Excessive excretion of cyclic beta-(1,2)-glucan by Rhizobium trifolii TA-1.

At 25 degrees C, the optimal temperature for growth of Rhizobium trifolii TA-1, extracellular and capsular polysaccharide (EPS and CPS) were the main carbohydrate products synthesized in mannitol-rich medium (10 g of mannitol and 1 g of glutamic acid per liter). In the same medium at 33 degrees C, EPS and CPS production was inhibited, and up to 3.9 g of cyclic beta-(1,2)-glucan was produced during an incubation period of 20 days with a total biomass of 0.55 g of protein. In a medium containing 50 g of mannitol and 10 g of glutamic acid per liter, high cell densities (3.95 g of protein) were obtained at 25 degrees C. This biomass excreted 10.9 g of cyclic beta-(1,2)-glucan within 10 days. Concomitantly, 4.8 g of EPS were synthesized, while CPS production was strongly suppressed. The excreted cyclic beta-(1,2)-glucans were neutral and had degrees of polymerization ranging from 17 to 25, with a degree of polymerization of 19 as the major glucan cycle.     

The ndvB locus of Rhizobium meliloti encodes a 319-kDa protein involved in the production of beta-(1----2)-glucan

The ndvB locus of Rhizobium meliloti was sequenced and found to encode a 319-kDa protein involved in the production of beta-(1----2)-glucan. Transposon Tn5 mutagenesis revealed that a large portion of the downstream half of this gene is not essential for symbiosis but is required for optimal production of beta-(1----2)-glucan. A high molecular weight inner membrane protein, believed to be the ndvB gene product, was absent from two different upstream ndvB::Tn5 mutants. This protein could be labeled in vitro with UDP-[U-14C]glucose in the wild type but not in the symbiotically defective mutants. Inner membrane preparations from the symbiotically competent downstream mutants labeled less well than did those from wild type with UDP-[U-14C] glucose and did not show distinct bands after polyacrylamide gel electrophoresis and fluorography, suggesting that C-terminal truncations of NdvB might affect the stability of this molecule. These downstream mutants had reduced amounts of periplasmic beta-(1----2)-glucan and exhibited several vegetative defects seen also in the upstream mutants. These included alterations in phage and antibiotic sensitivity, in motility, and in growth in low osmolarity media. Bacteroids produced by two of the downstream mutants were morphologically abnormal, indicating that ndvB is involved not only in invasion but also in bacteroid development.     

Role for [corrected] Agrobacterium tumefaciens ChvA protein in export of beta-1,2-glucan.

Functional chvA and chvB genes are required for attachment of Agrobacterium tumefaciens to plant cells, an early step in crown gall tumor formation. Strains defective in these loci do not secrete normal amounts of cyclic beta-1,2-glucan. Whereas chvB is required for beta-1,2-glucan synthesis, the role of chvA in glucan synthesis or export has not been clearly defined. We found that cultures of chvA mutants contained as much neutral beta-1,2-glucan in the cell pellets as did the wild type, with no detectable accumulation of glucan in the culture supernatant. The cytoplasm of chvA mutant cells contained over three times more soluble beta-1,2-glucan than did the cytoplasm of the wild-type parent. Unlike the wild type, chvA mutants contained no detectable periplasmic glucan. The amino acid sequence of chvA is highly homologous to the sequences of bacterial and eucaryotic export proteins, as observed previously in the case of ndvA, a rhizobial homolog of chvA. Strong sequence homology within this family of export proteins is concentrated in the carboxy-terminal portions of the proteins, but placement of consensus ATP-binding sites, internal signal sequences, and hydrophobic domains are conserved over their entire lengths. These data suggest a model for beta-1,2-glucan synthesis in A. tumefaciens in which glucan is synthesized inside the inner membrane with the participation of ChvB and transported across the inner membrane with the participation of ChvA.     

Excessive excretion of cyclic beta-(1,2)-glucan by Rhizobium trifolii TA-1

At 25 degrees C, the optimal temperature for growth of Rhizobium trifolii TA-1, extracellular and capsular polysaccharide (EPS and CPS) were the main carbohydrate products synthesized in mannitol-rich medium (10 g of mannitol and 1 g of glutamic acid per liter). In the same medium at 33 degrees C, EPS and CPS production was inhibited, and up to 3.9 g of cyclic beta-(1,2)-glucan was produced during an incubation period of 20 days with a total biomass of 0.55 g of protein. In a medium containing 50 g of mannitol and 10 g of glutamic acid per liter, high cell densities (3.95 g of protein) were obtained at 25 degrees C. This biomass excreted 10.9 g of cyclic beta-(1,2)-glucan within 10 days. Concomitantly, 4.8 g of EPS were synthesized, while CPS production was strongly suppressed. The excreted cyclic beta-(1,2)-glucans were neutral and had degrees of polymerization ranging from 17 to 25, with a degree of polymerization of 19 as the major glucan cycle.     

