Molecular architecture of a galactoglucan from Rhizobium meliloti.

Rhizobium meliloti mutants produce a linear, acidic exopolysaccharide with alternating galactopyranosyl and glucopyranosyl units having (1-->3) linkages. It has a 4,6-O-pyruvic cyclic acetal group on the alpha-galactosyl and 6-O-acetyl ester group on the beta-glucosyl units. X-ray diffraction patterns from polycrystalline and well oriented specimens of its potassium salt indicate that the polymer forms a 2-fold helix of pitch 15.89 A. The three-dimensional structure has been determined and refined by using the X-ray intensities and the linked-atom least-squares technique. The details of the antiparallel packing arrangement of two helices in an orthorhombic unit cell, a = 14.49, b = 9.79, and c = 15.89 A, reveal that each disaccharide repeating unit is associated with one potassium and three water molecules. The helices are interconnected by a series of ...COO- ...K+ ...W ...COO- ... interactions. Both pyruvyl and acetyl groups, which are on the periphery of the helix, are involved in the association of the polysaccharide chains and thus appear to be an integral component of the galactoglucan in the nodule invasion process.

Polysaccharide synthesis in relation to nodulation behavior of Rhizobium leguminosarum.

In this study, we characterized four Tn5 mutants derived from Rhizobium leguminosarum RBL5515 with respect to synthesis and secretion of cellulose fibrils, extracellular polysaccharides (EPS), capsular polysaccharides, and cyclic beta-(1,2)-glucans. One mutant, strain RBL5515 exo-344::Tn5, synthesizes residual amounts of EPS, the repeating unit of which lacks the terminal galactose molecule and the substituents attached to it. On basis of the polysaccharide production pattern of strain RBL5515 exo-344::Tn5, the structural features of the polysaccharides synthesized, and the results of an analysis of the enzyme activities involved, we hypothesize that this strain is affected in a galactose transferase involved in the synthesis of EPS only. All four mutants failed to nodulate plants belonging to the pea cross-inoculation group; on Vicia sativa they induced root hair deformation and rare abortive infection threads. All of the mutants appeared to be pleiotropic, since in addition to defects in the synthesis of EPS, lipopolysaccharide, and/or capsular polysaccharides significant increases in the synthesis and secretion of cyclic beta-(1,2)-glucans were observed. We concluded that it is impossible to correlate a defect in the synthesis of a particular polysaccharide with nodulation characteristics.

Influence of growth conditions on production of capsular and extracellular polysaccharides by Rhizobium leguminosarum.

The influence of growth rate and medium composition on exopolymer production by Rhizobium leguminosarum was studied. When grown in medium containing 10 g/l mannitol and 1 g/l glutamic acid, Rhizobium leguminosarum biovar trifolii TA-1 synthesized up to 2.0 g/l of extracellular polysaccharide (EPS), and up to 1.6 g/l of capsular polysaccharide (CPS). Under non-growing cell conditions in medium without glutamic acid, CPS synthesis by strain TA-1 could proceed to 2.1 g/l, while EPS-production remained relatively low (0.8 g/l). Maximal CPS-yield was 2.9 g CPS/l medium in a medium containing 20 g/l mannitol and 2 g/l glutamic acid. The EPS-deficient strain R. leguminosarum RBL5515, exo4::Tn5 was able to produce CPS to similar levels as strain TA-1, but CPS-recovery was easier because of the low viscosity of the medium and growth of the cells in pellets. With strain TA-1 in nitrogen-limited continuous cultures with a constant biomass of 500 mg cell protein/l, EPS was the most abundant polysaccharide present at every dilution rate D (between 0.12 and 0.02 h-1). The production rates were 50-100 mg/g protein/h for EPS and 15-20 mg/g protein/h for CPS. Only low amounts of cyclic beta-(1,2)-glucans were excreted (10-30 mg/l) over the entire range of growth rates.

Synthesis of cyclic beta-(1,2)-glucans by Rhizobium leguminosarum biovar trifolii TA-1: factors influencing excretion.

The synthesis of cyclic beta-(1,2)-glucans from UDP-[14C]glucose by a crude membrane preparation and whole cells of Rhizobium leguminosarum bv. trifolii TA-1 was investigated. The crude membrane system needed Mn2+, ATP, and NAD+ for optimal activity. Hardly any difference in biosynthetic activity between membrane fractions of TA-1 cells grown in the presence (200 mM) or absence of NaCl was observed. Whole TA-1 cells grown in the presence of NaCl excreted labeled, neutral cyclic beta-(1,2)-glucan during incubation with added UDP-[14C]glucose. With NaCl-free cultured TA-1 cells, no excretion was observed; however, after these cells were alternately frozen and thawed eight times, they excreted glucans. Glucan formation in vitro and glucan excretion by whole cells were strongly inhibited in the presence of 50 mg of cyclic glucan per ml (about 15 mM), indicating that biosynthesis of cyclic beta-(1,2)-glucans in strain TA-1 is controlled by end-product inhibition. These observations indicate that TA-1 cells become more permeable to cyclic glucans at high NaCl concentrations. The constant loss of glucans from cells grown in the presence of 200 mM NaCl prevented end-product inhibition and resulted in glucan accumulation of up to 1,600 mg/liter in the medium.

