-6 filtration and were radiolabeled by N-14C-acetylation. Glycopeptide pools C and D (0.45 less than Kd less than 0.77) were 95-100% bound to ConA-Sepharose and 7-12% bound to Lens culinaris (Lens)-Sepharose, but did not interact with any of the other lectins tested, suggesting major amounts of high mannose structures and minor amounts of fucosylated biantennary complex structures with terminal GlcNAc on the Man alpha 1-6 arm. Structures with greater than 3 branches were suggested for pools A and B (0 less than Kd less than 0.44) which were 34-65% unbound to ConA-Sepharose and 37-40% bound to Lens-Sepharose. Analysis of the ConA-unbound glycopeptides on RCA-Agarose before and after acid hydrolysis indicated variable amounts of terminal galactose and sialic acid residues. Major components of pool A were structures with terminal GlcNAc on all branches (22%) and fully sialylated structures (20%). In pool B, 20% of the radioactivity interacted with L-Phaseolus vulgaris agglutinin (PHA)-Agarose and with Ricinus communis (RCA)-Agarose, indicative of a fully galactosylated triantennary structure with branches at C-2 and C-6 of the Man alpha 1-6 arm. Half of the L-PHA-interacting material also bound to Lens-Sepharose, indicative of a core fucose residue. A fully galactosylated biantennary complex structure in pool A (13%) was identified by weak binding to ConA-Sepharose and strong interaction with RCA-Agarose. The presence of the polylactosamine sequence (Gal beta 1-4GlcNAc beta 1-3)n on this structure was suggested by a sialic acid independent interaction with wheat germ agglutinin (WGA)-Agarose. A sialic acid dependent WGA-interaction was observed in the ConA-unbound glycopeptides of pool A (5%). Some of the structures identified in this study may be associated with the malignant CLL phenotype and/or with a distinct stage of B cell differentiation.

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