Rawle I. Hollingsworth

The beneficial plant growth-promoting association of Rhizobium leguminosarum bv. trifolii with rice roots

his paper summarizes a multinational collaborative project to search for natural, intimate associations between rhizobia and rice (Oryza sativa L.), assess their impact on plant growth, and exploit those combinations that can enhance grain yield with less dependence on inputs of nitrogen (N) fertilizer. Diverse, indigenous populations of Rhizobium leguminosarum bv. trifolii (the clover root-nodule endosymbiont) intimately colonize rice roots in the Egyptian Nile delta where this cereal has been rotated successfully with berseem clover (Trifolium alexandrinum L.) since antiquity. Laboratory and greenhouse studies have shown with certain rhizobial strain-rice variety combinations that the association promotes root and shoot growth thereby significantly improving seedling vigour that carries over to significant increases in grain yield at maturity. Three field inoculation trials in the Nile delta indicated that a few strain-variety combinations significantly increased rice grain yield, agronomic fertilizer N-use efficiency and harvest index. The benefits of this association leading to greater production of vegetative and reproductive biomass more likely involve rhizobial modulation of the plants root architecture for more efficient acquisition of certain soil nutrients [e.g. N, phosphorus (P), potassium (K), magnesium (Mg), calcium (Ca), zinc (Zn), sodium (Na) and molybdenum (Mo)] rather than biological N 2 fixation.

Physiological Function of Alcohol Dehydrogenases and Long-Chain (C30) Fatty Acids in Alcohol Tolerance of Thermoanaerobacter ethanolicus

ABSTRACT A mutant strain (39E H8) of Thermoanaerobacter ethanolicus that displayed high (8% [vol/vol]) ethanol tolerance for growth was developed and characterized in comparison to the wild-type strain (39E), which lacks alcohol tolerance (<1.5% [vol/vol]). The mutant strain, unlike the wild type, lacked primary alcohol dehydrogenase and was able to increase the percentage of transmembrane fatty acids (i.e., long-chain C30 fatty acids) in response to increasing levels of ethanol. The data support the hypothesis that primary alcohol dehydrogenase functions primarily in ethanol consumption, whereas secondary alcohol dehydrogenase functions in ethanol production. These results suggest that improved thermophilic ethanol fermentations at high alcohol levels can be developed by altering both cell membrane composition (e.g., increasing transmembrane fatty acids) and the metabolic machinery (e.g., altering primary alcohol dehydrogenase and lactate dehydrogenase activities).

A preparation and screening strategy for glycosidase inhibitors

Abstract Here we present a one-pot procedure for the synthesis of a library of carbohydrate compounds belonging to four classes of glycosidase inhibitors: glycosides, aldonic acid lactams, aldonic acid lactones and dideoxy-iminoalditols. The complex mixture was separated into individual components that were screened for activity against α- and β-glucosidases. Among these were two 2- O -methyl glycosides that reduced the rate of the enzymatically-catalyzed hydrolysis of the p -nitrophenyl glucosides used in the assay. Relative inhibition indices normalized to the inhibition observed with deoxynojirimycin under similar conditions were determined. This methodology provides a straightforward, general and time-efficient way to synthesize, test and analyze an extensive number of glycosidase inhibitor candidates from several carbohydrate compound classes in one scheme. The direct reference to deoxynojirimycin gives an immediate indication of the concentration range in which a significant inhibitory activity can be obtained.

