Glucan Biosynthesis Research Articles

PCR-Amplification and Cloning of the Coding Region of a cDNA for a Reversibly Glycosylated Polypeptide from Rice with Possible Involvement in the Biosynthesis of Glucans

In an attempt to identify genes involved in glucan synthesis, we have amplified the coding region of a clone from a cDNA library prepared from rice inflorescence at the post-fertilization stage. This represents a single copy gene in the rice genome encoding a polypeptide of 41.3 kD, possibly involved in the synthesis of β-1,4 glucan linkages. The transcripts for the gene are more abundant in developing seeds of millets and stay for a longer period during temporal development as compared to rice. The targeted expression of the gene in the endosperm tissue of cereals like rice can help improve glucan content and nutrient status.

Detection of two loci involved in (1-->3)-beta-glucan (curdlan) biosynthesis by Agrobacterium sp. ATCC31749, and comparative sequence analysis of the putative curdlan synthase gene.

Genes essential for the production of a linear, bacterial (1-->3)-beta-glucan, curdlan, have been cloned for the first time from Agrobacterium sp. ATCC31749. The genes occurred in two, nonoverlapping, genomic fragments that complemented different sets of curdlan( crd )-deficient transposon-insertion mutations. These were detected as colonies that failed to stain with aniline blue, a (1-->3)-beta-glucan specific dye. One fragment carried a biosynthetic gene cluster (locus I) containing the putative curdlan synthase gene, crdS, and at least two other crd genes. The second fragment may contain only a single crd gene (locus II). Determination of the DNA sequence adjacent to several locus I mutations revealed homology to known sequences only in the cases of crdS mutations. Complete sequencing of the 1623 bp crdS gene revealed highest similarities between the predicted CrdS protein (540 amino acids) and glycosyl transferases with repetitive action patterns. These include bacterial cellulose synthases (and their homologs), which form (1-->4)-beta-glucans. No similarity was detected with putative (1-->3)-beta-glucan synthases from yeasts and filamentous fungi. Whatever the determinants of the linkage specificity of these beta-glucan synthases might be, these results raise the possibility that (1-->3)-beta-glucans and (1-->4)-beta-glucans are formed by related catalytic polypeptides.

Effects of Ionic and Osmotic Strength on the Glucosyltransferase of Rhizobium meliloti Responsible for Cyclic beta-(1,2)-Glucan Biosynthesis.

The cyclic beta-(1,2)-glucans of Rhizobium meliloti and Agrobacterium tumefaciens play an important role during hypoosmotic adaptation, and the synthesis of these compounds is osmoregulated. Glucosyltransferase, the enzyme responsible for cyclic beta-(1,2)-glucan biosynthesis, is present constitutively, suggesting that osmotic regulation of the biosynthesis of these glucans occurs through modulation of enzyme activity. In this study, we examined regulation of cyclic glucan biosynthesis in vitro with membrane preparations from R. meliloti. The results show that ionic solutes inhibit glucan synthesis, even when they are present at low concentrations (e.g., 10 mM). In contrast, neutral solutes (glucose, sucrose, and the compatible solutes glycine betaine and trehalose) were found to stimulate glucan synthesis in vitro when they were present at high concentrations (e.g., 1 M). Furthermore, high concentrations of these neutral solutes were shown to compensate for the inhibition of glucosyltransferase activity by ionic solutes. Consistent with their ionic character, the compatible solute potassium glutamate and the osmoprotectant choline chloride inhibited glucosyltransferase activity in vitro. The results suggest that intracellular ion concentrations, intracellular osmolarity, and intracellular concentrations of nonionic compatible solutes all act as important determinants of glucosyltransferase activity in vivo. Additional experiments were performed with an ndvA mutant defective for transport of cyclic glucans and an ndvB mutant that produces a C-terminal truncated glucosyltransferase. Cyclic beta-(1,2)-glucan biosynthesis, although reduced, was found to be osmoregulated in both mutants. These results reveal that NdvA and the C terminus of NdvB are not required for osmotic regulation of cyclic beta-(1,2)-glucan biosynthesis.

Molecular analysis of cellulose biosynthesis in Arabidopsis.

