Rhizobium trifolii mutant defective in the structure of lipopolysaccharide

Rhizobium trifolii AR182, a mutant resistant to rhizobiophages lysing the parental strain AR5, formed abortive nodules on the clover plant roots. The polyacrylamide gel electrophoresis of the isolated lipopolysaccharide revealed only one band. On the other hand, the lipopolysaccharide isolated from the non-mucoid mutant R. trifolii AR16 showed several, regularly spaced bands in the high molecular weight region. The results suggest that R. trifolii AR182 is a rough (R)-mutant.Abbreviations LPS lipopolysaccharide - EPS exopolysaccharide - CPS capsular polysaccharide - DOC sodium deoxycholate - PAGE polyacrylamide gel electrophoresis - GC-MS gas liquid chromatography-mass spectrometry - KDO 2-keto-3-deoxy-octonic acid - Rha rhamnose - Fuc fucose - Man mannose - Gal galactose - Glc glucose - UA uronic acid     

A core oligosaccharide component from the lipopolysaccharide of Rhizobium trifolii ANU843

The major oligosaccharide from the core region of the lipopolysaccharide from R. trifolii ANU843 was isolated and its structure determined. It is a trisaccharide consisting of two galacturonic acid residues and one 3-deoxy-D-manno-2-octulosonic acid (KDO) residue. The two galacturonic acid residues are terminally linked alpha to the C-4 and C-7 atoms of KDO. This structure was determined through use of 1H- and 13C-n.m.r. spectroscopy, f.a.b.-m.s., and g.l.c.-m.s. techniques. This oligosaccharide had not previously been reported to be present in the lipopolysaccharides from Gram-negative bacteria.     

A new core tetrasaccharide component from the lipopolysaccharide of Rhizobium trifolii ANU 843

A second core oligosaccharide fragment has been isolated and characterized from the lipopolysaccharide (LPS) of Rhizobium trifolii ANU 843. The oligosaccharide is a tetrasaccharide composed of galactose, galacturonic acid, mannose, and 3-deoxy-D-manno-2-octulosonic acid. The mannose residue is alpha-linked to the 4-position of 3-deoxy-D-manno-2-octulosonic acid and the galacturonic acid residue is alpha-linked to the 6-position of mannose. The galactose residue, which is acetylated at the 4-position, is attached to the 4-position of mannose by an alpha-linkage. All of the aldoses are in the pyranose form. The composition of the tetrasaccharide was determined by gas-liquid chromatography of the alditol acetate derivatives of the component monosaccharides. The configuration of anomeric linkages was determined by 1H NMR spectroscopy. Fast atom bombardment-mass spectrometry (FAB-MS) was performed on acetylated, per(trideutero)acetylated and underivatized tetrasaccharide giving sequence information in addition to information on the residue which was acetylated. Similar studies were performed on the oligosaccharide after reduction with sodium cyanoborohydride and peracetylation with labeled and unlabeled acetic anhydride as before. Further linkage and sequence analysis was obtained from methylation analysis, and from electron impact mass spectrometry of the per(trideutero)acetylated oligosaccharide and from collision-induced dissociation fast atom bombardment tandem mass spectrometry using linked scans at constant B/E on the cyanoborohydride-reduced, per (trideutero)acetylated oligosaccharide. The exact location of the acetyl group was deduced from 1H NMR double resonance experiments in conjunction with mass spectrometric data.     

Characterization of the lipopolysaccharide from a Rhizobium phaseoli mutant that is defective in infection thread development.

The lipopolysaccharide (LPS) from a Rhizobium phaseoli mutant, CE109, was isolated and compared with that of its wild-type parent, CE3. A previous report has shown that the mutant is defective in infection thread development, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that it has an altered LPS (K. D. Noel, K. A. VandenBosch, and B. Kulpaca, J. Bacteriol. 168:1392-1462, 1986). Mild acid hydrolysis of the CE3 LPS released a polysaccharide and an oligosaccharide, PS1 and PS2, respectively. Mild acid hydrolysis of CE109 LPS released only an oligosaccharide. Chemical and immunochemical analyses showed that CE3-PS1 is the antigenic O chain of this strain and that CE109 LPS does not contain any of the major sugar components of CE3-PS1. CE109 oligosaccharide was identical in composition to CE3-PS2. The lipid A's from both strains were very similar in composition, with only minor quantitative variations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of CE3 and CE109 LPSs showed that CE3 LPS separated into two bands, LPS I and LPS II, while CE109 had two bands which migrated to positions similar to that of LPS II. Immunoblotting with anti-CE3 antiserum showed that LPS I contains the antigenic O chain of CE3, PS1. Anti-CE109 antiserum interacted strongly with both CE109 LPS bands and CE3 LPS II and interacted weakly with CE3 LPS I. Mild-acid hydrolysis of CE3 LPS I, extracted from the polyacrylamide gel, showed that it contained both PS1 and PS2. The results in this report showed that CE109 LPS consists of only the lipid A core and is missing the antigenic O chain.     

