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Protozoa and the decline of Rhizobium populations added to soil.

A fall in Rhizobium abundance occurred in nonsterile soil inoculated with large numbers of the root-nodule bacteria, but many of the rhizobia still survived. No such decline was evident in sterile soil. Protozoa feeding on these bacteria were isolated from soil and other environments. As the abundance of Rhizobium meliloti and a cowpea Rhizobium strain in soil decreased, the protozoan density increased. The inability of the predators to eliminate their prey from soil was not the result of the presence of organisms feeding on the protozoa because many rhizobia survived in sterile soil inoculated with the prey and cultures of individual protozoa, nor was it the result of the rapid multiplication of the bacteria to replace those consumed because survivors were still numerous in essentially organic matter free soil in which the bacteria did not grow appreciably. The lack of elimination also was not associated with a protective effect of soil particles because survivors were still abundant in solutions inoculated with protozoa and bacteria. It is suggested that the size of the prey population diminishes until a density is attained at which the energy used by the predator in hunting for the survivors equals that obtained from the feeding.

Amoeba

Effects of interactions between different culture fractions of 'phosphobacteria' and Rhizobium on mycorrhizal infection, growth, and nodulation of Medicago sativa.

Interactions between cell-free culture supernatants, cells, and the whole cultures of Rhizobium and phosphobacteria with endomycorrhizal fungi and their effects on growth and nutrition of Medicago sativa grown in a low-phosphate soil were studied. A satisfactory nodulation was greatly dependent on the mycorrhizal symbiosis. Cell-free supernatants of Rhizobium and phosphobacteria improved plant growth, nodulation and mycorrhiza formation. The activity of phosphobacterial culture seemed to be due mainly to the supernatant and the possibility of plant hormones contained in this culture fraction being involved in such interactions is discussed. An increase of the overall pool of soluble P in soil by the inoculated phosphobacteria cells was not found in the conditions of this experiment. It was noteworthy that the best positive effect was achieved by the treatment which consisted of the whole cultures of Rhizobium, phosphobacteria, and the mycorrhizal fungi applied all together.

Fungi

[Deformation of lucerne root hairs caused by the growth substances and culture broth filtrates of Rhizobium meliloti].

Deformation of lucerne root hairs caused by the action of beta-indolylacetic acid (IAA) and alpha-naphthylacetic acid (NAA) differs from deformation induced by Rhizobium meliloti. High concentrations of IAA brought about abnormal deformation of root hairs wherease the action of average concentrations of IAA resulted in corkscrew-like winding and wavy structures. No deformation was observed under the action of low IAA concentrations or the cultural broth of Rhizobium. Infective filaments in root hairs were found only upon infection with pure cultures of Rhizobium.

Acetates

Salt tolerance of Rhizobium species in broth cultures.

Salt tolerance of five rhizobia strains was examined in broth cultures. Five levels of NaCl concentration were used and the optical density was taken as a measure for the vigour of bacterial growth. Rhizobium leguminosarum and R. meliloti were tolerant to high levels of salinity and growth curves in saline broth showed a similar pattern to the control level. Rhizobium japonicum, cowpea Rhizobium, and R. trifolii were intolerant to salt and showed a strong growth retardation with increasing salt concentration. Growth was inhibited at high levels of salinity. It is suggested that rhizobia sensitivity to salts may be partly responsible to the inhibition of nitrogen fixation by legumes growing under salt stress.

Culture Media

Carotenoids of rhizobia. II. The effect of nicotine on the carotenoid pattern of Rhizobium lupini.

With increasing concentrations in the growth medium of the cyclization inhibitors nicotine or 2-(4-chlorophenylthio)-triethylamine hydrochloride (CPTA) the previously identified bicyclic carotenoids of Rhizobium lupini (2,3,2',3'-tetrahydroxy-beta,beta-caroten-4-one and 2,3,2',3'-tetrahydroxy-beta,beta-carotene) were successively replaced by hitherto unknown monocyclic carotenoids. By application of mass and nuclear magnetic resonance spectroscopy 3 carotenoids were identified as 2,3-trans-dihydroxy-beta,psi-caroten-4-one, 2,3-trans-dihydroxy-beta,psi-carotene, and 3-hydroxy-beta,psi-caroten-4-one. A further compound was tentatively established as (2- or 3-)monohydroxy-beta,psi-carotene. It was found that other inhibitors such as diphenylamine or 4-chloro-5-(dimethylamino)-2-alpha,alpha,alpha(trifluoro-m-tolyl)-3-(2H)-pyridazinone (San 6706) did not affect the pigment pattern. The results are discussed in relation to carotenoid biosynthesis in Rhizobium lupini.

Carotenoids

Immunological evidence for the capability of free-living Rhizobium japonicum to synthesize a portion of a nitrogenase component.

