Organisation of nodulation and nitrogen fixation genes on a Rhizobium trifolii symbiotic plasmid

A Rhizobium trifolii symbiotic plasmid specific gene library was constructed and the physical organisation of regions homologous to nifHDK, nifA and nod genes was determined. These symbiotic gene regions were localised to u 25 kb region on the sym-plasmid, pPN1. In addition four copies of a reiterated sequence were identified on this plasmid, with one copy adjacent to nifH. No rearrangement of these reiterated sequences was observed between R. trifolii bacterial and bacteroid DNA. Analysis of a deletion derivative of pPN1 showed that these sequences were spread over a 110 kb region to the left of nifA.     

Phylogenetic analyses of symbiotic nodulation genes support vertical and lateral gene co-transfer within the Bradyrhizobium genus

Symbiotic nitrogen fixing bacteria-known as rhizobia-harbour a set of nodulation (nod) genes that control the synthesis of modified lipo-chitooligosaccharides, called Nod factors that are required for legume nodulation. The nodA gene, which is essential for symbiosis, is responsible for the attachment of the fatty acid group to the oligosaccharide backbone. The nodZ, nolL, and noeI genes are involved in specific modifications of Nod factors common to bradyrhizobia, i.e., the transfer of a fucosyl group on the Nod factor core, fucose acetylation and fucose methylation, respectively. PCR amplification, sequencing and phylogenetic analysis of nodA gene sequences from a collection of diverse Bradyrhizobium strains revealed the monophyletic character with the possible exception of photosynthetic Bradyrhizobium, despite high sequence diversity. The distribution of the nodZ, nolL, and noeI genes in the studied strains, as assessed by gene amplification, hybridization or sequencing, was found to correlate with the nodA tree topology. Moreover, the nodA, nodZ, and noeI phylogenies were largely congruent, but did not closely follow the taxonomy of the strains shown by the housekeeping 16S rRNA and dnaK genes. Additionally, the distribution of nodZ, noeI, and nolL genes suggested that their presence may be related to the requirements of their legume hosts. These data indicated that the spread and maintenance of nodulation genes within the Bradyrhizobium genus occurred through vertical transmission, although lateral gene transfer also played a significant role.     

Negative effects of agrocin 84-encoding Agrobacterium plasmids on symbiotic properties of Rhizobium meliloti

Two plasmids, pAgK84::Tn5-Mob from Agrobacterium radiobacter carrying genes for the production of agrocin 84, and RP4-4 from E. coli were inserted either separately or together into a strain of Rhizobium meliloti. Each of these plasmid-containing R. meliloti transconjugants was less effective than the wild type strain in their ability to fix nitrogen in Medicago tornata. The pAgK84::Tn5-Mob-containing transconjugant was significantly less effective than that containing RP4-4. The transconjugant strains were inferior to the wild type strain in their ability to nodulate seedlings and to compete for nodulation.     

The formation of R-prime deletion mutants and the identification of the symbiotic genes in Rhizobium fredii strain USDA191

Summary R-prime plasmids were formed between the plasmid of Rhizobium fredii strain USDA191 containing nodulation and nitrogen-fixation genes, pRjaUSDA191c, and pRL180, and RP1 derivative. R. fredii USDA191 contains four HindIII fragments that hybridize with an 8.7 kb EcoRI fragment that contains nodulation genes from R. meliloti. These four fragments are on pRjaUSDA191c and are 15.5 kb, 12.5 kb, 6.8 kb, and 5.2 kb in size. A series of R-primes generated in E. coli of pRjaUSDA191c were transferred into a Nod^- Nif^- derivative of strain USDA191 to determine which nodulation region is necessary for nodule formation. Transconjugants containing the 12.5 kb and the 6.8 kb HindIII fragments on segments of pRjaUSDA191c produced nodules on soybean plants. However, transconjugants containing the 12.5 kb HindIII fragment alone were unable to form nodules, suggesting that the 6.8 kb HindIII fragment or the 6.8 kb and the 12.5 kb HindIII fragments together were needed for nodule formation. The 6.8 kb HindIII fragment was subcloned into the vector pVK102 and transferred into transconjugants containing no sequences homologous to R. meliloti nodulation DNA or to transconjugants containing only the 12.5 kb HindIII fragment. Nodules were formed on soybeans only when both the 12.5 kb and the 6.8 kb HindIII fragments were present in R. frediistrain USDA191.     