Characterization of an aromatic amino acid aminotransferase from Rhizobium leguminosarum biovar trifolii.

The most abundant aromatic amino acid aminotransferase of Rhizobium leguminosarum biovar trifolii was partially purified. The molecular mass of the enzyme was estimated to be 53 kDa by gel filtration. The enzyme transaminated aromatic amino acids and histidine. It used aromatic keto acids and alpha-ketoglutaric and oxalacetic acids as amino-group acceptors. The optimum temperature was 35 degrees C. Using phenylalanine and alpha-ketoglutaric acid as substrates the activation energy was 46.2 kJ.mol-1 and for the couple tryptophan:alpha-ketoglutaric acid it was 70.3 kJ.mol-1. The optimum pH was different for each substrate: 7.3 for phenylalanine, 7.9 for histidine and 8.7 for tryptophan.     

Symbiotic effects of deltamatB Rhizobium leguminosarum bv. trifolii mutant on clovers

The role of malonate in symbiotic nitrogen metabolism has long been controversial, although it is known to occur in legume roots, especially in the nodules. Here we report that malonate metabolism plays a key role in the differentiation of bacteroids Rhizobium leguminosarum bv. trifolii in clover nodules. An operon, mat, that consists of three consecutive genes (matABC) has been discovered. Mat encodes enzymes that catalyze the uptake and conversion of malonate to acetyl-CoA through malonyl-CoA. A mutant bacteria, which replaced matB that encodes malonyl-CoA synthetase with a kanamycin resistant gene, was generated and infected with white clover. Clover growth was considerably reduced, even though nodules were formed. However, the nodules were filled with vacuoles, but not with bacteroids. This indicates that malonate metabolism is an important requirement for the formation of mature nodules that are filled with bacteroids.     

Characterization of sialic acids containing lipopolysaccharide from Rhizobium meliloti M 11 S

Abstract The lipopolysaccharide (LPS) of Rhizobium meliloti strain M 11 S was isolated and analyzed. It contained fatty acids (3-hydroxymyristic, palmitic, stearic, arachidic acids) and sugars: glucose, galactose, glucosamine, 3-deoxy- d -mannooctulosonic acid and sialic acids (NeuAc, 9- O -acetyl-NeuAc) identified by combined gas-liquid-chromatography/ mass spectrometry (GLC-MS).     

Bacteriocinogeny of Rhizobium trifolii.

Rhizobium trifolii strains differing in cell and colony morphology, streptomycin resistance, phage sensitivity pattern and infectivity to clover plants produced bacteriocins sensitive to proteases. Elimination of bacteriocin production ability wtih SDS and rifampicin treatment indicates that this feature is plasmid controlled. Elimination of bacteriocinogenic plasmid did not influence other features of R. trifolii.     

Isolation and Partial Characterization of DNA in Capsular Preparations of Rhizobium trifolii

The capsular polysaccharides from thymidine-(methyl-³H) labeled cultures of Rhizobium trifolii; strain 162S7 (Nitragin Co.) were centrifuged from bacterial cells and collected by ethanol precipitation. Following the addition of unlabeled carrier nucleic acids, labeled DNA, termed cap-DNA, was isolated from the capsular polysaccharides. Cap-DNA absorbed maximally at H-260 nm and was DNase sensitive. Approximately 11 μg of ³H-cap-DNA were consistently isolated per liter of 48 h cultures. Cap-DNA production was generally synchronized with the synthesis of the capsular polysaccharide and bacterial growth, attaining maximum recoverable amounts in 48 h cultures. By five days of culture growth, significant decreases in the amount of recoverable cap-DNA were noted. The presence of label in the cap-DNA demonstrated that the cap-DNA originated via de novo synthesis by the Rhizobium cells rather than from an anomalous source. The cap-DNA and intracellular Rhizobium DNA had similar buoyant densities of p= 1.719, indicating that cap-DNA arose specifically from the intracellular DNA. In 48-h cultures the specific activity of the cap-DNA was about one-third that of the intracellular DNA. This implies intracellular DNA was released during early growth with a relatively low specific activity which diluted the isotopic label of DNA released later. The evidence suggests lysogeny was the principal release mechanism.     

