Michael Batley

Clovers secrete specific phenolic compounds which either stimulate or repress nod gene expression in Rhizobium trifolii

Rhizobium trifolii mutants containing Escherichia coli lac gene fusions to specific nodulation (nod) genes were used to characterise phenolic compounds secreted from the roots of white clover (Trifolium repens) plants. These compounds either had stimulatory or inhibitory effects upon the induction of the nod genes. The stimulatory compounds were hydroxylated flavones and the most active compound was 7,4′‐dihydroxyflavone. The inhibitory compounds present in white clover root exudates were umbelliferone (a coumarin) and formononetin (an isoflavone). Transcriptional activation of nod gene promoters in response to short exposures (3 h) of 7,4′‐dihydroxyflavone was growth phase dependent; cells in early log phase were highly responsive to flavone additions in vitro and nod gene induction could be detected within 20 min of exposure at 5 x 10−7 M. Cells in other growth phases were generally unresponsive. A 10‐fold molar excess of umbelliferone to 7,4′‐dihydroxyflavone resulted in complete inhibition of nod gene induction. Some commercially‐obtained flavones were found to have weak stimulatory activity but could also inhibit nod gene induction by more effective stimulatory compounds. Strong stimulatory and inhibitory compounds all possessed a 7‐hydroxy moiety and showed other structural similarities. This suggested that there was one binding site for these compounds. Because the response to these compounds was rapid, we propose that these phenolics act at the bacterial membrane or that an active uptake system is involved.

Alteration of the Effective Nodulation Properties of a Fast-growing Broad Host Range Rhizobium due to Changes in Exopolysaccharide Synthesis

Summary Strain NGR234 is a broad host range Rhizobium which can effectively nodulate a broad spectrum of legumes. Ninety mutants with altered exopolysaccharide production were isolated after strain NGR234 was subjected to transposon Tn5 mutagenesis. These mutants were classified on the basis of their physiological properties into 9 groups. Their symbiotic porperties were tested on four legumes which form spherical (determinate) nodules ( Macroptilium atropurpureum, Desmodium intortum, Desmodium uncinatum and Lablab purpureus ) and on the legume Leucaena leucocephala which forms cylindrical (indeterminate) nodules. On these plants strain NGR234 forms nitrogen-fixing nodules (Nod + Fix + ). The results from the testing of the various mucoid-defective mutants on these plants show that it is possible to alter the synthesis of the surface polysaccharides of strain NGR234 and produce a narrow host range Nod + Fix + Rhizobium .

Analytical studies of lipopolysaccharide and its derivatives from Salmonella minnesota R595. I. Phosphorus magnetic resonance spectra

Abstract The 31P nuclear magnetic resonance spectrum of lipopolysaccharide from Salmonella minnesota R595 shows that the p K a and chemical shift values of the phosphate groups are very sensitive to changes in molecular structure. The presence of 3′ and / or 6′ substituents can be determined from the spectra of the 4′-phosphates and the type of 4′ or 6′ substituent affects the phosphates. When cells are grown in nutrient broth, the 4′ position in the lipopolysaccharide is substituted with as much as 91% aminoarabinose phosphate, with the remaining 9% as monophosphate (with 3′ acyl substituent). When L.B. medium is used, 52% of the 4′ position is substituted with phosphomonoester (with accompanying 3′-acyl group), with smaller amounts of monoester (with no 3′ substituent) and phosphodiester. Phosphate substitution at the 1 position is incomplete when cells are grown in nutrient broth. A detailed analysis is made of the chemical and physical parameters which influence p K a and chemical shift of phosphates. The conversion of 4′-phosphodiester to 4′-phosphomonoester and 4′-dephospho products with different hydrazine treatments was also studied.

The structure of the exopolysaccharide from Rhizobium sp. strain ANU280 (NGR234)

Abstract The structure of an exopolysaccharide from Rhizobium sp. strain ANU280 (derivative of the broad host-range strain NGR234) has been determined. Fragments generated by partial acid hydrolysis were fractionated by sequential ion-exchange ang gel chromatography, and their structures were assigned by 13 C-n.m.r. spectroscopy. Extensive overlap of structure between the fragments, together with the results of periodate oxidation and colorimetric analyses, permitted assignment of the nonsaccharide repeating-unit shown below. The terminal galactosyl group carries a 4,6- O -(1-carboxyethylidene) group and, probably, an acetyl group.

The reactions of 3-deoxy-d-manno-oct-2-ulosonic acid (KDO) in dilute acid

Abstract On heating in dilute acid, 3-deoxy- d -manno-oct-2-ulosonic acid (KDO) is converted into 2,7-anhydro-3-deoxy-α- d -manno-2-octulofuranosonic acid and 5-( d -erythro-1,2,3-trihydropropyl)-2-furoic acid. The former is unreactive to periodic acid-thiobarbituric acid and to semicarbazide, and its formation explains the depressed estimates of KDO in lipopolysaccharides. Formation of the furoic acid can lead to high estimates using the semicarbazide assay. Neither product can be formed from 5-O-glycosyl-KDO.