Bacterial cyclic beta-(1,2)-glucan acts in systemic suppression of plant immune responses

Although cyclic glucans have been shown to be important for a number of symbiotic and pathogenic bacterium-plant interactions, their precise roles are unclear. Here, we examined the role of cyclic beta-(1,2)-glucan in the virulence of the black rot pathogen Xanthomonas campestris pv campestris (Xcc). Disruption of the Xcc nodule development B (ndvB) gene, which encodes a glycosyltransferase required for cyclic glucan synthesis, generated a mutant that failed to synthesize extracellular cyclic beta-(1,2)-glucan and was compromised in virulence in the model plants Arabidopsis thaliana and Nicotiana benthamiana. Infection of the mutant bacterium in N. benthamiana was associated with enhanced callose deposition and earlier expression of the PATHOGENESIS-RELATED1 (PR-1) gene. Application of purified cyclic beta-(1,2)-glucan prior to inoculation of the ndvB mutant suppressed the accumulation of callose deposition and the expression of PR-1 in N. benthamiana and restored virulence in both N. benthamiana and Arabidopsis plants. These effects were seen when cyclic glucan and bacteria were applied either to the same or to different leaves. Cyclic beta-(1,2)-glucan-induced systemic suppression was associated with the transport of the molecule throughout the plant. Systemic suppression is a novel counterdefensive strategy that may facilitate pathogen spread in plants and may have important implications for the understanding of plant-pathogen coevolution and for the development of phytoprotection measures.     

A novel cyclic beta-1,2-glucan mutant of Rhizobium meliloti.

The periplasmic cyclic beta-1,2-glucans produced by bacteria within the Rhizobiaceae family provide functions during hypo-osmotic adaptation and plant infection. In Rhizobium meliloti, these molecules are highly modified with phosphoglycerol and succinyl substituents, and it is possible that the anionic character of these glucans is important for their functions. In the present study, we have used a thin-layer chromatographic screening method to identify a novel R. meliloti mutant specifically blocked in its ability to transfer phosphoglycerol substituents to the cyclic beta-1,2-glucan backbone. Further analysis revealed that the cyclic glucans produced by this mutant contained elevated levels of succinyl substituents. As a result, the overall anionic charge on the cyclic beta-1,2-glucans was found to be similar to that of wild-type cells. Despite this difference in cyclic beta-1,2-glucan structure, the mutant was shown to effectively nodulate alfalfa and to grow as well as wild-type cells in hypo-osmotic media.     

Osmosensitivity phenotypes of Agrobacterium tumefaciens mutants that lack periplasmic beta-1,2-glucan.

The periplasmic cyclic beta-1,2-glucan of Agrobacterium tumefaciens is believed to maintain high osmolarity in the periplasm during growth of the bacteria on low-osmotic-strength media. Strains with mutations in the chvA or chvB gene do not accumulate beta-1,2-glucan in their periplasm and exhibit pleiotropic phenotypes, including inability to form crown gall tumors on plants. We examined the effects of medium osmolarity to determine whether some or all of these phenotypes result from suboptimal periplasmic osmolarity. The mutants grew more slowly than wild-type cells and exhibited altered periplasmic and cytoplasmic protein content when cultured in low-osmotic-strength media, but not when cultured in high-osmotic-strength media. These observations support a role for periplasmic glucan in osmoadaptation. However, the mutants were avirulent and exhibited reduced motility regardless of the osmolarity of the medium. Therefore, beta-1,2-glucan may play roles in virulence and motility that are unrelated to its role in osmoadaptation.     

Formation in Rhizobium and Agrobacterium spp. of a 235-kilodalton protein intermediate in beta-D(1-2) glucan synthesis.

beta-D(1-2) Glucan was synthesized by Agrobacterium and Rhizobium spp. in vitro with enzymes from the internal membranes upon the addition of UDF glucose and Mg2+ or Mn2+. An intermediate containing protein and beta-D(1-2) glucan was formed during the reaction. It could be precipitated with trichloroacetic acid or separated by polyacrylamide gel electrophoresis under denaturing conditions. After detection with Coomassie blue or a radioactive substrate, the intermediate appeared as a 235-kilodalton protein. The radioactivity could be chased with a nonradioactive substrate. All strains that formed beta-D(1-2) glucan in vitro formed the 235-kilodalton protein, whereas avirulent, beta-D(1-2) glucan-negative mutants did not synthesize it. Transposon insertions in the chvB locus of strains ME2 and ME116 did not alter the virulence of the strains. These strains were able to form beta-D(1-2) glucan in vitro and synthesize the 235-kilodalton protein.     

