Descripción: The chvA gene product of Agrobacterium tumefaciens is required for virulence and attachment of bacteria to plant cells. Three chvA mutants were studied. In vivo, they were defective in the synthesis, accumulation, and secretion of beta-(1-2)glucan; however, the 235-kilodalton (kDa) protein known to be involved in the synthesis of beta-(1-2)glucan (A. Zorreguieta and R. Ugalde, J. Bacteriol. 167:947-951, 1986) was present and active in vitro. was present and active in vitro. Two molecular forms of cyclic beta-(1-2)glucan, designated types I and II, were resolved by gel chromatography. Type I beta-(1-2)glucan was substituted with nonglycosidic residues, and type II beta-(1-2)glucan was nonsubstituted. Wild-type cells accumulated type I beta-(1-2)glucan, and chvA mutant cells accumulated mainly type II beta-(1-2)glucan and a small amount of type I beta-(1-2)glucan. Inner membranes of wild-type and chvA mutants formed in vitro type II nonsubstituted beta-(1-2)glucan. A 75-kDa inner membrane protein is proposed to be the chvA gene product. chvA mutant inner membranes had increased levels of 235-kDa protein; partial trypsin digestion patterns suggested that the 235-kDa protein (the gene product of the chvB region) and the gene product of the chvA region form a complex in the inner membrane that is involved in the synthesis, secretion, and modification of beta-(1-2)glucan. All of the defects assigned to the chvA mutation were restored after complementation with plasmid pCD522 containing the entire chvA region.

Descripción: Three wild type strains of Rhizobium fredii, USDA 191, USDA 257 and HH 303, do not synthesize in vivo or in vitro beta(1-3), beta(1-6) cyclic glucans, all strains form in vitro and in vivo cyclic beta(1-2) glucans. Approximately 80% of the recovered R. fredii cellular cyclic beta(1-2) glucans were anionic and the substituent was identified as phosphoglycerol. Inner membranes prepared from these R. fredii strains have a beta(1-2) glucan-intermediate-protein with apparent molecular mass undistinguishable from Agrobacterium tumefaciens beta(1-2) glucan intermediate protein. Studies of the degree of polymerization of the oligosaccharides recovered from the protein-intermediate after short pulse incubations with UDP-14C-glucose suggested that the rate limiting step in the biosynthesis of cyclic glucan is cyclization. Kinetic studies revealed that the K(m) for UDP-glucose was 0.33 mM. No difference was detected between the K(m) for initiation/elongation and cyclization reactions. Nodulation studies of a ndvB R. fredii mutant with Mc Call and Peking soybean cultivars, revealed that beta(1-2) glucans do not seem to be required for normal nodule invasion of these soybean cultivars.

Descripción: Fil:Zorreguieta, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Descripción: Potato (Solanum tuberosum L.) tuber UDP-glucose:protein transglucosylase (UPTG) (EC 2.4.1.112) is involved in the first of a two-step mechanism proposed for protein-bound α-glucan synthesis by catalyzing the covalent attachment of a single glucose residue to an acceptor protein. The resulting glucosylated 38-kilodalton polypeptide would then serve as a primer for enzymic glucan chain elongation during the second step. In the present report, we describe the fast protein liquid chromatography purification of UPTG from a membrane pellet of potato tuber. An apparently close association of UPTG, phosphorylase, and starch synthase was observed under native conditions during different purification steps. Enrichment of a 38-kilodalton polypeptide was found throughout enzyme purification. It is now shown that the purified UPTG, with an apparent molecular mass of 38 kilodaltons, undergoes self-glucosylation in a UDP-glucose- and Mn2+-dependent reaction. Therefore, it is concluded that UPTG is the enzyme and at the same time the priming protein required for the biogenesis of protein-bound α-glucan in potato tuber.

Descripción: A mutant of Rhizobium meliloti that elicited the formation of inactive nodules in alfalfa was found not to form β-(1→2) glucan in vivo or in vitro. It was nonmotile because it lacks flagella. The 235-kilodalton protein which acts as an intermediate in β-(1→2) glucan synthesis was undetectable in the mutant. These properties of the mutant are common to those of chvB mutants of Agrobacterium tumefaciens. Exopolysaccharide formation by the R. meliloti mutant was about double that by the wild type.

Descripción: The polysaccharides from cystocarpic Iridaea undulosa, soluble and insoluble in 2M potassium chloride, Cs and Ci, respectively, were treated with alkali and fractionated by precipitation with increasing concentrations of KCl. They were later separated by ion-exchange chromatography, to yield fractions enriched in an α-(1→6)-glucan, agaroids and carrageenans.

Descripción: In organello starch biosynthesis was studied using intact chloroplasts isolated from spinach leaves (Spinacia oleracea). Immunoblot analysis using a specific antiserum against the mitochondrial adenylate (ADP/ATP) translocator of Neurospora crassa shows the presence of an adenylate translocator protein in the chloroplast envelope membranes, similar to that existing in mitochondria and amyloplasts from cultured cells of sycamore (Acer pseudoplatanus). The double silicone oil layer-filtering centrifugation technique was employed to study the kinetic properties of adenylate transport in the purified chloroplasts; ATP, ADP, AMP, and most importantly ADP-Glc were shown to be recognized by the adenylate translocator. Similar to the situation with sycamore amyloplasts, only ATP and ADP-Glc uptake was inhibited by carboxyatractyloside, an inhibitor of the mitochondrial adenylate translocator. Evidence is presented to show that the ADP-Glc transported into the chloroplast stroma is utilized for starch synthesis catalyzed by starch synthase (ADP-Glc: 1,4-α-d-glucan 4-α-d-glucosyltransferase). The high activity of sucrose synthase producing ADP-Glc observed in the extrachloroplastic fractions suggests that starch biosynthesis in chloroplasts may be coupled with the direct import of ADP-Glc from the cytosol.