Levels of trehalose and glycogen in Arthrobacter globiformis under conditions of nutrient starvation and osmotic stress.

Cells of Arthrobacter globiformis grown in carbohydrate-rich media were found to contain large quantities of low-Mr carbohydrates (800 micrograms/mg protein) and only small amounts of amino acids, in addition to high amounts of glycogen (2 mg/mg protein). At increasing osmotic values of the medium, low-Mr carbohydrate levels increased to 1300 micrograms/mg protein. Low-Mr pools were extracted from the cells with hot 75% ethanol, and subjected to thin layer, gel and gas-liquid chromatography. They turned out to consist mainly of alpha,alpha-trehalose. Levels of trehalose in Arthrobacter cells have the tendency to remain constant, both during nutrient exhaustion (resulting in glycogen consumption), and on addition of excess of carbon source to the medium (resulting in an increased glycogen content of the cells). The stress-tolerant properties of Arthrobacter (resistance to nutrient starvation, desiccation and high salt concentration) are discussed with respect to the high glycogen and trehalose contents of the cells.

Unusual structure of the exopolysaccharide of Rhizobium leguminosarum bv. viciae strain 248.

The exopolysaccharide from R. leguminosarum bv. viciae strain 248 differs from those of other Rhizobium strains with similar symbiotic behavior. 13C-N.m.r. spectroscopy of fragments generated by partial hydrolysis, together with methylation analysis and 13C-n.m.r. spectroscopy of the enzymically depolymerised exopolysaccharide, indicated the following nonasaccharide repeating-unit: [formula: see text] The locations of the acetyl and 3-hydroxybutanoyl substituents in the exopolysaccharide are assigned provisionally. R. leguminosarum bv. viciae strain 248, cured of its Sym plasmid pRL1JI, synthesised an exopolysaccharide in which the sites and degree of substitution were unchanged. A Tn5 mutant, derived from strain 248 and unable to induce nodules, synthesised small amounts of EPS that lacked galactose.

Effect of the concentration of sodium chloride in the medium on the relative proportions of poly- and oligo-saccharides excreted by Rhizobium meliloti strain YE-2SL.

Rhizobium meliloti mutant strains have been found which, in the presence of low concentrations of NaCl, produce a galactoglucan instead of the usual succinoglycan. When grown in a mannitol-glutamic acid-salts medium, the principal products secreted by R. meliloti YE-2S1 were comparable quantities of succinoglycan repeating-units and galactoglucan. As NaCl was added progressively to the culture medium, the repeating units nearly completely disappeared and the galactoglucan was gradually replaced by a succinoglycan.

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.

Re-examination of cellular cyclic beta-1,2-glucans of Rhizobiaceae: distribution of ring sizes and degrees of glycerol-1-phosphate substitution.

Gel-filtration and thin layer chromatography of low molecular weight carbohydrates from culture filtrates of Agrobacterium radiobacter, Isolate II, have shown, that next to the neutral beta-1,2-glucan fraction a major acidic fraction was present which was found to be glycerophosphorylated cyclic beta-1,2-glucans. Re-examination of cyclic beta-1,2-glucan preparations which had been obtained by extraction of Rhizobium cells with hot phenol-water also showed these acidic modified beta-1,2-glucans to be present. Cyclic beta-1,2-glucans from R. leguminosarum (9 strains) and of R. phaseoli (1 strain) had ring size distribution with degrees of polymerisation (DPs) of 19 and 20 as major ring sizes of which a minor part was glycerophosphorylated; beta-1,2-glucans of R. trifolii (3 strains) had ring sizes with DPs measuring 19-22 as prominent components which were largely unsubstituted, and R. meliloti (7 strains) had beta-1,2-glucans with ring size distributions extending to still higher DPs of 19-25 of which the major part appeared to be glycerophosphorylated.

Polysaccharides production by Rhizobium phaseoli and the typing of their excreted anionic polysaccharides.