Influence ofRhizobium leguminosarum biovartrifolii host specific nodulation genes on the ontogeny of clover nodulation

Summary The infection of white clover seedlings byRhizobium strains with different host range properties was assessed using various microscopic techniques. Several wild-type andRhizobium leguminosarum biovarvicias hybrid strains containing definedR. l. bv.trifolii host range genes were used. The morphological changes in the root tissue of uninoculated and rhizobia inoculated white clovers were identified and compared. In particular, changes were observed in the induction of inner cortical cell division, alterations to nodule development and lateral root formation. The responses of the infected roots and the types of structures formed support the hypothesis that lateral roots and nodules may be physiologically homologous structures. To establish a normal pattern of nodulation on white clover roots, both sets of known host specific nodulation genes (operonsnod FERL andnod MNX) ofR. l. bv.trifolii were required. However, some nodule development occurred when only thenod FERL genes were present in the hybrid strain.     

Characterization of a large plasmid of Rhizobium meliloti involved in enhancing nodulation

Summary The plasmid pattern of Rhizobium meliloti strain GR4 was studied and a gene bank of one of the large plasmids (pRmeGR4) of 140 Mdal, was constructed using the broad host range vector pRK290. A restriction map was established with EcoRI. Two regions of this plasmid involved in the infectivity of GR4 on Medicago sativa were identified. An EcoRI fragment hybridizing with the PstI-nif fragment of pID1 was also identified. However, no homology to the cloned Klebsiella pneumoniae nitrogenase genes (pSA30) was detected.     

Pigment production from in vitro cultures of Alkanna tinctoria Tausch

Summary An in vitro culture of Alkanna tinctoria Tausch cells was set up in order to investigate the possibility of producing alkannin, a red naphthoquinone naturally present in the root bark of this plant. Furthermore, an in vitro culture of callusderived roots was established and the production of alkannin evaluated. In the different experimental conditions investigated, differences in the production of alkannin derivatives as well as in the type of pigments produced, were observed. The potential use of this technology is discussed.     

Organization of the nif genes in cyanobacteria in symbiotic association with Azolla and Anthoceros

The sizes of endonuclease digestion fragments of DNA from cyanobacteria in symbiotic association with Azolla caroliniana or Anthoceros punctatus, or in free-living culture, were compared by Southern hybridization using cloned nitrogenase (nif) genes from Anabaena sp. PCC 7120 as probes. The restriction fragment pattern produced by cyanobacteria isolated from A. caroliniana by culture through symbiotic association with Anthoceros differed from that of the major symbiotic cyanobacterium freshly separated from A. caroliniana. The results indicate that minor cyanobacterial symbionts occur in association with Azolla and that the dominant symbiont was not cultured in the free-living state. Both the absence of hybridization to an xisA gene probe and the mapping of restriction fragments indicated a contiguous nifHDK organization in all cells of the symbiont in association with Azolla. On the other hand, in the cultured isolate from Azolla and in Nostoc sp. 7801, the nifD and nifK genes are nominally separated by an interval of unknown length, compatible with the interruption of the nifHDK operon by a DNA element as observed in Anabaena sp. PCC 7120. In the above cultured strains, restriction fragments consistent with a contiguous nifHDK operon were also present at varying hybridization intensities, especially in Nostoc sp. 7801 grown in association with Anthoceros, presumably due to gene rearrangement in a fraction of the cells.Non-standard abbreviations bp base pairs - kb kilobase pairs - kd kilodaltons     

Characterization of heat resistant mutant strains of Rhizobium sp. [Cajanus] for growth, survival and symbiotic properties

Fourteen heat resistant mutant strains were isolated from a wild-type strain (PP201, Nod⁺ Fix⁺) of Rhizobium sp. (Cajanus) by giving it a heat shock of 43°C. These mutant strains showed a greater increase in optical density (O.D.) and a higher viable cell count in both rhizospheric and non-rhizospheric soil at high temperature. Symbiotic studies showed that pigeon pea plants inoculated with a few mutant strains had ineffective nodules (Nod⁺ Fix⁻) under controlled temperature (43°C) conditions, but under natural high temperature (40–45°C) conditions, the host plants infected with all the mutant strains showed higher total shoot nitrogen than the plants inoculated with the parent strain. Four mutant strains (HR-3, HR-6, HR-10 and HR-12) were found to be highly efficient for all the symbiotic parameters, and thus have the potential to be used as bioinoculants in the North-Western regions of India during the summer season.     

