Lipopolysaccharide profiles from nodules as markers of bradyrhizobium strains nodulating wild legumes.

To develop the use of electrophoretic lipopolysaccharide profiles for Bradyrhizobium strain identification, we studied the feasibility of using electrophoresis of whole legume nodule homogenates to obtain distinctive lipopolysaccharide profiles. The electrophoretic patterns were the same whether we used nodule extracts, bacteroids, or cultured bacteria as samples, and there was no evidence of changes in the ladder-like pattern during the nodulation process. To assess the reliability of using lipopolysaccharide profiling performed with individual nodules for studying the diversity and microdistribution of the rhizobia nodulating wild shrub legumes, we used a population of Adenocarpus foliolosus seedlings. We obtained 75 different profiles from the 147 nodules studied. There was no dominant profile in the sample, and a plant with different nodules generally produced different profiles. Electrophoresis of legume root nodules proved to be a fast and discriminating technique for determining the diversity of a bradyrhizobial population, although it did not allow the genetic relationships among the nodulating strains to be studied.

Transient energy coupling between rhizobia and legume cells mediated by the peribacteroid membrane ATPase proton pump.

We present a model for the metabolic coupling between rhizobia and plant cell in the nitrogen-fixing legume root nodules. The symbiosome, an organelle-like structure formed by the modified rhizobia (the bacteroids) enclosed by a plant cell derived peribacteroid membrane, is an unique structure in which two energized membranes are closely packed: the inner bacteroid membrane and the peribacteroid membrane that possesses an ATPase proton pump. The model is based on the following points: (i) The permeability for hydrogen ions of the outer membrane of the rhizobia. (ii) The reversibility of the ATPase proton pump of the peribacteroid membrane [Szafran, M.M. and Haaker, H. (1995) Plant Physiol. 108, 1227-1232]. (iii) The relative affinites for oxygen of the bacteroid and plant mitochondria terminal oxidases, and the prevailing oxygen concentration inside the nodule, which results in aerobic metabolism for the bacteroid, but in quite fermentative catabolism for the host plant cell. We propose that the bacteroid can transiently supply free energy to the plant cell in the form of protonmotive force by the movement of hydrogen ions from the bacteroid periplasmic space to the plant cytoplasm through the peribacteroid membrane ATPase. The proposed hydrogen ion flux could be dependent on the phosphorylation potential in both the plant cell cytoplasm and the bacteroid, and the simultaneous ion movements to avoid the development of opposite delta psi. It could be important in situations of transient ATP depletion inside plant cell, which involves the block of ammonia assimilation and, subsequently, the inhibition of bacteroid nitrogenase.

Electrochemical measurement of trace concentrations of biological hydrogen produced by Enterobacteriaceae.

Accurate measurements of the hydrogen gas produced by Escherichia coli and Hafnia alvei pure cultures during glucose metabolism were performed under different growth conditions: stagnant, with magnetic stirring or with vibrational shaking. These measurements were carried out using an electrochemical hydrogen sensor based on a platinum-coated solid polymer electrolyte membrane (Pt-SPE). The results obtained were dependent on the hydrodynamic conditions of the growth, with greater hydrogen production being associated with the stagnant conditions. These measurements will eventually enable us to elucidate whether the pathway used for glucose metabolism is either strictly or mainly anaerobic and to modify experimental conditions so as to influence the reaction.

Precipitation of Metallic Cations by the Acidic Exopolysaccharides from Bradyrhizobium japonicum and Bradyrhizobium (Chamaecytisus) Strain BGA-1.