Cyclic beta-glucans of members of the family Rhizobiaceae.

Cyclic beta-glucans are low-molecular-weight cell surface carbohydrates that are found almost exclusively in bacteria of the Rhizobiaceae family. These glucans are major cellular constituents, and under certain culture conditions their levels may reach up to 20% of the total cellular dry weight. In Agrobacterium and Rhizobium species, these molecules contain between 17 and 40 glucose residues linked solely by beta-(1,2) glycosidic bonds. In Bradyrhizobium species, the cyclic beta-glucans are smaller (10 to 13 glucose residues) and contain glucose linked by both beta-(1,6) and beta-(1,3) glycosidic bonds. In some rhizobial strains, the cyclic beta-glucans are unsubstituted, whereas in other rhizobia these molecules may become highly substituted with moieties such as sn-1-phosphoglycerol. To date, two genetic loci specifically associated with cyclic beta-glucan biosynthesis have been identified in Rhizobium (ndvA and ndvB) and Agrobacterium (chvA and chvB) species. Mutants with mutations at these loci have been shown to be impaired in their ability to grow in hypoosmotic media, have numerous alterations in their cell surface properties, and are also impaired in their ability to infect plants. The present review will examine the structure and occurrence of the cyclic beta-glucans in a variety of species of the Rhizobiaceae. The possible functions of these unique molecules in the free-living bacteria as well as during plant infection will be discussed.     

Hypoosmotic adaptation in Rhizobium meliloti requires beta-(1----2)-glucan.

beta-(1----2)-Glucan, an unusual cyclic oligosaccharide, can be isolated from the periplasm of bacteria belonging to the family Rhizobiaceae. Data presented here suggest that the periplasmic beta-(1----2)-glucan of Rhizobium meliloti plays a major role in osmotic adaptation. First, growth of R. meliloti in a low-osmolarity medium causes a large accumulation of periplasmic beta-(1----2)-glucan. Second, mutations in the ndv genes, which prevent this accumulation of beta-(1----2)-glucan, reduce cell growth rates under low-osmolarity conditions and cause several other phenotypic changes indicative of an altered or stressed surface. Third, growth of the ndv mutants can be restored by raising the osmolarity of the medium with the addition of a variety of ionic or nonionic compounds. The phenotypic changes associated with the cell surface of the mutants can also be substantially suppressed by increasing the medium osmolarity. On the basis of these data and general considerations about the periplasmic space in gram-negative bacteria, we suggest a mechanism of hypoosmotic adaptation in R. meliloti in which beta-(1----2)-glucan plays an essential role.     

The site of cellulose synthesis. Hormone treatment alters the intracellular location of alkali-insoluble beta-1,4-glucan (cellulose) synthetase activities

Membrane preparations from growing regions of 8-day old Pisum sativum epicotyls contain multiple beta-1,4-glucan (cellulose) synthetase activities (UDP- or GDP-glucose: beta-1,4-glucan-glucosyl transferase), and the levels of some of these are influenced by treatments with the growth hormone, indoleacetic acid (IAA). When membranes from control epicotyl segments (zero time) are fractionated by isopycnic sedimentation in sucrose density gradients, all of the synthetase activities are associated mainly with Golgi membrane (density 1.55 g/cm3). After decapitation and treatment of epicotyls with IAA, synthetases also appear in a smooth vesicle fraction (density 1.11 g/cm3) which is rich in endoplasmic reticulum (ER) marker enzyme. Major fractions of these synthetases are not recovered in association with plasma membrane or washed cell walls. When [14-C]sucrose is supplied in vivo to segments +/- IAA, radioactive cellulose is deposited only in the wall. Cellulose or cellodextrin precursors do not accumulate in those membranes in which synthetase activities are recovered in vitro. In experiments where tissue slices containing intact cells are supplied with [14C]sugar nucleotide in vitro, alkali-insoluble beta-1,4-glucan is synthesized (presumably outside the protoplast) at rates which greatly exceeded (20-30 times) those obtained using isolated membrane preparations. Progressive disruption of cell structure results in increasing losses of this high activity. These results are consistent with the interpretation that Golgi and ER-associated synthetases are not themselves loci for cellulose synthesis in vivo, but represent enzymes in transit to sites of action at the wall:protoplast omterface. There they operate only if integrity of cellular organization is maintained.     

Mechanism of action of cyclic beta-1,2-glucan synthetase from Agrobacterium tumefaciens: competition between cyclization and elongation reactions.