Identification and cloning of nodulation genes from the stem-nodulating bacterium ORS571

Summary After random Tn5 mutagenesis of the stem-nodulating Sesbania rostrata symbiont strain ORS571, Nif^-, Fix^- and Nod^- mutants were isolated. The Nif^- mutants had lost both free-living and symbiotic N₂ fixation capacity. The Fix^- mutants normally fixed N₂ in the free-living state but induced ineffective nodules on S. rostrata. They were defective in functions exclusively required for symbiotic N₂ fixation. A further analysis of the Nod^- mutants allowed the identification of two nod loci. A Tn5 insertion in nod locus 1 completely abolished both root and stem nodulation capacity. Root hair curling, which is an initial event in S. rostrata root nodulation, was no longer observed. A 400 bp region showing weak homology to the nodC gene of Rhizobium meliloti was located 1.5 kb away from this nod Tn5 insertion. A Tn5 insertion in nod locus 2 caused the loss of stem and root nodulation capacity but root hair curling still occurred. The physical maps of a 20.5 kb DNA region of nod locus 1 and of a 40 kb DNA region of nod locus 2 showed no overlaps. The two nod loci are not closely linked to nif locus 1, containing the structural genes for the nitrogenase complex (Elmerich et al. 1982).     

The role of Rhizobium conserved and host specific nodulation genes in the infection of the non-legume Parasponia andersonii

Summary Rhizobium and Bradyrhizobium bacteria gain intercellular entry into roots of the non-legume Parasponia andersonii by stimulating localized sites of cell division which disrupt the epidermis. Infection threads are then initiated from intercellular colonies within the cortex. Infection via the information of infection threads within curled root hairs, which commonly occurs in legumes, was not observed in Parasponia. The conserved nodulation genes nodABC, necded for the curling of legume root hairs, were not essential for the initiation of infection, however, these genes were required for Parasponia prenodule development. In contrast, the nodD gene of Rhizobium strain NGR234 was essential for the initiation of infection. In addition, successful infection required not only nodD but a region of the NGR234 symbiotic plasmid which is not needed for the nodulation of legumes. Agrobacterium tumefaciens carrying this Parasponia specific region, as well as legume nod genes, was able to form nodules on Parasponia which reached an advanced stage of development.     

豆科植物共生结瘤的分子基础和调控研究进展

豆科植物与根瘤菌共生互作的结果导致了一个新的植物器官――根瘤的形成, 根瘤菌生活在根瘤中, 它们具有将氮气转化为能被植物同化的氨的能力。该文阐述了根瘤的形成过程和类型, 并主要以模式豆科植物蒺藜苜蓿(Medicago truncatula)和日本百脉根(Lotus japonicus)为例, 对近年来共生结瘤过程中宿主植物对根瘤菌结瘤因子的识别和信号传递、侵入线形成和固氮的分子基础, 以及宿主植物对根瘤形成的自主调控机制、环境中氮素营养对结瘤的影响研究进行了综述, 指出当前豆科植物与根瘤菌共生互作研究存在的问题, 并对今后的研究方向作了分析与展望。     

Selection of Rhizobium leguminosarum bv. viceae strains for inoculation of Pisum sativum L. cultivars: Analysis of symbiotic efficiency and nodulation competitiveness