Cellulose, an abundant, crystalline polysaccharide, is central to plant morphogenesis and to many industries. Chemical and ultrastructural analyses together with map-based cloning indicate that the RSW1 locus of Arabidopsis encodes the catalytic subunit of cellulose synthase. The cloned gene complements the rsw1 mutant whose temperature-sensitive allele is changed in one amino acid. The mutant allele causes a specific reduction in cellulose synthesis, accumulation of noncrystalline beta-1,4-glucan, disassembly of cellulose synthase, and widespread morphological abnormalities. Microfibril crystallization may require proper assembly of the RSW1 gene product into synthase complexes whereas glucan biosynthesis per se does not.

Characterization of a lipopeptide-resistant strain ofCandida albicans

Lipopeptides are antifungal agents that inhibit cell wall beta-(1,3)-glucan biosynthesis in fungal organisms. A mutant resistant to lipopeptides was generated by UV mutagenesis and characterized. The Candida albicans mutant (LP3-1) was stable and showed resistance specificity to a broad range of lipopeptides and certain glycolipid inhibitors. Other antifungal agents with diverse modes of action had a normal minimum inhibitory concentration profile for LP3-1 compared with the wild-type strain (CCH 442). In the in vitro beta-(1,3)-glucan synthase assay, both the lipopeptides and papulacandin-related agents had considerably higher 50% inhibitory concentration values in the LP3-1 strain than in the wild-type strain. In reconstitution assays, the resistance factor was associated with the integral membrane pellet rather than the peripheral GTP-binding protein. The LP3-1 strain had a membrane lipid profile similar to that of the parent strain and was virulent in a murine model of systemic candidiasis. Taken together, these results indicate that the resistance factor is associated with the integral membrane component of beta-(1,3)-glucan synthase. Lipopeptides are common antifungal agents encountered during screening of natural products. The LP3-1 strain was resistant to natural product extracts known to contain various lipopeptides. Thus, LP3-1 can be used in a dereplication assay.

Localization of Cytoplasmic Water-Soluble Reserve (1->3)-beta-Glucans in Achlya with Immunostaining

The cytoplasmic water-soluble reserve (1→3)-β-glucans of Achlya are of two types: a small neutral and a large phosphorylated. These glucans have been localized using indirect immunolabelling with a commercial polyclonal antibody specific for(1→3)-β-D-glucopyranose linkages. The glucans were localized in large vesicles in the organelle-rich areas of the hypha, and in the large central vacuole in more mature areas. Preabsorption of the antibody with either purified neutral or phosphoglucan completely eliminated subsequent labelling of hyphal sections, indicating that this antibody can also be used to identify phosphorylated (1→3)-β-glucans. No labelling of the large population of apical vesicles was found suggesting that these reserve glucans are not directly involved in apical growth. Since the labelling occurred in association with vesicles and the central vacuole in the mature areas of the hypha and no other cytoplasmic sites showed conjugation, the vesicle and vacuole membranes probably contain the glucan synthase responsible for the biosynthesis of the reserve glucans. The hyphal walls, known to contain a large amount of insoluble (1→3)-β-glucans, were labelled as expected.

Localization of cytoplasmic water-soluble reserve (1→3)-β-glucans in Achlya with immunostaining

The cytoplasmic water-soluble reserve (1→3)-β-glucans of Achlya are of two types: a small neutral and a large phosphorylated. These glucans have been localized using indirect immunolabelling with a commercial polyclonal antibody specific for(1→3)-β-D-glucopyranose linkages. The glucans were localized in large vesicles in the organelle-rich areas of the hypha, and in the large central vacuole in more mature areas. Preabsorption of the antibody with either purified neutral or phosphoglucan completely eliminated subsequent labelling of hyphal sections, indicating that this antibody can also be used to identify phosphorylated (1→3)-β-glucans. No labelling of the large population of apical vesicles was found suggesting that these reserve glucans are not directly involved in apical growth. Since the labelling occurred in association with vesicles and the central vacuole in the mature areas of the hypha and no other cytoplasmic sites showed conjugation, the vesicle and vacuole membranes probably contain the glucan synthase responsible for the biosynthesis of the reserve glucans. The hyphal walls, known to contain a large amount of insoluble (1→3)-β-glucans, were labelled as expected.

Osmoadaptation by rhizosphere bacteria.