The interaction between the acidic exopolysaccharides produced by two Bradyrhizobium strains and several metal cations has been studied. Aqueous solutions in the millimolar range of Fe but not of Fe precipitated the exopolysaccharides from Bradyrhizobium (Chamaecytisus) strain BGA-1 and, to a lesser extent, Bradyrhizobium japonicum USDA 110. The precipitation was pH dependent, with a maximum around pH 3. The precipitate was redissolved by changing the pH and by Fe reduction or chelation. Deacetylation of B. japonicum polysaccharide increased its precipitation by Fe. At pH near neutrality, the polysaccharide from Bradyrhizobium (Chamaecytisus) strain BGA-1 stabilized Fe solutions, despite the insolubility of Fe(OH)(3). Aluminum precipitated Bradyrhizobium (Chamaecytisus) polysaccharide but not the polysaccharide produced by B. japonicum. The precipitation showed a maximum at about pH 4.8, and the precipitate was redissolved after Al chelation with EDTA. Precipitation was inhibited by increases in the ionic strength over 10 mM. Bradyrhizobium (Chamaecytisus) polysaccharide was also precipitated by Th, Sn, Mn, and Co. The presence of Fe increased the exopolysaccharide precipitation by aluminum. No precipitation, gelation, or increase in turbidity of polysaccharide solutions occurred when K, Na, Ca, Mg, Cu, Cd, Pb, Zn, Hg, or U was added at several pH values. The results suggest that the precipitation is based on the interaction between carboxylate groups from different polysaccharide chains and the partially hydrolyzed aquoions of Fe, Al, Th, and Sn.

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.

Alcian blue fixation allows silver staining of the isolated polysaccharide component of bacterial lipopolysaccharides in polyacrylamide gels.

The effect of the cationic dye Alcian Blue on the silver staining of bacterial lipopolysaccharide and its polysaccharide and lipid A portions in polyacrylamide gels was investigated. The polysaccharide was only stained when the gel was previously treated with the dye. The polysaccharide moiety was found to be responsible for the lipopolysaccharide staining with silver, whereas the lipid A seemed unimportant. Treatment with Alcian Blue may prove useful to detect hydrophilic components of lipopolysaccharide samples that could not be stained by the usual silver staining procedures.

Mode of action of netzahualcoyone.

The activity of netzahualcoyone on Bacillus subtilis and Escherichia coli was studied. The product inhibited the respiration of intact cells of B. subtilis but had no effect on the respiration of E. coli. However, when preparations of sonically disrupted cells were examined, inhibitory activity on both bacteria was observed.

L-form-like colonies of Staphylococcus aureus induced by an extracellular lytic enzyme from Pseudomonas aeruginosa.

An extracellular enzyme produced by Pseudomonas aeruginosa had a lytic effect on lyophilized Staphylococcus aureus cells. It was purified from the culture supernatant by ammonium sulfate fractionation followed by column chromatography with P cellulose and Sephadex G-50. The molecular weight of the enzyme was estimated to be 19,000 +/- 1,750 with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pI of the enzyme was estimated to be 8.5 with isoelectric focusing. The enzyme was inactive in 4% NaC1-40 mM sodium phosphate buffer or at pH values lower than 6.0 or higher than 11.0; however, it was not affected by 1 M sucrose or 0.25% heat-denatured horse serum. The action of the enzyme on cultures of S. aureus resulted in the presence of many cells lacking cell walls. In addition, when cultivation was carried out on osmotically stabilized solid media, these cell wall-deficient cell developed in L-form colonies.

Purification and peptidase activity of a bacteriolytic extracellular enzyme from Pseudomonas aeruginosa.

A bacteriolytic enzyme excreted by Pseudomonas aeruginosa Paks I was purified: samples were found to be homogeneous by gel filtration chromatography, ion exchange chromatography using CM-cellulose, immunoelectrophoresis, PAGE and SDS-PAGE. The molecular weight of the lytic enzyme was estimated to be 15,000-19,000. The enzyme was active on Gram-positive bacteria with glycine-containing interpeptide bridges in their murein layers. In addition, this lytic enzyme showed peptidase activity catalysing the hydrolysis of pentaglycine peptides into tri- and diglycine peptides.

Effects of staphylolytic enzymes from Pseudomonas aeruginosa on the growth and ultrastructure of Staphylococcus aureus.

Pseudomonas aeruginosa produces two extracellular staphylolytic enzymes able to lyse Staphylococcus aureus cells when they are added to liquid cultures of S. aureus. In addition, when cultivation was carried out in the presence of both lytic enzymes and 1 M sucrose, the staphylococci either lacked cell walls or showed damaged walls. Lytic activity-resistant cells of S. aureus were also detected.