We have examined some aspects of the mechanism of cyclic beta-1,2-glucan synthetase from Agrobacterium tumefaciens (235-kDa protein, gene product of the chvB region). The enzyme produces cyclic beta-1,2-glucans containing 17 to 23 glucose residues from UDP-glucose. In the presence of added cyclic beta-1,2-glucans (> 0.5 mg/ml) (containing 17 to 23 glucose residues), the enzyme instead synthesizes larger cyclic beta-1,2-glucans containing 24 to 30 glucose residues. This is achieved by de novo synthesis and not by disproportion reactions with the added product. This is interpreted as inhibition of the specific cyclization reaction for the synthesis of cyclic beta-1,2-glucans containing 17 to 23 glucose residues but with no concomitant effect on the elongation (polymerization) reaction. Temperature and detergents both affect the distribution of sizes of cyclic beta-1,2-glucans, but glucans containing 24 to 30 glucose residues are not produced. We suggest that the size distribution of cyclic beta-1,2-glucan products depends on competing elongation and cyclization reactions.     

Kre6 protein essential for yeast cell wall beta-1,6-glucan synthesis accumulates at sites of polarized growth

Saccharomyces cerevisiae Kre6 is a type II membrane protein with amino acid sequence homology with glycoside hydrolase and is essential for β-1,6-glucan synthesis as revealed by the mutant phenotype, but its biochemical function is still unknown. The localization of Kre6, determined by epitope tagging, is a matter of debate. We raised anti-Kre6 rabbit antiserum and examined the localization of Kre6 and its tagged protein by immunofluorescence microscopy, subcellular fractionation in sucrose density gradients, and immunoelectron microscopy. Integration of the results indicates that the majority of Kre6 is in the endoplasmic reticulum; however, a small but significant portion is also present in the secretory vesicle-like compartments and plasma membrane. Kre6 in the latter compartments is observed as strong signals that accumulate at the sites of polarized growth by immunofluorescence. The truncated Kre6 without the N-terminal 230-amino acid cytoplasmic region did not show this polarized accumulation and had a severe defect in β-1,6-glucan synthesis. This is the first evidence of a β-1,6-glucan-related protein showing the polarized membrane localization that correlates with its biological function.     

Observations on the enzymatic synthesis of a beta-1,4-glucan by a soluble preparation form mung bean(Phaseolus aureus)

An enzyme extract of mung bean roots and hypocotyls (Phaseolus aureus) that catalyzes the synthesis of a β-1,4-glucan from guanosine 5′-diphosphate-d-glucose was prepared by a modification of the method of T.-Y. Liu and W. Z. Hassid (1970, J. Biol. Chem.245, 1922–1925). Its activity was not increased by any of those factors that have contributed to the marked improvement in the performance of various cell-free polysaccharide-synthesizing systems from other organisms. Evidence is presented to suggest that in the mung bean system a stable precursor of the cell wall polysaccharide or intermediate in its synthesis is formed by incubation of the enzyme with guanosine 5′-diphosphate-d-mannose.     

Cloning of the RHO1 gene from Candida albicans and its regulation of beta-1,3-glucan synthesis.

The Saccharomyces cerevisiae RHO1 gene encodes a low-molecular-weight GTPase. One of its recently identified functions is the regulation of beta-1,3-glucan synthase, which synthesizes the main component of the fungal cell wall (J. Drgonova et al., Science 272:277-279, 1996; T. Mazur and W. Baginsky, J. Biol. Chem. 271:14604-14609, 1996; and H. Qadota et al., Science 272:279-281, 1996). From the opportunistic pathogenic fungus Candida albicans, we cloned the RHO1 gene by the PCR and cross-hybridization methods. Sequence analysis revealed that the Candida RHO1 gene has a 597-nucleotide region which encodes a putative 22.0-kDa peptide. The deduced amino acid sequence predicts that Candida albicans Rho1p is 82.9% identical to Saccharomyces Rho1p and contains all the domains conserved among Rho-type GTPases from other organisms. The Candida albicans RHO1 gene could rescue a S. cerevisiae strain containing a rho1 deletion. Furthermore, recombinant Candida albicans Rho1p could reactivate the beta-1,3-glucan synthesis activities of both C. albicans and S. cerevisiae membranes in which endogenous Rho1p had been depleted by Tergitol NP-40-NaCl treatment. Candida albicans Rho1p was copurified with the beta-1,3-glucan synthase putative catalytic subunit, Candida albicans Gsc1p, by product entrapment. Candida albicans Rho1p was shown to interact directly with Candida albicans Gsc1p in a ligand overlay assay and a cross-linking study. These results indicate that Candida albicans Rho1p acts in the same manner as Saccharomyces cerevisiae Rho1p to regulate beta-1,3-glucan synthesis.