From an analysis of 481 Rhizobium leguminosarum bv. viceae strains with 7 pea cultivars in pot and field experiments, we demonstrated that effective strains could be isolated from a rich medium-acid grey forest soil of the Oröl area (Central region of the European part of Russia) but not from a poor acid podzolic soil of the St. Petersburg area (North-West Russia). The proportion of the isolates significantly increasing N accumulation in pea plants (10.2%) is higher than that of strains increasing the shoot dry mass (4.6%) in the pot experiments. The mean values of the increase for N accumulation (33.8%) upon inoculation are also higher than for shoot mass (27.0%) in these experiments. N accumulation in the inoculated pea plants in the pot experiments was significantly correlated with seed yield and seed N accumulation in field experiments, while for shoot dry mass these correlations were either weak or not significant. Two-factor analysis of variance demonstrated that the contribution of plant cultivars to the variation of the major symbiotic efficiency parameters is higher (30.8–31.6%) and contributions of cultivar-strain specificity is lower (5.4–8.8%) than the contributions of strain genotypes (13.4–14.9%). We identified an ineffective R. leguminosarum bv. viceae strain 50 which can be used as a tester for assessing the nodulation competitiveness of the effective strains by an indirect method (analysis of dry mass and N accumulation in pea plants inoculated with the mixture of the tested effective strains and the tester strain). The relative competitive ability (RCA) determined by this method was 75.7–82.8% for strain 52 but only 10.5–13.8% for strain 250a; this difference was confirmed by a direct method (use of the streptomycin-resistant mutants). Results of screening of the diverse collection of 53 effective R. leguminosarum bv. viceae strains by the indirect method permits us to divide them into 3 groups (32 high-competitive, 10 medium-competitive and 11 low-competitive strains) but reveals no correlation between the competitiveness and symbiotic efficiency. N accumulation in the pea shoots is demonstrated to be a much more suitable criterion than the shoot mass for selection either of the highly-effective or of highly-competitive (by the indirect estimation) R. leguminosarum bv. viceae strains in the pot experiments.     

Rhizobium nodulation genes involved in root hair curling (Hac) are functionally conserved

Summary Five specific transposon-induced nodulation defective (Nod⁻) mutants from different fast-growing species ofRhizobium were used as the recipients for the transfer of each of several endogenous Sym(biosis) plasmids or for recombinant plasmids that encode early nodulation and host-specificity functions. The Nod⁻ mutants were derived fromR. trifolii, R. meliloti and from a broad-host-rangeRhizobium strain which is able to nodulate both cowpea (tropical) legumes and the non-legumeParasponia. These mutants had several common features (a), they were Nod⁻ on all their known plant hosts, (b), they could not induce root hair curling (Hac⁻) and (c), the mutations were all located on the endogenous Sym-plasmid of the respective strain. Transfer to these mutants of Sym plasmids (or recombinant plasmids) encoding heterologous information for clover nodulation (pBR1AN, pRt032, pRt038), for pea nodulation (pJB5JI, pRL1JI::Tn1831), for lucerne nodulation (pRmSL26), or for the nodulation of both tropical legumes and non-legumes (pNM4AN), was able to restore root hair curling capacity and in most cases, nodulation capacity of the original plant host(s). This demonstrated a functional conservation of at least some genes involved in root hair curling. Positive hybridization between Nod DNA sequences fromR. trifolii and from a broad-host-rangeRhizobium strain (ANU240) was obtained to other fast-growingRhizobium strains. These results indicate that at least some of the early nodulation functions are common in a broad spectrum ofRhizobium strains.     

Phenotypic and genetic characterization of rhizobia associated with alfalfa in the Hokkaido and Ishigaki regions of Japan

Twenty five rhizobial isolates were obtained from root nodules of Medicago sativa inoculated with soil samples collected from the Sapporo region and Ishigaki Island in Japan. To study their diversity and characterize them in relation to the climatic conditions of their soils of origin, a polyphasic approach analyzing stress tolerance, symbiotic and genetic properties was used. Stress tolerance assays revealed marked variations in salinity, pH and temperature tolerance. Isolates originating from a sub-tropical climate in alkaline soil (Ishigaki Island) tolerated high temperature, salinity and pH levels. Moreover, isolates recovered from a temperate climate in acidic soil (Sapporo) were sensitive to high temperature and salinity, and tolerated acidic pH. Phylogenetic analysis of conserved 16S rRNA and recA genes, and symbiotic nodA and nifDK revealed 25 isolates to be closely related to Ensifer meliloti. Furthermore, the branch patterns of phylogenetic trees constructed from different genes revealed the existence of at least two E. meliloti types in the soils studied. These results may be relevant to programs directed towards improving crop productivity through biofertilization with locally adapted and genetically defined strains.     