Immunodiffusion tests conducted under aerobic conditions demonstrated that cross-reactive material to antiserum prepared against the Mo-Fe protein component of nitrogenase from soybean nodule bacteroids was detectable in extracts of free-living Rhizobium japonicum cells cultured in a standard medium under: aerobic conditions; aerobic conditions with nitrate; aerobic conditions with ammonia; anaerobic conditions with nitrate; and anaerobic conditions with nitrate and ammonia. The most intense precipitin bands resulted from cross-reaction of the antiserum with extracts of cells cultured anaerobically with nitrate or anaerobically with ammonia and nitrate. Immunodiffusion experiments with crude bacteroid extract and purified Mo-Fe protein revealed a greater number of precipitin bands in tests conducted under aerobic conditions than those conducted under anaerobic conditions. These results indicate that some of the cross-reactive material observed under aerobic conditions resulted from breakdown of the Mo-Fe protein. Bacteroid extracts of nodules from plants supplied with ammonia exhibited only a trace of nitrogenase activity. The addition of an excess of the Fe protein component of nitrogenase, however, resulted in 270-fold enhancement of activity indication the presence of active Mo-Fe protein in these extracts. Our experiments together with results published elsewhere provide evidence that the genetic information for synthesis of a part of the Mo-Fe component of nitrogenase is carried by Rhizobium.

Aerobiosis

Effect of lindane on radio-carbon (14C) incorporation by Rhizobium japonicum.

Experiments conducted in vitro with three levels (1, 2, and 5 ppm active ingredient) of the insecticide lindane (gamma-BHC) showed no effect on the growth of Rhizobium japonicum, but altered the incorporation of radio-carbon (14C-glucose) into the different constituents of the growing cells. While all the three levels of the insecticide significantly depressed the incorporation of radiocarbon in the alcohol-extractable fraction, with no effect on the alcohol-ether soluble fraction of the cells, the 2 and 5 ppm levels enhanced the incorporation rate in the cold-TCA soluble fraction, but reduced it in the hot-TCA soluble fraction. Only with the 5ppm level of the insecticide treatment an increase in the specific activity of the insoluble protein fraction of the cells was observed. The results indicated that lindane, at various concentrations, affected the carbon (glucose) metabolism of the Rhizobium cells.

Glucose

Re-examination of transformation within different species of Rhizobium.

Investigations of the phenomenon of transformation in Rhizobium were carried out. Streptomycin resistance (str) was the genetic marker used in all experiments, with the exception of auxotrophic strains. Twenty-one experiments were performed on nine different Rhizobium strains. Some of these strains were previously reported to be transformed, while others had no prior history of transformation. Different conditions which are thought to affect the development of competence were used. In these experiments no positive results were obtained. The possibility that the experiments failed, due to inactivation of donor DNA during its preparation, was ruled out by comparison with results obtained with strains of Bacillus subtilis.

Bacillus subtilis

The Rhizobium--legume symbiosis.

The rhizobia are soil microorganisms that can interact with leguminous plants to form root nodules within which conditions are favourable for bacterial nitrogen fixation. Legumes allow the development of very large rhizobial populations in the vicinity of their roots. Infections and nodule formation require the specific recognition of host and Rhizobium, probably mediated by plant lectins. Penetration of the host by a compatible Rhizobium species usually provokes host root cell division to form the nodule, and a process of differentiation by both partners then ensues. In most cases the rhizobia alter morphologically to form bacteroids, which are usually larger than the free-living bacteria and have altered cell walls. At all stages during infection, the bacteria are bounded by host cell plasmalemma. The enzyme nitrogenase is synthesized by the bacteria and, if leghaemoglobin is present, nitrogen fixation will occur. Leghaemoglobin is a product of the symbiotic interaction, since the globin is produced by the plant while the haem is synthesized by the bacteria. In the intracellular habitat the bacteria are dependent upon the plant for supplies of energy and the bacteroids, in particular, appear to differentiate so that they are no longer able to utilize the nitrogen that they fix. Regulation of the supply of carbohydrate and the use of the fixed nitrogen thus appear to be largely governed by the host.

Leghemoglobin

Adsorption of a phage tail-like bacteriocin to isolated lipopolysaccharide of Rhizobium.

Purified lipopolysaccharide (LPS) from the bacteriocin sensitive strain Rhizobium lupini i6-2 was shown to neutralize the killing activity of the bacteriocin. In the electron microscopical preparation the phage tail-like bacteriocin appears to be adsorbed to the LPS; the tail sheath is contracted and the fibres are oriented towards the LPS ribbon. In contrast, no interaction was observed between the bacteriocin and the LPS of two resistant strains of Rhizobium (16-2/Ii and 16-3). The inactivation of the bacteriocin by LPS depends on salt concentration, pH, and temperature. The receptor activity of LPS was destroyed by mild acid hydrolysis and by treatment with deoxycholate, which indicates that the micellar structure of the LPS is necessary for bacteriocin adsorption. The chemical composition of the 16-2 LPS was compared to that of the LPS of two resistant strains. In the case of 16-2/ii LPS minor modifications suffice to confer resistance against the bacteriocin.