Conserved Nodulation Genes in Rhizobium meliloti and Rhizobium trifolii

Plasmids which contained wild-type or mutated Rhizobium meliloti nodulation (nod) genes were introduced into Nod⁻R. trifolii mutants ANU453 and ANU851 and tested for their ability to nodulate clover. Cloned wild-type and mutated R. meliloti nod gene segments restored ANU851 to Nod⁺, with the exception of nodD mutants. Similarly, wild-type and mutant R. meliloti nod genes complemented ANU453 to Nod⁺, except for nodCII mutants. Thus, ANU851 identifies the equivalent of the R. meliloti nodD genes, and ANU453 specifies the equivalent of the R. meliloti nodCII genes. In addition, cloned wild-type R. trifolii nod genes were introduced into seven R. meliloti Nod⁻ mutants. All seven mutants were restored to Nod⁺ on alfalfa. Our results indicate that these genes represent common nodulation functions and argue for an allelic relationship between nod genes in R. meliloti and R. trifolii.     

Growth-phase-dependent immunodeterminants of Rhizobium trifolii lipopolysaccharide which bind trifoliin A, a white clover lectin.

The lipopolysaccharide (LPS) from Rhizobium trifolii 0403 was isolated at different stages of growth and was examined for its (i) ability to bind a white clover lectin (trifoliin A), (ii) immunochemical properties, and (iii) composition. There was significantly more binding of trifoliin A to purified LPS and cells in the early stationary phase than to cells in the exponential phase. Immunofluorescence and enzyme-linked immunosorbent assays indicated that new antigenic determinants of the LPS appeared for brief periods on cells at the end of the lag phase and again at the beginning of the stationary phase. These new antigens were not detected on cells in midexponential or late stationary phase. Monovalent fragments of immunoglobulin G antibodies raised against the unique antigenic determinants in the LPS competitively blocked the binding of trifoliin A to cells in the early stationary phase. Gas chromatographic analysis showed that the relative quantity of several glycosyl components in the LPS increased as the culture advanced from the midexponential to the early stationary phase. In addition, LPS from cells in the early stationary phase had a higher aggregate molecular weight. Quinovosamine (2-amino-2,6-dideoxyglucose) was identified by combined gas chromatography-mass spectrometry as a sugar component of the LPS which had not been previously reported. D-Quinovosamine, N-acetyl-D-quinovosamine, and its n-propyl-beta-glycoside were effective hapten sugars which inhibited the binding of trifoliin A, anti-clover root antibody, and homologous antibody to these new determinants in the LPS. White clover plants had more infected root hairs after incubation with an inoculum of cells in the early stationary phase than after incubation with cells in the midexponential phase. The profound influence of the growth phase on the composition of lectin-binding polysaccharides of Rhizobium may be a major underlying cause of conflicting data among laboratories testing the lectin-recognition hypothesis. In addition, these chemical modifications may reflect mechanisms which regulate Rhizobium-root hair recognition in this nitrogen-fixing symbiosis.     

A Rhizobium leguminosarum AcpXL mutant produces lipopolysaccharide lacking 27-hydroxyoctacosanoic acid

The structure of the lipid A from Rhizobium etli and Rhizobium leguminosarum lipopolysaccharides (LPSs) lacks phosphate and contains a galacturonosyl residue at its 4' position, an acylated 2-aminogluconate in place of the proximal glucosamine, and a very long chain omega-1 hydroxy fatty acid, 27-hydroxyoctacosanoic acid (27OHC28:0). The 27OHC28:0 moiety is common in lipid A's among members of the Rhizobiaceae and also among a number of the facultative intracellular pathogens that form chronic infections, e.g., Brucella abortus, Bartonella henselae, and Legionella pneumophila. In this paper, a mutant of R. leguminosarum was created by placing a kanamycin resistance cassette within acpXL, the gene which encodes the acyl carrier protein for 27OHC28:0. The result was an LPS containing a tetraacylated lipid A lacking 27OHC28:0. A small amount of the mutant lipid A may contain an added palmitic acid residue. The mutant is sensitive to changes in osmolarity and an increase in acidity, growth conditions that likely occur in the nodule microenvironment. In spite of the probably hostile microenvironment of the nodule, the acpXL mutant is still able to form nitrogen-fixing root nodules even though the appearance and development of nodules are delayed. Therefore, it is possible that the acpXL mutant has a host-inducible mechanism which enables it to adapt to these physiological changes.