The osmolality of rhizosphere soil water is expected to be elevated in relation to bulk-soil water osmolality as a result of the exclusion of solutes by plant roots during water uptake, the release of plant root exudates, and the production of exopolymers by plant roots and rhizobacteria. In contrast, the osmolality of water within highly hydrated bulk soil is low (less than 50 Osm/kg); thus the ability to adapt to elevated osmolality is likely to be important for successful rhizosphere colonization by rhizobacteria. The present review focuses on the osmoadaptive responses of three gram-negative rhizobacterial genera: Rhizobium, Azospirillum, and Pseudomonas. Specifically, we examine the compatible solutes and osmoprotectants utilized by various species within these genera. The adaptation of rhizobacteria to hypoosmotic environments is also examined in the present review. In particular, we focus on the biosynthesis and accumulation of periplasmic glucans by rhizobacteria. Finally, the relationship between rhizobacterial osmoadaptation and selected plant-microbe interactions is considered.

Identification of the aminocatechol A-3253 as an in vitro poison of DNA topoisomerase I from Candida albicans.

The aminocatechol A-3253 is active against several pathogenic fungi, including Candida albicans, Cryptococcus albidus, and Aspergillus niger. A-3253 interferes with both the in vitro biosynthesis of (1,3)-beta-glucan and the activity of topoisomerases I isolated from Candida spp. It is likely that one or more of the enzymes involved in glucan biosynthesis rather than topoisomerase I is the primary intracellular target of A-3253, since a strain of Saccharomyces cerevisiae lacking topoisomerase I is as susceptible to A-3253 as cells containing wild-type levels of topoisomerase I. However, the interaction of A-3253 with topoisomerase I in vitro is of interest since the Candida topoisomerase is more susceptible to A-3253 than is the topoisomerase I isolated from human HeLa cells. A-3253 is both a reversible inhibitor of topoisomerase I catalysis and a reversible poison of topoisomerase I, and in both reactions the fungal topoisomerase I is more susceptible than the human topoisomerase I to A-3253. In contrast, an earlier study found that the human topoisomerase I is more susceptible than the fungal topoisomerase to camptothecin (J. M. Fostel, D. A. Montgomery, and L. L. Shen, Antimicrob. Agents Chemother. 36:2131-2138, 1992). Taken together with the response to camptothecin, the greater susceptibility of the Candida topoisomerase I to A-3253 suggests that there are structural differences between the human and fungal type I topoisomerases which can likely be exploited to allow for the development of antifungal agents which act against the fungal topoisomerase and which have minimal activity against the human enzyme.

Synthesis of glycerophosphorylated cyclic beta-(1,2)-glucans by Rhizobium meliloti ndv mutants.

The periplasmic cyclic beta-(1,2)-glucans of Rhizobium spp. are believed to provide functions during hypoosmotic adaptation and legume nodulation. In Rhizobium meliloti, cyclic beta-(1,2)-glucans are synthesized at highest levels when cells are grown at low osmolarity, and a considerable fraction (> or = 35%) of these glucans may become substituted with phosphoglycerol moieties. Thus far, two chromosomally encoded proteins, NdvA and NdvB, have been shown to function during cyclic beta-(1,2)-glucan biosynthesis; however, the precise roles for these proteins remain unclear. In the present study, we show that R. meliloti mutants lacking up to one-third of the downstream region of ndvB synthesize cyclic beta-(1,2)-glucans similar to those produced by wild-type cells with respect to size and phosphoglycerol substituent profile. In contrast, no phosphoglycerol substituents were detected on the cyclic beta-(1,2)-glucans synthesized by an R. meliloti ndvA mutant.

Identification and cloning of a cyclic beta-(1-->3), beta-(1-->6)-D-glucan synthesis locus from Bradyrhizobium japonicum.

A cosmid clone complementing a cyclic beta-(1-->2)-glucan biosynthesis (ndvB) mutant of Rhizobium meliloti was isolated from a Bradyrhizobium japonicum gene library. This clone specified synthesis of beta-(1-->3), beta-(1-->6)-linked glucans in R. meliloti. The complemented strain was osmotically tolerant and symbiotically competent on alfalfa. Thus, beta-(1-->3), beta-(1-->6)-glucans can substitute functionally for beta-(1-->2)-glucans in R. meliloti.