Acetates

Adsorption of bacteria to roots as related to host specificity in the Rhizobium-clover symbiosis.

Quantitative microscope techniques were utilized to examine the adsorption of rhizobial cells to clover root hairs. Adsorption of cells of noninfective strains of Rhizobium trifolii or infective R. meliloti strains to clover root hairs was four to five times less than that of the infective R. trifolii strains. Attachment of the rod-shaped bacteria to clover root cells occurred in a polar, end-on fashion. Viable or heat-killed R. trifolii cells precoated with a clover lectin having 2-deoxyglucose specificity had increased adsorption to clover roots. Adsorption of bacteria to roots was not increased if the clover lectin was inactivated by heat or 2-deoxyglucose treatment prior to incubation with R. trifolii. Adsorption of R. trifolii to clover root hairs was inhibited by 2-deoxyglucose (30 mM) but not by 2-deoxygalactose or alpha-D-glucose. Adsorption of R. meliloti cells to alfalfa root hairs was not affected by 2-deoxyglucose at that concentration. These results suggest that expression of host specificity in the Rhizobium-clover symbiosis involves a preferential adsorption of infective cells to clover root hairs through a 2-deoxyglucose-sensitive receptor site.

Adsorption

Resistance of Rhizobium strains to phygon, spergon, and thiram.

Strains of Rhizobium meliloti, Rhizobium sp. nodulating cowpeas, and R. phaseoli derived from cultures susceptible to tetramethylthiuram disulfide (thiram), 2,3-dichloro-1,4-naphthoquinone (phygon), and 2,3,5,6-tetrachloro-p-benzoquinone (spergon), respectively, grew in the presence of high concentrations of the fungicides and converted them to products not toxic to the sensitive rhizobia. The results of chemical assays demonstrated that the pesticides were destroyed by the resistant bacteria but not by the susceptible parent rhizobia. Resting cells of thiram-metabolizing R. meliloti formed large quantities of dimethyldithiocarbamate, dimethylamine, and CS2 from the pesticide. The products were characterized by gas and thin-layer chromatography, colorimetric reactions, and ultraviolet spectrometry. Dimethylamine and CS2 were formed spontaneously from dimethyldithiocarbamate, but the yield was higher in the presence of R. meliloti. The phygon-resistant bacterium converted the fungicide to five metabolites and thereby rendered the chemical nontoxic to a test fungus. The resistant strain of R. phaseoli generated at least one organic product and released about one-third of the chlorine during its detoxication of spergon.

Biodegradation, Environmental

Transfer of R factors to and between genetically marked sublines of Rhizobium japonicum.

Plasmids R1822 and pRD1 of the P-1 incompatibility group, for which Rhizobium japonicum had not previously been shown to serve as host, were introduced into a strain of R. japonicum. Acquisition of R68 and R68.45 plasmids by this Rhizobium was equivocal. Transfer of R1822 from Pseudomonas aeruginosa and of pRD1 from Escherichia coli to R. japonicum was unambiguous, because the exconjugants subsequently cotransferred the three R-factor resistance determinants (kanamycin, tetracycline, and penicillin) between genetically marked sublines of strain I-110. Under optimal conditions the transfer of R1822 and pRD1 occurred at frequencies of approximately 10(-3) in plate matings of strains bearing as many as five dissimilar genetic markers. In matings with R1822 on membrane filters, recombinants were formed at incidences as high as 4%.

Anti-Bacterial Agents

Cross-reactive antigens and lectin as determinants of symbiotic specificity in the Rhizobium-clover association.