Synthetic routes to l-carnitine and l-gamma-amino-beta-hydroxybutyric acid from (S)-3-hydroxybutyrolactone by functional group priority switching

Abstract ( R )-3-Hydroxy-4-trimethylaminobutyric acid ( l -carnitine) and ( R )-4-amino-3-hydroxybutyric acid (GABOB) are two compounds with a very high level of medical significance. They can be prepared from ( R )-3-hydroxy-γ-butyrolactone which is not readily available in significant quantities. The corresponding ( S )-lactone is available in large quantities but attempts at inverting the stereochemistry of the hydroxyl group lead to elimination to give the furanone. Here we describe a straightforward route to these two compounds, starting from ( S )-3-hydroxy-γ-butyrolactone by adding a highly oxidized carbon at one end whilst removing one carbon from the other, thus switching the functional group priorities. In this method, the lactone is transformed to an ( R )-4-cyano-3-hydroxybutyric acid ester which is then converted to an acyl hydrazide by treatment with hydrazine. This stable, crystalline hydrazide has not been described before. It is readily converted to ( R )-4-amino-3-hydroxybutyronitrile, a precursor of l -carnitine and GABOB, by Curtius rearrangement under conditions that do not result in deamination.

The structure of the O-antigenic chain of the lipopolysaccharide of Rhizobium trifolii 4s

The structure of the O-antigen chain of the lipopolysaccharide (LPS) of Rhizobium trifolii 4s has been determined by a combination of chemical and spectroscopic methods. The glycosyl components were found to be L-rhamnose, N-acetyl-D-glucosamine, and N-acetyl-D-mannosamine in 3:1:1 molar proportion, as determined by gas chromatography and gas chromatography-mass spectrometry of alditol acetate and persilylated (R)-2-hydroxybutyl glycoside derivatives. The linkage positions and configurations of the glycosyl residues were obtained by 1D and 2D NMR spectroscopy. The polymer has a pentasaccharide repeating-unit containing rhammose and N-acetylglucosamine in the main chain and N-acetylmannosamine as the sole-side chain component. This latter residue is linked to a main-chain rhamnose residue. This result was suggested by NMR spectroscopy and confirmed by periodate oxidation. The sequence was deduced by 1D and 2D NMR NOE experiments and by partial hydrolysis studies. The repeating unit of the polysaccharide is shown. This constitutes the first complete structure of an O-antigenic chain of the lipopolysaccharide of any strain of Rhizobium trifolii.

Mutation or Increased Copy Number of nodE Has No Effect on the Spectrum of Chitolipooligosaccharide Nod Factors Made By Rhizobiumleguminosarum bv. trifolii

The bacterial gene nodE is the key determinant of host specificity in the Rhizobium leguminosarum-legume symbiosis and has been proposed to determine unique polyunsaturated fatty acyl moieties in chitolipooligosaccharides (CLOS) made by the bacterial symbiont. We evaluated nodE function by examining CLOS structures made by wild-type R. leguminosarum bv. trifolii ANU843, an isogenic nodE::Tn5 mutant, and a recombinant strain containing multiple copies of the pSym nod region of ANU843. 1H-NMR, electrospray ionization mass spectrometry, fast atom bombardment mass spectrometry, flame ionization detection-gas chromatography, gas chromatography/mass spectrometry, and high performance liquid chromatography/UV photodiode array analyses revealed that these bacterial strains made the same spectrum of CLOS species. We also found that ions in the mass spectra which were originally assigned to nodE-dependent CLOS species containing unique polyunsaturated fatty acids (Spaink, H. P., Bloemberg, G. V., van Brussel, A. A. N., Lugtenberg, B. J. J., van der Drift, K. M. G. M., Haverkamp, J., and Thomas-Oates, J. E.(1995) Mol. Plant-Microbe Interact. 8, 155-164) were actually due to sodium adducts of the major nodE-independent CLOS species. No evidence for nodE-dependent CLOSs was found for these strains. These results indicate a need to revise the current model to explain how nodE determines host range in the R. leguminosarum- legume symbiosis.