A simple method for cloning genes involved in glucan biosynthesis: isolation of structural and regulatory genes for glycogen synthesis in Escherichia coli

A simple and widely applicable method for cloning genes involved in glucan biosynthesis is described. An Escherichia coli genomic library was prepared in the low-copy plasmid, pLG339, and E. coli transformants from this library were screened by staining with iodine vapor. Colonies that stained darker than the control were isolated and characterized. The three classes of clones that were identified included: ( i) plasmids encoding E. coli glycogen biosynthetic ( glg) structural genes, ( ii) clones that resulted in elevated glycogen levels, but did not encode glg structural genes or enhance the level of the first enzyme of the pathway, ADPglucose pyrophosphorylase (AGPP), and ( iii) clones that enhanced the level of AGPP, but did not encode this enzyme. Two clones from the latter class also enhanced glgC'-' lacZ-encoded β-galactosidase activity, and may encode factors that regulate the expression of glg structural genes. It should be possible to readily clone glycogen biosynthetic genes from other bacterial species via this method. The method could be made specific for a desired glg gene by using a recipient strain that is defective in the gene of interest.

Biosynthesis and excretion of cyclic glucans by Rhizobium meliloti 1021

Cyclic beta-1,2-glucans produced by Agrobacterium and Rhizobium species play an important role in the interaction of these bacteria with plant hosts. In this study, we show that (i) the neutral cyclic glucans are the biosynthetic precursors of anionic cyclic glucans; (ii) the conversion of neutral to anionic glucans is much more rapid and more extensive in exponentially growing cultures than in cultures in the stationary phase, although the latter synthesize large amounts of glucan; and (iii) the excretion of glucan, as well as the total amount synthesized, is strongly influenced by the medium.

Microbial epimerization of 1-deoxynojirimycin to 1-deoxymannojirimycin.

We attempted to epimerize 1-deoxynojirimycin to produce 1-deoxymannojirimycin, a potent mannosidase inhibitor which blocks conversion of high-mannose to complex saccharide on N-asparagine linked glucan biosynthesis. The data reported describes the microbial conversion of 1-deoxynojirimycin to the manno-form and its glycosidase inhibitory activity

Expression of nodule-specific genes in alfalfa root nodules blocked at an early stage of development.

To help dissect the molecular basis of the Rhizobium-legume symbiosis, we used in vitro translation and Northern blot analysis of nodule RNA to examine alfalfa-specific genes (nodulins) expressed in two types of developmentally defective root nodules elicited by Rhizobium meliloti. Fix- nodules were elicited by R. meliloti nif mutants; these nodules were invaded by rhizobia and contained differentiated bacteroids. 'Empty' nodules were elicited by R. meliloti exo and ndv mutants and by Agrobacterium tumefaciens strains carrying the R. meliloti nod genes; these nodules contained a nodule meristem but lacked infection threads, intracellular bacteria, and bacteroids. Fix- nodules contained a spectrum of nodulins similar to wild-type nodules. In contrast, only two nodulins, Nms-30 and a nodulin homologous to ENOD2 of soybean, were detected in empty nodules. Although R. meliloti ndv and exo mutants elicited nodules with the same defective phenotype, ndv and exo mutants (except for exoC mutants) had distinct biochemical phenotypes. R. meliloti ndvA and ndvB mutants were deficient in cyclic glucan production but not the acidic exopolysaccharide; the converse was true for exoA, exoB, and exoF mutants. exoC mutants were defective in both exopolysaccharide and cyclic glucan biosynthesis. Our results support the model that the R. meliloti nod genes produce a signal that results in nodule meristem induction. Both the exopolysaccharide and cyclic glucan, however, appear to act at the next step in the developmental process and are involved in the production of a signal (or structure) that allows infection thread formation and invasion of the nodule.

Managing fungal and viral infection in the immunocompromised host.

