Inducing effect of plant cells on nitrogenase activity by Spirillum and Rhizobium in vitro

Eleven different plant cell tissue cultures of both legume and non-legume origin have been grown in direct association, and in separate but close proximal association with both Spirillum lipoferum and Rhizobium sp. 32H1. Basic similarities were found in the nutritional requirement for the induction of nitrogenase activity (C2H2) in both organisms. In the absence of plant cell cultures both organisms need to be provided with a pentose sugar and a tricarboxylic acid to induce high levels of nitrogen-fixing activity. Plant cell callus tissue appears only capable of supplying the tricarboxylic acid to induce high levels of nitrogen-fixing activity. Plant cell callus tissue appears only capable of supplying the tricarboxylic acids needed but not the sugar component. The plant tissue, however, seems able to activate certain carbohydrates, which in themselves are incapable of substituting for the pentose additive.     

Transfer of the Rhizobium leguminosarum biovar trifolii symbiotic plasmid pRtr5a to a strain of Rhizobium sp. that nodulates on Hedysarum coronarium

The Rhizobium leguminosarum biovar trifolii symbiotic plasmid pRtr5a was transferred to the Rhizobium sp. (Hedysarum coronarium) strain RB16. Transconjugants carrying pRtr5a ineffectively nodulated Trifolium repens, T. pratense and T. alexandrinum and were unable to nodulate H. coronarium plants. Agarose gel electrophoresis of transconjugants showed that all had lost an indigenous plasmid (230 Md). These results suggest that this plasmid harbours the symbiotic determinants for nodulation on H. coronarium.     

Role of bacterial polysaccharides in the derepression of ex-planta nitrogenase activity with rhizobia

Abstract Since bacterial polysaccharides may limit the availability of oxygen to the cells, we have investigated the role of rhizobial extracellular polysaccharides (EPS) and the non-rhizobial polyscharide, xanthan, in the depression of ex-planta nitrogenase activity with rhizobia in liquid medium. Two rhizobial strains known to exhibit ex-planta nitrogenase activity on solid media were used; the slow-growing Bradyrhizobium japonicum USDA 110 and the arctic Rhizobium strain N31, both being prolific EPS producers. In low nitrogen mannitol (LNM) liquid medium strain N31 exhibited nitrogenase activity only after 15 days, when sufficient EPS had accumulated in the medium, and activity was correlated with EPS production. When rhizobial EPS from an old culture was added to the LNM medium, nitrogenase activity was detected after 48 h incubation, indicating that EPS of the medium decreased oxygen diffusion to cells to a level that depressed nitrogenase activity. In modified LNM medium with xanthan nitrogenase activity was readily depressed. In both strains activity increased with increased xanthan concentration, but decreased sharply at higher concentrations. Strain N31 exhibited a narrower range of polysaccharide concentration for nitrogenase activity than the slow strain USDA 110. Thus, the condition for derepression of nitrogenase might be a careful balancing of the oxygen concentration surrounding the cells, and this condition is met when a balancing of polsaccharide, either synthesized by the rhizobia or added to the medium, can permit oxygen diffusion to within the narrow range required for the depression and expression of nitrogenase.     

The nodulation and nitrogenase activity of natural stands of mangrove legumes in a Nigerian swamp

Nodule characteristics and nitrogenase activity of Dalbergia ecastophyllum (L.) Taub, Drepanocarpus (Machearum) lunatus (L.) Mey, Pterocarpus sontelinoides (L.) and the characteristics of nodules of Baphia pubescens (Hook. f) were studied in natural mangrove stands in Nigeria. Nodules were generally spherical but occasionally elongate in Pterocarpus and Baphia; a few were also lobate in Baphia. Nodule sizes, numbers and weights varied widely between vegetations, and between sites of the same vegetation. Total nodule fresh-weight (g·m⁻²) ranged from 0.11 to 9.80, represented by the extreme values for Drepanocarpus. The plants' habitats had pH and salinity respectively in the ranges 4.8–5.49 and 0.1–25‰. Nodule acetylene reduction rates decreased in the order Pterocarpus, Drepanocarpus, Dalbergia with their corresponding aerobic rates (n moles C₂H₄ g⁻¹ freshweight) respectively as follows: 312, 23.39 and 16.07. These rates were higher than in anaerobic incubation by between 4.95 and 100%.     