The pattern of polysaccharide production amongst strains of Rhizobium phaseoli appear very varied: some strains produce anionic exopolysaccharides (EPS) as major polysaccharides (EPS) as major polymer without any other product, but most strains exhibit greater polysaccharide diversity. Apart from EPS they excrete capsular polysaccharides (CPS) and accumulate poly-beta-hydroxybutyric acid (PHB) and/or glycogen in their cells. The latter can then be used as C-sources for further synthesis of EPS and CPS. Some strains are only very poor producers or do not produce at all. Nine strains of R. phaseoli have been analysed and shown to possess the K-36 type of polysaccharide (EPS), as do strains of R. leguminosarum (6 strains) and R. trifolii (9 strains). Three strains of R. phaseoli have been found to possess the K-87 type of polysaccharide and types K-38 and K-44 polysaccharides have only been found in their own type strains.

Congo red absorption and cellulose synthesis by Rhizobiaceae.

Congo red uptake by Rhizobium colonies from yeast extract-mannitol-mineral salts-Congo red-agar plates was related with the cellulose content in the cell capsule of the bacteria.

Cellular glycogen, beta-1,2,-glucan, poly beta-hydroxybutyric acid and extracellular polysaccharides in fast-growing species of Rhizobium.

Synthesis of acidic exopolysaccharides, neutral cellular polysaccharides and poly-beta-hydroxybutyric acid (PHB) by Rhizobium is strongly dependent on cultural conditions and the strains used. Exopolysaccharide production by R. leguminosarum, R. Phaseoli and R. trifolii closely parallels growth, whereas R. meliloti mainly excretes (low mol wt) polysaccharides when cell propagation is limited by lack of a necessary growth element (nitrogen) and an excess of carbon source is still present in the medium. In all strains, accumulation of cellular glycogen, beta-1,2-glucan and PHB is initiated only under growth-limiting conditions. When the external carbon source is exhausted, glycogen and PHB are metabolized by the cells, sustaining their longevity and thus act as true reserve materials; on the other hand, beta-1,2-glucan and excreted polysaccharides are not utilized on further incubation of the culture. Differences exist in the nature and relative amounts of the products synthesized by strains of different species of Rhizobium. R. leguminosarum, R. phaseoli and R. trifolii synthesize a uronic containing exopolysaccharide, PHB and/or glycogen, non-metabolizable capsular polysaccharide and low amounts of beta-1,2-glucan. R. meliloti synthesizes a uronic acid-free exopolysaccharide, PHB and /or glycogen and high concentrations of beta-1,2-glucan. Exopolysaccharides, beta-1,2-glucan and glycogen preparations were obtained by isolation and purification from cells of fast-growing species of Rhizobium and chemically characterized.

Surface carbohydrates of Rhizobium. I. Beta-1, 2-glucans.

Because of increased interest in surface carbohydrates of Rhizobium in relation to host specificity, phenol-water extractions were carried out of whole cells of Rhizobium strains of the species R. leguminosarum, R. phaseoli, R. trifolii and R. meliloti. Fractionation of the crude extracts with cetavlon afforded polysaccharide mixtures, which were essentially free of RNA and acidic exopolysaccharide (EPS). They could be separated into a high molecular weight heteropolysaccharide fraction of lipopolysaccharide (LPS) nature and a low molecular weight glucan fraction. Glucan turned out to be the principal polysaccharide component of the cells (up to 10% of the dry cell weight), when cultivated in carbohydrate-rich media, and to be present as firmly attached capsular material. Glucan (mol wt 3000) structure was elucidated by methylation and periodate oxidation techniques. Methylation yielded 3, 4, 6-tri-O-methyl-D-glucose, characterized by GLC-MS, as the only product of hydrolysis of the fully methylated glucan. The glucan consumed 1 mole of periodate per mole anhydroglucose unit and gave sophorose on partial hydrolysis. From these data a linear beta-1,2-linked glucan structure was deduced. The occurrence of beta-1,2-glucan and the implications for the specific binding properties of Rhizobium cells are discussed.

Inhibitory effects of copper on bacteria related to the free ion concentration.

Cu(2+) ion determinations were carried out in complex and in inorganic salts-glycerol media, to which increasing amounts of Cu(II) had been added, with the ion-specific Cu(II)-Selectrode. Likewise, complexing capacity of bacterial suspensions was estimated by titration with CuSO4.Copper-sensitive bacteria, e.g.,Klebsiella aerogenes, were inhibited in their growth and survival in the range of 10(-8)-10(-6) M Cu(2+) ion concentrations. In copper-buffered complex media, high copper loads could be tolerated, as growth proceeded with most of the copper bound to medium components. In low-complexing mineral salts media, in which high Cu(2+) ion concentrations exist at low copper loads, there was competition of Cu(2+) for binding sites of the cells. Total allowed copper was then determined by the ratio of copper to biomass.Copper-resistant bacteria could be isolated from a stock solution of CuSO4, containing 100 ppm Cu(II). They were of thePseudomonas type and showed a much higher tolerance towards Cu(2+), up to 10(-3) M.