The increasing number of opportunistic fungal infections in neutropenic cancer patients has become of major concern. Postmortem examinations have shown that more than half of the deaths attributed to infection were due to disseminated fungal infections. Candida species are the major pathogens. Unfortunately, the early diagnosis of serious fungal invasion is exceedingly difficult in these patients, since the manifestations of infection are ill-defined and the organisms can only rarely be isolated from blood cultures. Therefore, empirical antifungal therapy in febrile patients early in the course of disease appears necessary to prevent fungal superinfections and to control clinically undetected fungal invasion. Even if a fungal infection is diagnosed, treatment options are limited to a small number of drugs, and the chance of successful treatment is slim. Amphotericin B remains the most effective drug for systemic fungal infection; however, it is also the most toxic antimicrobical in use. Reduction of its toxicity and, simultaneously, improvement of its effectiveness can be achieved by incorporation into liposomes, although liposomal amphotericin B is not yet available commercially. The newer azoles, such as ketoconazole, vibunazole, and itraconazole are orally active and less toxic. Azoles have been successfully used in the treatment of superficial candidal infections; they are not very active in systemic candidiasis. Inhibitors of the cell wall biosynthesis of candida species include inhibitors of chitin and glucan biosynthesis. Echinocandins and papulacandins interfer with the glucan synthesis and show good anticandidal activity. Their therapeutic potential is currently being explored in clinical trials. Neutropenic patients and particularly bone marrow transplant (BMT) recipients are, in addition to fungal infections, at very high risk for herpesvirus infections: herpes simplex virus (HSV) 0-20 days after BMT, cytomegalovirus (CMV) 40-80 days after BMT, varicella zoster virus (VZV) 100-160 days after BMT, and Epstein-Barr virus (EBV) 14-21 days after BMT. Acyclovir and vidarabine are, at present, the only antiviral drugs available for HSV and VZV infections, acyclovir apparently being the more effective. It is also the drug of choice for prevention of HSV infections in severely immunocompromised, i.e., BMT, patients. Bromovinyldeoxyuridine seems to be suitable for systemic treatment of HSV-1 and VZV infections in these patients. Its potency in inhibiting HSV-1 and VZV replication is superior to that of ACV, and in therapeutic doses it is nontoxic to hematopoietic progenitor cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Glucan biosynthesis in red beet root microsomes and tissue slices

Glucan biosynthesis in red beet root microsomes and tissue slices

An intermediate in cyclic β1–2 glucan biosynthesis

Incubation of UDP-[ 14C]Glc with the inner membranes of Agrobacterium tumefaciens leads to the formation of cyclic β1–2 glucan and trichloroacetic acid-insoluble compounds. The proteolysis products of the latter show a positive charge in acid and a negative charge in alkaline buffers. The cyclic β1–2 glucan and the trichloroacetic acid insoluble compounds yield the same products on partial acid hydrolysis. Addition of excess non-radioactive UDP-Glc to the reaction mixture nearly stops the formation of radioactive β1–2 glucan and leads to a rapid fall of radioactivity in the trichloroacetic acid precipitate. Alkaline treatment of the insoluble compounds under conditions of β-elimination leads to the partial release of free saccharides (about 30%). It is concluded that β1–2 glucan chains are built up joined to a protein and then released as free cyclic β1–2 glucan.

Biosynthesis of Alkali-insoluble Cell-wall Glucan in Schizophyllum commune Protoplasts

SUMMARY: Biosynthesis of cell-wall glucans was studied during regeneration of protoplasts from Schizophylhum commune. Double-labelling experiments using [14C]glucose and [3H]glucose indicated a larger pool size of precursors for an alkali-insoluble (1 → 3)-β-glucan than for the other cell-wall components, (1 → 3)-α-glucan and chitin. Pulse-chase experiments established the existence of a water-soluble, partly alkali-soluble (1 → 3)-β-glucan as a precursor for the alkali-insoluble wall glucan, containing only (1 → 3)-β-linkages. Polyoxin D, an inhibitor of chitin synthase, completely arrested accumulation of alkali-insoluble (1 → 3)-β-glucan. This antibiotic did not inhibit the synthesis of the water-soluble glucan, but prevented the incorporation of this material into the alkali-insoluble glucan/chitin complex. Cycloheximide added at the start of regeneration prevented the synthesis of the alkali-insoluble (1 → 3)-β-glucan and of the water-soluble glucan precursor, whereas no effect on the formation of the alkali-insoluble glucan was observed when cycloheximide was added 3 h after the onset of the regeneration.

THE BIOSYNTHESIS OF STORAGE GLUCANS IN PROKARYOTIC AND EUKARYOTIC ALGAE

THE BIOSYNTHESIS OF STORAGE GLUCANS IN PROKARYOTIC AND EUKARYOTIC ALGAE