The regulatory status of the fixL- and fixJ-like genes in Bradyrhizobium japonicum may be different from that in Rhizobium meliloti

Summary The cloning, sequencing and mutational analysis of the Bradyrhizobium japonicum symbiotic nitrogen fixation genes fixL and fixJ are reported here. The two genes were adjacent and probably formed an operon, fixLJ. The predicted FixL and FixJ proteins, members of the two-component sensor/regulator family, were homologous over almost their entire lengths to the corresponding Rhizobium meliloti proteins (approx. 50% identity). Downstream of the B. japonicum fixJ gene was found an open reading frame with 138 codons (ORF138) whose product shared 36% homology with the N-terminal part of FixJ. Deletion and insertion mutations within fixL and fixJ led to a loss of approximately 90% wildtype symbiotic nitrogen fixation (Fix) activity, whereas an ORF138 mutant was Fix⁺. In fixL, fixJ and ORF138 mutant backgrounds, the aerobic expression of the fixR-nifA operon was not affected. NifA itself did not regulate the expression of the fixJ gene. Thus, the B. japonicum FixL and FixJ proteins were neither involved in the regulation of aerobic nifA gene expression nor in the anaerobic NifA-dependent autoregulation of the fixRnifA operon; rather they appeared to control symbiotically important genes other than those whose expression was dependent on the NifA protein. The fixL and fixJ mutant strains were unable to grow anaerobically with nitrate as the terminal electron acceptor. Therefore, some of the FixJ-dependent genes in B. japonicum may be concerned with anaerobic respiration.     

Molecular organisation of the malate synthase genes of Aspergillus nidulans and Neurospora crassa

Summary A soybean nodulin cDNA clone (E41) hybrid-selected mRNA for three in vitro translation products with apparent molecular weights of 26 kDa, 25 kDa and 24 kDa. Based on Southern analysis of soybean genomic DNA, combined with mapping and sequencing of genomic clones, we identified four genes that are related to E41, one of which was identified to be the previously characterized N-20 gene. Our data indicate the linkage of three of the genes, of which one is a truncated version and suggest that they originated by gene duplication combined with deletion and conversion. The genes are highly expressed and we postulate that the sequence conservation in the 5 and 3 flanking regions of all four genes, has a functional role in their expression. Hybrid-selected translation products of E41 are not immunoprecipitable with antibody to the soluble fraction of nodules suggesting that they are membrane associated. The N-20 gene, which is a member of this gene subfamily, showed sequence similarity to four previously characterized nodulin genes and a phylogenetic tree is proposed based on the extent of sequence similarity.     

Nucleotide sequence of the fixABC region of Azorhizobium caulinodans ORS571: similarity of the fixB product with eukaryotic flavoproteins, characterization of fixX, and identification of nifW.

Summary The nucleotide sequence of a 4.1 kb DNA fragment containing the fixABC region of Azorhizobium caulinodans was established. The three gene products were very similar to the corresponding polypeptides of Rhizobium meliloti. The C-terminal domains of both fixB products displayed a high degree of similarity with the -subunits of rat and human electron transfer flavoproteins, suggesting a role for the FixB protein in a redox reaction. Two open reading frames (ORF) were found downstream of fixC. The first ORF was identified as fixX on the basis of sequence homology with fixX from several Rhizobium and Bradyrhizobium strains. The second ORF potentially encoded a 69 amino acid product and was found to be homologous to a DNA region in the Rhodobacter capsulatus nif cluster I. Insertion mutagenesis of the A. caulinodans fixX gene conferred a Nif^– phenotype to bacteria grown in the free-living state and a Fix^– phenotype in symbiotic association with the host plant Sesbania rostrata. A crude extract from the fixX mutant had no nitrogenase activity. Furthermore, data presented in this paper also indicate that the previously identified nifO gene located upstream of fixA was probably a homologue of the nifW gene of Klebsiella pneumoniae and Azotobacter vinelandii.     

Nutrient transfer between the root zones of soybean and maize plants connected by a common mycorrhizal mycelium

The objective of the study was to determine whether nutrient fluxes mediated by hyphae of vesicular-arbuscular mycorrhizal (VAM) fungi between the root zones of grass and legume plants differ with the legume's mode of N nutrition. The plants, nodulating or nonnodulating isolines of soybean [ Glycine max (L.) Merr.], were grown in association with a dwarf maize ( Zea mays L.) cultivar in containers which interposed a 6-cm-wide root-free soil bridge between legume and grass container compartments. The bridge was delimited by screens (44 μm) which permitted the passage of hyphae, but not of roots and minimized non VAM interactions between the plants. All plants were colonized by the VAM fungus Glomus mosseae (Nicol. & Gerd.) Gerd. and Trappe. The effects of N input to N-sufficient soybean plants through N₂-fixation or N-fertilization on associated maize-plant growth and nutrition were compared to those of an N-deficient (nonnodulating, unfertilized) soybean control. Maize, when associated with the N-fertilized soybean, increased 19% in biomass, 67% in N content and 77% in leaf N concentration relative to the maize plants of the N-deficient association. When maize was grown with nodulated soybean, maize N content increased by 22%, biomass did not change, but P content declined by 16%. Spore production by the VAM fungus was greatest in the soils of both plants of the N-fertilized treatment. The patterns of N and P distribution, as well as those of the other essential elements, indicated that association with the N-fertilized soybean plants was more advantageous to maize than was association with the N₂-fixing ones.     