Cross-reactive antigens of clover roots and Rhizobium trifolii were detected on their cell surfaces by tube agglutination, immunofluorescent, and radioimmunoassay techniques. Anti-clover root antiserum had a higher agglutinating titer with infective strains of R. trifolii than with noninfective strains. The root antiserum previously adsorbed with noninfective R. trifolii cells remained reactive only with infective cells, including infective revertants. When adsorbed with infective cells, the root antiserum was reactive with neither infective nor noninfective cells. Other Rhizobium species incapable of infecting clover did not demonstrate surface antigens cross-reactive with clover. Radioimmunoassay indicated twice as much antigenic cross-reactivity of clover roots and R. trifolii 403 (infective) than R. trifolii Bart A (noninfective). Immunofluorescence with anti-R. trifolii (infective) antiserum was detected on the exposed surface of the root epidermal cells and diminished at the root meristem. The immunofluorescent crossreaction on clover roots was totally removed by adsorption of anti-R. trifolii (infective) antiserum with encapsulated infective cells but not with noninfective cells. The cross-reactive capsular antigens from R. trifolii strains were extracted and purified. The ability of these antigens to induce clover root hair deformation was much greater when they were obtained from the infective than noninfective strains. The cross-reactive capsular antigen of R. trifolii 403 was characterized as a high-molecular-weight (greater than 4.6 times 10(6) daltons), beta-linked, acidic heteropolysaccharide containing 2-deoxyglucose, galactose, glucose, and glucuronic acid. A soluble, nondialyzable, substance (clover lectin) capable of binding to the cross-reactive antigen and agglutinating only infective cells of R. trifolii was extracted from white clover seeds. This lectin was sensitive to heat, Pronase, and trypsin. inhibition studies indicated that 2-deoxyglucose was the most probable haptenic determinant of the cross-reactive capsular antigen capable of binding to the root antiserum and the clover lectin. A model is proposed suggesting the preferential adsorption of infective versus noninfective cells of R. trifolii on the surface of clover roots by a cross-bridging of their common surface antigens with a multivalent clover lectin.

Antigens, Bacterial

Bacterial polysaccharide which binds Rhizobium trifolii to clover root hairs.

Immunofluorescence, quantitative immunoprecipitation, and inhibition of bacterial agglutination and passive hemagglutination indicate that cross-reactive antigenic determinants are present on the surface of Rhizobium trifolii and clover roots. These determinants are immunochemically unique to this Rhizobium-legume cross-inoculation group. The multivalent lectin trifoliin and antibody to the clover root antigenic determinants bind competitively to two acidic heteropolysaccharides isolated from capsular material of R. Trifolii 0403. The major polysaccharide is an antigen which lacks heptose, 2-keto-3-deoxyoctulosonic acid, and endotoxic lipid A. The minor polysaccharide in the capsular material of R. Trifolii 0403 contains the same antigen in addition to heptose, 2-keto-3-deoxyoctonate, and lipid A. The acidic polysaccharides of two strains of R. trifolii share the clover r-ot cross-reactive antigenic determinant despite other differences in their carbohydrate composition. Studies with monovalent antigen-binding fragments of anti-clover root antibody and Azotobacter vinelandii hybrid transformants carrying the unique antigenic determinant suggest that these polysaccharides bind R. trifolii to the clover root hair tips which contain trifoliin.

Azotobacter

The role of 6-phosphogluconate dehydrogenase in Rhizobium.

A nicotinamide adenine dinucleotide (NAD) linked 6-phosphogluconate (6-PG)dehydrogenase has been detected in Rhizobium. The enzyme activity is similar in both slow- and fast-growing rhizobia. The nicotinamide adenine dinucleotide phosphate (NADP) dependent 6-PG dehydrogenase was detected only in the fast growers and was more than twice as active as the NAD-linked enzyme. Partial characterization of the products of 6-PG oxidation in Rhizobium suggests that the NADP-linked enzyme is the decarboxylating enzyme of the pentose phosphate (PP) pathway (EC 1.1.1.44) whereas a phosphorylated six-carbon compound, containing ketonic group(s), is the product of the oxidation catalyzed by the NAD-linked enzyme.

Cell-Free System

Thymidine incorporation into Rhizobium meliloti.

Thymidine is rapidly catabolized to thymine, beta-aminoisobutyric acid, and carbon dioxide by Rhizobium meliloti cells. The incorporation of labelled thymidine into the DNA of R. meliloti cells can be enhanced by the addition of low concentrations (10-20 micrograms/mL) of deoxyadenosine or other nucleosides (adenosine, uridine, guanosine). However, at high concentrations ( greater than 50 micrograms/mL) these compounds inhibit thymidine incorporation. Conditions to obtain highly radioactive DNA of Rhizobium are described.

Carbon Radioisotopes

Fingerprinting bacterial chromosomal DNA with restriction endonuclease EcoRI: comparison of Rhizobium spp. and identification of mutants.

Total cellular DNA from Rhizobium trifolii, R. melitoti, and R. japonicum strains 110 and 117 were prepared. DNA fragments generated with restriction endonuclease EcoRI from these DNA samples were compared in agarose gels after electrophoresis. DNA cleavage patterns generated from R. japonicum strain 110, R. trifolii, and R. meliloti were clearly distinguishable from each other. Restriction endonuclease cleavage patterns of DNA from R. japonicum strain 110 and presumptive R. trifolii mutant strains that nodulate soybean were found to be similar. Rhizobium trifolii mutant strains were also lysed by a phage specific for R. japonicum strain 110. These results show that "R. trifolii mutant strains" are indeed derivatives of R. japonicum strain 110 and not R. trifolii.

Chromosomes, Bacterial