Structural and functional analysis of nitrogenase genes from the broad-host-range Rhizobium strain ANU240

The genes encoding the structural components of nitrogenase, nifH, nifD and nifK, from the fast-growing, broad-host-range Rhizobium strain ANU240 have been identified and characterized. They are duplicated and linked in an operon nifHDK in both copies. Sequence analysis of the nifH gene from each copy, together with partial sequence analysis of the nifD and nifK genes, and restriction endonuclease analysis suggested that the duplication is precise. Comparison of the Fe-protein sequence from strain ANU240 with that from other nitrogen-fixing organisms revealed that, despite its broad host range and certain physiological properties characteristic of Bradyrhizobium strains, ANU240 is more closely related to the narrow-host-range Rhizobium strains than to the broad-host-range Bradyrhizobium strains. The promoter regions of both copies of the nif genes contain the consensus sequence characteristic of nif promoters, and functional analysis of the two promoters suggested that both nif operons are transcribed in nodules.     

The induction of nitrogenase activity in Rhizobium by non-legume plant cells

Summary Nitrogen fixation was induced in a strain of cowpea rhizobia, 32Hl, when it was grown in association with cell cultures of the non-legume, tobacco (Nicotiana tabacum). Rhizobia grown alone on the various media examined did not show nitrogenase activity, indicating the involvement of particular plant metabolites in nitrogenase induction. Nitrogenase activity, as measured by C₂H₂ reduction, was maximized at an O₂ concentration of 20% and at an assay temperature of 30°C, the conditions under which the plant cell-rhizobia associations developed. Glutamine, as a nitrogen source, could be replaced by other organic nitrogen sources, but NH₄ ⁺ and NO₃ ^- repressed nitrogenase activity. Nitrogenase activity induced in rhizobia when cultured adjacent to, but not in contact with, the plant cells could be stimulated by providing succinate in the medium. At least 12 other strains of rhizobia also reduced C₂H₂ in association with tobacco cells; the highest levels of activity were found among cowpea strains.     

Respiratory control determines respiration and nitrogenase activity of Rhizobium leguminosarum bacteroids.

The relationship between the O2 input rate into a suspension of Rhizobium leguminosarum bacteroids, the cellular ATP and ADP pools, and the whole-cell nitrogenase activity during L-malate oxidation has been studied. It was observed that inhibition of nitrogenase by excess O2 coincided with an increase of the cellular ATP/ADP ratio. When under this condition the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added, the cellular ATP/ADP ratio was lowered while nitrogenase regained activity. To explain these observations, the effects of nitrogenase activity and CCCP on the O2 consumption rate of R. leguminosarum bacteroids were determined. From 100 to 5 microM O2, a decline in the O2 consumption rate was observed to 50 to 70% of the maximal O2 consumption rate. A determination of the redox state of the cytochromes during an O2 consumption experiment indicated that at O2 concentrations above 5 microM, electron transport to the cytochromes was rate-limiting oxidation and not the reaction of reduced cytochromes with oxygen. The kinetic properties of the respiratory chain were determined from the deoxygenation of oxyglobins. In intact cells the maximal deoxygenation activity was stimulated by nitrogenase activity or CCCP. In isolated cytoplasmic membranes NADH oxidation was inhibited by respiratory control. The dehydrogenase activities of the respiratory chain were rate-limiting oxidation at O2 concentrations (if >300 nM. Below 300 nM the terminal oxidase system followed Michaelis-Menten kinetics (Km of 45 +/- 8 nM). We conclude that (i) respiration in R. leguminosarum bacteroids takes place via a respiratory chain terminating at a high-affinity oxidase system, (ii) the activity of the respiratory chain is inhibited by the proton motive force, and (iii) ATP hydrolysis by nitrogenase can partly relieve the inhibition of respiration by the proton motive force and thus stimulate respiration at nanomolar concentrations of O2.     