Effect of salinity on growth of four strains of Rhizobium and their infectivity and effectiveness on two species of Acacia

Two Rhizobium strains (WU1001 and WU1008) were isolated from nodules of Acacia redolens growing in saline areas of south-west Australia, and two strains selected from the University of Western Australia's culture collection (WU429 isolated from A. saligna and WU433 from A. cyclops). The growth of each in buffered, yeast extract mannitol broth culture was largely unaffected by salt up to 300 mM NaCl. A slight increase in lag time occurred at concentrations of 120 mM NaCl and above, but cell number at the static phase was not affected. Each of the four Rhizobium strains tested accumulated Na⁺ but showed decreasing levels of sugar with increasing salt in the external medium. Amino acid levels also increased, in some cases by more than tenfold. However, the relative proportion of each remained fairly constant in the bacteria, irrespective of salt treatment. Only trace quantities of proline were detected and there was no increase in this amino acid with salt. Acidic amino acids (glutamate and aspartate) remained as a constant proportion.Rhizobium strains WU429, WU1001 and WU1008 produced effective nodules on both A. cyclops and A. redolens grown in sand with up to 80 mM NaCl (added in nutrient solutions free of nitrogen). Strain WU433 was highly infective on both Acacia species tested at low salt concentrations (2–40 mM NaCl), but infection was sensitive to salt levels at 120 mM NaCl and above. Nodules formed with strain WU433 were, however, ineffective on both A. redolens and on A. cyclops and showed nil or negligible rates of acetylene reduction at all salt concentrations. Strains WU429, WU1001 and WU1008 in combination with a highly salt-tolerant provenance of A. redolens formed symbioses which did not vary significantly in nodule number and mass, specific nodule activity or total N content irrespective of salt level up to 160 mM NaCl. On a more salt sensitive provenance of A. redolens and on A. cyclops the infectivity and effectivity of the Rhizobium strains tested usually decreased as the external salt concentration increased. These data are interpreted to indicate that tolerance of the legume host was the most important factor determining the success of compatible Rhizobium strains in forming effective symbioses under conditions of high soil salinity.     

Nodulation of lucerne (Medicago sativa L.) in an acid soil: Effects of inoculum size and lime-pelleting

The effects of inoculum level and lime-pelleting were studied in an acid soil with respect to the nodulation and growth of lucerne (Medicago sativa cv Resis) and the population dynamics of Rhizobium meliloti. In small root-boxes (rhizotrons), the in-situ survival of inoculated rhizobia was studied in the micro-environment around the seed for a period of 12 days after sowing. During the initial 24 hours, a strong increase in rhizobial numbers was measured, concomitantly with the development of roots. As a result of lime-pelleting, rhizobial numbers were higher only at 3 days after sowing (P<0.05). Later, this difference diminished steadily. Addition of lime did not increase the adhesion of the rhizobia to the seedling tap root. Plant responses to inoculation were studied in pots. To obtain optimal nodulation, the soil had to be neutralized around the seed with lime and at least 10⁵ cells of R. meliloti were required. With more than 10⁵ rhizobia per seed, lime-pelleting increased the number of crown-nodulated seedlings from 24% to 77%. Higher numbers of rhizobia could not compensate the effect of lime. A strong correlation was found between crown nodulation, nitrogen content and dry weight of the shoots.     

Biology of nitrogen-fixing Rhizobacteria

In non-legumes associative nitrogen-fixing system, several genera of rhizobacteria have been reported. The object of this paper is to summarize the current understanding of how rhizobacteria adhere to the root surface of non-legumes especially rice and other cereal crops. Evidence for involvement of rice lectin in adhesion will be reviewed. An emphasis will be placed on theKlebsiella R15 ammonium assimilation system in free-living state and in associative state with rice seedlings. Nitrogenase and glutamine synthetase (GS) activities of associativeKlebsiella increased significantly in the rhizosphere of rice comparing to the free-living state. In rice, the soluble form of GS specific activity appear to be slightly lower than in rice root in the absence of bacteria. These results suggest that nitrogen-fixing activity has been enhanced during association. The dinitrogen fixed should be changed to amino acids via GS-GOGAT pathway in bacteria. Transfer of fixed nitrogen and assimilation in the rice plant is the problem that needs to be solved in order to improve the efficiency of associative nitrogen fixation.     

Microfilaments in the preprophase band of freeze substituted tobacco root cells

Summary The organization and distribution of microfilaments (MFs) in the preprophase bands (PPBs) of tobacco (Nicotiana tabacum L. var. Maryland Mammoth) root tip cells were studied with high pressure freezing and freeze-substitution methods. MFs were present predominantly as single filaments interspersed among microtubules (MTs) throughout the PPB. Some MFs appeared to be associated with MTs, whereas others were not. This is the first time that MFs have been demonstrated in the PPB at the electron microscope level.     

Characterization of rhizobia fromLeucaena

Seventy-six rhizobia were isolated from the nodules ofLeucaena plants of various genotypes growing in a wide range of soil types and climatic regions. The isolates were fast-growing and acid-producing. In establishing a serological grouping for the isolates, the intrinsic antibiotic resistance (IAR) patterns to low concentrations of eight antibiotics was helpful for selecting the strains for immunization purposes. Eight distinct somatic serogroups ofLeucaena rhizobia were identified by using strain-specific fluorescent antibodies. The results indicated that use of serological markers is a more specific technique than IAR pattern for strain identification. Strains from some different serogroups had the same IAR patterns. The immunofluorescence cross-reactions ofLeucaena rhizobia serogroups among themselves and with other species of fast- and slow-growing rhizobia were very low. Sero-grouping is ideal for use in further ecological studies in field inoculation trials.