Synthesis of methyl pyranosides and furanosides of 3-deoxy-d-manno-oct-2-ulosonic acid (KDO) by acid-catalysed solvolysis of the acetylated derivatives

Treatment of methyl 2,4,5,7,8-penta-O-acetyl-3-deoxy-alpha-D-manno-oct- 2-ulopyranosonic acid, or its methyl ester, with refluxing methanolic 0.1 M hydrogen chloride for 16 h gave 95% of methyl (methyl 3-deoxy-alpha-D-manno-oct-2-ulopyranosid)onate. Acetylation of the methyl ester of 3-deoxy-D-manno-oct-2-ulosonic acid (KDO) gave mainly methyl 2,4,6,7,8-penta-O-acetyl-3-deoxy-alpha,beta-D-manno-oct-2-ulofuranoso nate. Treatment of this mixture with methanolic 0.02 M hydrogen chloride at room temperature gave methyl (methyl 3-deoxy-alpha, beta-D-manno-oct-2-ulofuranosid)onate and the corresponding 4-acetates which were isolated by reverse-phase column chromatography of their 7,8-O-isopropylidene derivatives. Confirmation of the position of the isopropylidene group was obtained by acetylation to give methyl (methyl 4,6-di-O-acetyl-3-deoxy-7,8-O-isopropylidene-alpha,beta-D-manno-oct-2-ul ofuranosid)onate. The furanose anomers were differentiated primarily by J3,4 values (alpha approximately 6.1 Hz, beta approximately 2.2 Hz). The anomeric configuration in the furanose series has been assigned on the basis of optical rotation.

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.

Characterisation of glycerophosphorylated cyclic β-1,2-glucans from a fast-growing Rhizobium species

Abstract A family of cyclic β-1,2-glucans, substituted with sn -glycerol-1-phosphate at the C6 position of some of the glucose residues, has been found in the culture supernatant of the fast-growing, broad host range Rhizobium species, NGR234. The dynamic behaviour of the molecules, studied by 13 C nuclear magnetic resonance spectroscopy, showed that they are disc-shaped with significant, but limited internal motion.

Molecular analysis of the phospholipids of Escherichia coli K12

Phospholipids from Escherichia coli K12 were converted to 1,2-diacylglycerols with phospholipase C from Bacillus cereus. High-pressure liquid chromatography of 1,2-diacylglycerol p-methoxybenzoates on LiChrosorb RP-18 using 2-propanol/acetonitrile (35:65) as eluant permitted separation of 14 molecular species. The main combinations of fatty acids were 1-16:0-2-16:1, 1-16:0-2-cyclo-17:0 and 1-16:0-2-18:1. Positional isomers were not present. The 1,2-di-16:0 compound was present at a significant level (7-10 mol%). Proportions of molecular species varied between phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. Phospholipid from the outer membrane of E. coli K12 contained a lower level of molecules with two unsaturated chains than was present in the cytoplasmic membrane. The method is sensitive, has good resolving power and employs readily available equipment.

Five genetic loci involved in the synthesis of acidic exopolysaccharides are closely linked in the genome of Rhizobium sp strain NGR234

SummaryR-prime plasmids were constructed from a derivative of Rhizobium strain NGR234 (ANU280) and were shown to contain overlapping genomic DNA segments involved in biosynthesis of exopolysaccharides (EPS). The R-primes originally constructed carried the mutant allele from Tn5-induced EPS-deficient (Exo−) mutant ANU2811. This plasmid-located mutant allele was dominant to the corresponding wild-type allele as merodiploid strains were Exo−. Exo+ revertants occurred at a low rate (1×10-7) and these were shown to result from double reciprocal recombination events, which led to the isolation of R-prime plasmids carrying functional wild-type exo alleles. R-prime plasmids that carry overlapping segments of DNA from parental strain ANU280 complemented 28 of the 30 group 2 Exo− mutants of strain ANU280. Complementation of these Exo− mutants also restored their symbiotic abilities of effective nodulation. Subsequent in vivo recombination between the wild-type alleles located on the R-prime and the corresponding mutated allele on the genome, was used to generate a new family of R-primes, which carried mutations in the exo genes. The 30 group 2 Exo− mutants were classified into 7 distinct genetic groups based upon complementation and physical mapping data. Five of the seven exo loci were gentically linked and located on a 15-kb region of DNA. Mutations at two loci were dominant only when the mutations were R-prime plasmid-located while a mutation at a second locus was cis-dominant to two other exo loci. At least five genes involved in the synthesis of acidic exopolysaccharide synthesis have been identified.