Control by light of whole-cell nitrogenase activity and of nitrogenase and bacteriochlorophyll a formation in Rhodobacter capsulatus strain B10S

Control of nitrogenase and bacteriochlorophyll a (BChl) by light was studied under steady-state conditions with continuous cultures of Rhodobacter capsulatus B10S supplied with malate and growth-limiting amounts of ammonium. Consumption of malate and, correspondingly, the C/N ratio at which malate and ammonium were consumed increased when illumination was increased from 3 to approximately 20 klx and became constant at higher illuminations of up to 40 klx. Essentially the same kinetics were observed with respect to nitrogenase activity of cells, contents of nitrogenase polypeptides, and nifH promoter activity. Substrate consumption was half-maximal at 8 klx and was independent of the presence of nitrogenase. Therefore, it is concluded that light controls the C/N ratio (a quantitative measure of the nitrogen status of cells), which in turn is involved in the control of nitrogenase at the level of nif promoter activity. Post-translational regulation of nitrogenase activity by ADP-ribosylation was not observed under steady-state conditions, but it took place when illumination was suddenly decreased to the range where malate consumption and, consequently, the C/N ratio decreased. Irrespective of the presence or absence of nitrogenase, specific BChl contents of the cultures were constant above 20 klx, and they increased at lower illuminations. These results do not confirm a recently proposed link between nitrogen fixation and photosynthesis as represented by BChl. Received: 29 October 1998 / Accepted: 30 December 1998     

Identification of a nodD-dependent locus in the Rhizobium strain NGR234 activated by phenolic factors secreted by soybeans and other legumes

Transfer of the strain NGR234nodD 1 gene into the narrow host range R. trifolii strain ANU843 on either a 6.7-kb HindIII or 17-kb XhoI fragment broadens the host range of this bacterium to include the tropical legumes Vigna unguiculata, Glycine ussuriensis, Leucaena leucocephala, and siratro (Macroptilium atropurpureum). Contrary to previous data (Bassam et al. 1986), mutagenesis of the 17-kb XhoI fragment with a mini-Mu lac transposon (Mu dII1734) showed that a functional nodD 1 gene was essential for extended host range. Gene expression studies using both Mu dII1734 fusions and a promoter-cloning vector indicated that several loci, including the nodD 1 gene, are constitutively expressed. No evidence was found for regulation of the strain NGR234 nodD 1 gene by its product. Another locus nod-81, was induced only in the presence of exudates from various plant species, including soybean (Glycine max). Whereas the expression of nod-81 was dependent on the presence of a functional nodD 1 gene product, a regulatory nod-box DNA sequence was not detected 5' to this gene by using available oligonucleotide hybridization probes. The nod-81 locus was induced by genistein, daidzein, naringenin, and coumestrol from both cotyledon and root tissue of freshly germinated soybean seedlings. A broad spectrum of commercially available phenolic compounds stimulated induction of the nod-81 locus, including some that antagonize nod gene induction in other Rhizobium species. The nodD 1 gene product from strain NGR234 was shown to determine the spectrum of compounds that induce nod-81 expression.     

A new root-nodulating symbiont of the tropical legume Sesbania, Rhizobium sp SIN-1, is closely related to R. galegae, a species that nodulates temperate legumes

Abstract Rhizobium sp. SIN-1, isolated in India from root nodules on the tropical legume Sesbania aculeata , also induces nitrogen-fixing nodules on roots of S. macrocarpa, S. speciosa, S. procumbens, S. punicea, S. rostrata , and Vigna unguiculata . Unlike Azorhizobium caulinodans , SIN-1 does not induce stem nodules on S. rostrata . The nodules induced by SIN-1 develop exclusively at the bases of secondary roots. Electron microscopic studies of mature nodule sections revealed rhizobia within intercellular spaces, indicating a 'crack entry' mechanism of root infection. SIN-1 is a fast-growing, acid-producing, salt-tolerant Rhizobium that utilizes a wide variety of carbon sources. The nodulation ( nod ) genes of this strain are located on a 300-MDa symbiosis ( sym ) plasmid. Fatty acid profile and sequence comparison of a 260-bp conserved region of the 16S rRNA gene demonstrated that SIN-1 is phylogenetically closely related to R. galegae , a species that nodulates temperate legumes.     