Characterization of structural defects in the lipopolysaccharides of symbiotically impaired Rhizobium leguminosarum biovar viciae VF-39 mutants

The lipopolysaccharides (LPS) of a wild type strain of Rhizobium leguminosarum biovar viciae (strain VF-39) and two symbiotically defective Tn5 mutants (VF-39-32 and VF-39-86) have been studied. The LPS of the mutants reflected impaired synthesis of the O-antigen. In the LPS of one mutant, the core tetrasaccharide was lacking and in that of the other it was truncated to a disaccharide containing mannose and 3-deoxy-D-manno-oct-2-ulosonic acid (KdO). The latter mutant also synthesized an unusual carbohydrate component containing mannose, galactose, and an unidentified saccharide. The lipid A composition was similar to that found in other strains of R. leguminosarum biovar viciae. The O-antigen of the wild-type bacterium contained 2-O-methylfucose, fucose, 3,6-dideoxy-3-(methylamino)hexose, glucose, 2-amino-2,6-dideoxyhexose, and heptose. This study clearly defines a role for the bacterial LPS in the proper functioning of the Rhizobium legume symbiosis.

Synthesis of C-glycosides of N-acetylglucosamine by direct alkylation of 2-amino-2-deoxy-2,3,4,6-tetra-O-acetyl glucopyranosyl chloride

Abstract The β-isomer of C-glycoside, ethyl 2-acetamino-2-deoxy-3,4,6-tri-O-acetyl-β-D-glucopyranosylacetate( 3 ) was prepared stereoselectively in two steps by reaction of 2-acetamino-2-deoxy-3,4,6-tri-O-acetyl-α-D-glucopyranosyl chloride( 4 ) with potassium diethylmalonate and 18-crown-6, followed by decarboxylation.

The structures of the lipopolysaccharide core components from rhizobium leguminosarum biovar phaseoli CE3 and two of its symbiotic mutants, CE109 and CE309

The structures for the core regions of the lipopolysaccharides (LPSs) from R. leguminosarum bv. phaseoli CE3 and two symbiotic mutants were determined by g.l.c.-m.s., proton nuclear magnetic resonance spectroscopy (n.m.r.), fast-atom-bombardment mass spectrometry (f.a.b.-m.s.), and by comparison with known structures from the LPS of R. leguminosarum bv. trifolii ANU843. The core oligosaccharides were separated into two components, P2-2 and P2-3, by gel-filtration chromatography using Bio-Gel P2. The P2-2 oligosaccharide from CE3 is a tetrasaccharide consisting of 3-deoxy-D-manno-2-octulosonic acid (Kdo), mannose, galactose and galacturonic acid. The mannosyl residue is alpha-linked to O-4 of Kdo, and the galactosyl and galactosyluronic residues are alpha-linked to O-4 and O-6, respectively, of the mannosyl residue. The P2-2 oligosaccharide from mutant CE109 is missing the galactosyluronic residue, while that from mutant CE309 is missing both the galactosyl and galactosyluronic residues. The P2-3 oligosaccharide from CE3 LPS is a trisaccharide consisting of two galactosyluronic residues alpha-linked to the O-4 and O-7 of Kdo. Fraction P2-3 from mutant CE309 has the same structure as CE3 P2-3. Fraction P2-3 from mutant CE109 contains galacturonic acid and Kdo, but its structure differs from that of CE3 P2-3.

A simple three-step method for preparing homochiral 5-trityloxymethyl-2-oxazolidinones from optically active 3-hydroxy-γ-butyrolactones

Abstract A simple high-yield three-step route to O -tritylated optically active 5-hydroxymethyl oxazolidinones from optically active 3-hydroxy-γ-butyrolactones is described. The key intermediate is the 4- O -trityl ether of homochiral 3,4-dihydroxybutyramide, which is obtained in quantitative yield from 3-hydroxy-γ-butyrolactone by treatment with ammonia. It is readily transformed to the oxazolidinone by Hoffmann rearrangement in a two-phase system. The carbonyl group in the oxazolidinone is derived from C-1 of the amide, and a separate carbonylation reaction is not required. Oxazolidinones are key compounds in drug synthesis especially in the areas of antibacterials and behavior disorder therapy.