Descripción: Although the existence of O-linked oligosaccharide residues in glycoproteins of Plasmodium falciparum has been shown, the existence of N- linked glycoproteins is still a matter of controversy and skepticism. This report demonstrates the unequivocal presence of N-linked glycoproteins in P. falciparum, principally in the ring and young trophozoite stages of the intraerythrocytic cycle. These glycoproteins lose their capacity to bind to concanavalin A-Sepharose after treatment of cultures with tunicamycin under conditions that do not affect protein synthesis. When the glycoproteins were treated with N-Glycanase®, oligosaccharides were released. It was possible to identify an N-linked glycoprotein of >200 kDa in the ring stage and also N-linked glycoproteins in the range of 200-30 kDa in the trophozoite stage. Treatment of trophozoites with 12 μM tunicamycin inhibited differentiation to the schizont stage. To our knowledge, this is the first report in the literature unequivocally showing N-linked glycoproteins in trophozoites of P. falciparum as well as their importance for the differentiation of the intraerythrocytic stages of this parasite.

Descripción: Plants have evolved sophisticated mechanisms to sense and respond to pathogen attacks. Resistance against necrotrophic pathogens generally requires the activation of the jasmonic acid (JA) signaling pathway, whereas the salicylic acid (SA) signaling pathway is mainly activated against biotrophic pathogens. SA can antagonize JA signaling and vice versa. Here, we report that the necrotrophic pathogen Botrytis cinerea exploits this antagonism as a strategy to cause disease development. We show that B. cinerea produces an exopolysaccharide, which acts as an elicitor of the SA pathway. In turn, the SA pathway antagonizes the JA signaling pathway, thereby allowing the fungus to develop its disease in tomato (Solanum lycopersicum). SA-promoted disease development occurs through Nonexpressed Pathogen Related1. We also show that the JA signaling pathway required for tomato resistance against B. cinerea is mediated by the systemin elicitor. These data highlight a new strategy used by B. cinerea to overcome the plant's defense system and to spread within the host. © 2011 American Society of Plant Biologists. All rights reserved.

Descripción: Xanthan is the major exopolysaccharide secreted by Xanthomonas spp. Despite its diverse roles in bacterial pathogenesis of plants, little is known about the real implication of this molecule in Xanthomonas pathogenesis. In this study we show that in contrast to Xanthomonas campestris pv campestris strain 8004 (wild type), the xanthan minus mutant (strain 8397) and the mutant strain 8396, which is producing truncated xanthan, fail to cause disease in both Nicotiana benthamiana and Arabidopsis (Arabidopsis thaliana) plants. In contrast to wild type, 8397 and 8396 strains induce callose deposition in N. benthamiana and Arabidopsis plants. Interestingly, treatment with xanthan but not truncated xanthan, suppresses the accumulation of callose and enhances the susceptibility of both N. benthamiana and Arabidopsis plants to 8397 and 8396 mutant strains. Finally, in concordance, we also show that treatment with an inhibitor of callose deposition previous to infection induces susceptibility to 8397 and 8396 strains. Thus, xanthan suppression effect on callose deposition seems to be important for Xanthomonas infectivity. © 2006 American Society of Plant Biologists.

Descripción: To understand how the Rhizobium leguminosarum rail-raiR quorum-sensing system is regulated, we identified mutants with decreased levels of RaiI-made N-acyl homoserine lactones (AHLs). A LuxR-type regulator, ExpR, is required for raiR expression, and RaiR is required to induce rail. Since raiR (and rail) expression is also reduced in cinI and cinR quorum-sensing mutants, we thought CinI-made AHLs may activate ExpR to induce raiR. However, added CinI-made AHLs did not induce raiR expression in a cinI mutant. The reduced raiR expression in cinI and cinR mutants was due to lack of expression of cinS immediately downstream of cinI. cinS encodes a 67-residue protein, translationally coupled to CinI, and cinS acts downstream of expR for raiR induction. Cloned cinS in R. leguminosarum caused an unusual collapse of colony structure, and this was delayed by mutation of expR. The phenotype looked like a loss of exopolysaccharide (EPS) integrity; mutations in cinI, cinR, cinS, and expR all reduced expression of plyB, encoding an EPS glycanase, and mutation of plyB abolished the effect of cloned cinS on colony morphology. We conclude that CinS and ExpR act to increase PlyB levels, thereby influencing the bacterial surface. CinS is conserved in other rhizobia, including Rhizobium etli; the previously observed effect of cinI and cinR mutations decreasing swarming in that strain is primarily due to a lack of CinS rather than a lack of CinI-made AHL. We conclude that CinS mediates quorum-sensing regulation because it is coregulated with an AHL synthase and demonstrate that its regulatory effects can occur in the absence of AHLs. Copyright © 2009, American Society for Microbiology.