Phylogeny of theCyclanthaceae

The affinities ofCyclanthaceae are discussed, and it is concluded that the sister-group to this family is most probablyPandanaceae. A hypothesis of generic relationships inCyclanthaceae, based on cladistic methods, is presented. Bootstrap analysis and Bremer support (decay index) have been used to test the strength of individual clades, and the result is compared with previously made phylogenetic analyses. TheSphaeradenia group (Chorigyne, Stelestylis, Sphaeradenia, andLudovia) is supported as monophyletic and acceptably resolved, while theAsplundia group (remaining genera inCarludovicoideae) may be paraphyletic, with largely uncertain relationships. A formal recognition of these groups is therefore not justified. The probable character evolution inCarludovicoideae is discussed.     

Ensifer meliloti bv. lancerottense establishes nitrogen-fixing symbiosis with Lotus endemic to the Canary Islands and shows distinctive symbiotic genotypes and host range

Eleven strains were isolated from root nodules of Lotus endemic to the Canary Islands and they belonged to the genus Ensifer, a genus never previously described as a symbiont of Lotus. According to their 16S rRNA and atpD gene sequences, two isolates represented minority genotypes that could belong to previously undescribed Ensifer species, but most of the isolates were classified within the species Ensifer meliloti. These isolates nodulated Lotus lancerottensis, Lotus corniculatus and Lotus japonicus, whereas Lotus tenuis and Lotus uliginosus were more restrictive hosts. However, effective nitrogen fixation only occurred with the endemic L. lancerottensis. The E. meliloti strains did not nodulate Medicago sativa, Medicago laciniata Glycine max or Glycine soja, but induced non-fixing nodules on Phaseolus vulgaris roots. nodC and nifH symbiotic gene phylogenies showed that the E. meliloti symbionts of Lotus markedly diverged from strains of Mesorhizobium loti, the usual symbionts of Lotus, as well as from the three biovars (bv. meliloti, bv. medicaginis, and bv. mediterranense) so far described within E. meliloti. Indeed, the nodC and nifH genes from the E. meliloti isolates from Lotus represented unique symbiotic genotypes. According to their symbiotic gene sequences and host range, the Lotus symbionts would represent a new biovar of E. meliloti for which bv. lancerottense is proposed.     

Multilocus sequence analysis of bradyrhizobia isolated from Aeschynomene species in Senegal

This study reports the multilocus sequence analysis (MLSA) of nine house-keeping gene fragments (atpD, dnaK, glnA, glnB, gltA, gyrB, recA, rpoB and thrC) on a collection of 38 Bradyrhizobium isolated from Aeschynomene species in Senegal, which had previously been characterised by several phenotypic and genotypic techniques, allowing a comparative analysis of MLSA resolution power for species delineation in this genus. The nifH locus was also studied to compare house-keeping and symbiotic gene phylogenies and obtain insights into the unusual symbiotic properties of these Aeschynomene symbionts. Phylogenetic analyses (maximum likelihood, Bayesian) of concatenated nine loci produced a well-resolved phylogeny of the strain collection separating photosynthetic bradyrhizobial strains (PB) from non-photosynthetic bradyrhizobial (NPB) ones. The PB clade was interpreted as the remains an expanding ancient species that presently shows high diversification, giving rise to potential new species. B. denitrificans LMG8443 and BTAi1 strains formed a sub-clade that was identified as recently emerging new species. Congruence analyses (by Shimodaira–Hasegawa (S–H) tests) identified three gene-fragments (dnaK, glnB and recA) that should be preferred for MLSA analyses in Bradyrhizobium genus. The nine loci or nifH phylogenies were not correlated with the unusual symbiotic properties of PB (nod-dependent/nod-independent). Advantages and drawbacks of MLSA for species delineation in Bradyrhizobium are discussed.     

Host-specific nodulation is encoded on a 14kb DNA fragment in Rhizobium trifolii

Summary The Rhizobium trifolii genes necessary for nodule induction and development have been isolated on a 14.0kb fragment of symbiotic (Sym) plasmid DNA. When cloned into a broad-host-range plasmid vector, these sequences confer a clover nodulation phenotype on a derivative of R. trifolii which has been cured of its endogenous Sym plasmid. Furthermore, these sequences encode both host specificity and nodulation functions since they confer the ability to recognize and nodulate clover plants on Agrobacterium and a fast-growing cowpea Rhizobium. This indicates that the bacterial genes essential for the initial, highly-specific interaction with plants are closely linked.