Extracellular polysaccharide composition, ex planta nitrogenase activity, and DNA homology in Rhizobium japonicum

The composition of the major acidic extracellular polysaccharide (EPS) of 25 strains of Rhizobium japonicum was determined. Eight strains synthesized an acidic EPS containing rhamnose and 4-O- methylglucuronic acid and were closely related according to DNA homology. These same strains also expressed high levels of ex planta nitrogenase activity. Sixteen strains produced an acidic EPS containing glucose, mannose, galacturonic acid, and galactose and were also related by DNA homology. These strains developed little or no nitrogenase activity under the experimental conditions employed.     

Carbon-nitrogen requirements for the expression of nitrogenase activity in cultured Parasponia-Rhizobium strain ANU 289

Nutritional factors controlling derepression of nitrogenase activity in Parasponia-Rhizobium strain ANU 289 were studied in stationary and agitated liquid cultures. Altering type and/or concentrations of the constituents of the derepression medium in respect of carbon and nitrogen sources influenced both derepression kinetics as well as the maximal level of activity. Hexose sugars and disaccharides stimulated nitrogenase activity three to six-fold compared to pentose sugars. Activity was also modulated by combining sugars with some organic acids such as succinate, fumarate and pyruvate but not with others (e.g. -ketoglutarate, malate, malonate). Of the range of nitrogen sources tested, either casamino acids (at 0.05%, but not at 0.1%), glutamate, proline or to a lesser extent histidine (each at 5 mM N) supported significant derepression of nitrogenase activity. Notably glutamine, urea, alanine, ammonium sulfate, nitrate, nitrite (each at 5 mM N) and yeast extract (0.05%) failed to derepress or support nitrogenase activity. Ammonium (5 mM) abolished established nitrogenase activity of rapidly agitated cultures within 15 h after addition. This inhibitory effect was alleviated by the addition of methionine sulfoximime (10 mM). Thus, in view of strong glutamine effects, ammonium repression appears to be mediated by glutamine and not by ammonium itself.Abbreviations HEPES [4-(2-hydroxyethyl)-1-piperazine-ethane; sulfonic acid] - MOPS [3-(N-morpholino) propane sulphonic acid] - MSX Methionine sulfoximine     

Nodulation of Trifolium repens with modified Bradyrhizobium and the nodulation of Parasponia with Rhizobium leguminosarum biovar trifolii

Thirty one strains of Rhizobium leguminosarum biovar trifolii isolated from the North and South American continents, New Guinea, USSR, Turkey and Australia, nodulated P. andersonii ineffectively when grown in plant growth tubes and in Leonard jars. Nodules were slow to form, sometimes taking over 100 days. Reisolates of R. leguminosarum biovar trifolii from P. andersonii nodulated Trifolium repens and their identity was confirmed using serological techniques. Dual occupation of nodules by Rhizobium and Bradyrhizobium in P. andersonii is reported. The reduced effectiveness of the Bradyrhizobium symbiosis depended on the relative numbers of Rhizobium occupants in this dual system. R. leguminosarum biovar trifolii and Bradyrhizobium strains from Parasponia were able to co-exist in nodules on P. andersonii and maintain similar populations in the rhizosphere and on culture media. Bradyrhizobium strains, separated from R. leguminosarum biovar trifolii, were able to initiate and form nodule-like structures on T. repens. Bradyrhizobium bacteria were identified as the sole occupants of the cells of the nodule-like structures on Trifolium repens using an immunogold labelling technique applied to ultrathin sectins. The re-isolates of Bradyrhizobium obtained from these nodule-like structures on T. repens were able to effectively nodulate P. andersonii.