Complete genome structure of the nitrogen-fixing symbiotic bacterium Mesorhizobium loti.

The complete nucleotide sequence of the genome of a symbiotic bacterium Mesorhizobium loti strain MAFF303099 was determined. The genome of M. loti consisted of a single chromosome (7,036,071 bp) and two plasmids, designated as pMLa (351,911 bp) and pMLb (208, 315 bp). The chromosome comprises 6752 potential protein-coding genes, two sets of rRNA genes and 50 tRNA genes representing 47 tRNA species. Fifty-four percent of the potential protein genes showed sequence similarity to genes of known function, 21% to hypothetical genes, and the remaining 25% had no apparent similarity to reported genes. A 611-kb DNA segment, a highly probable candidate of a symbiotic island, was identified, and 30 genes for nitrogen fixation and 24 genes for nodulation were assigned in this region. Codon usage analysis suggested that the symbiotic island as well as the plasmids originated and were transmitted from other genetic systems. The genomes of two plasmids, pMLa and pMLb, contained 320 and 209 potential protein-coding genes, respectively, for a variety of biological functions. These include genes for the ABC-transporter system, phosphate assimilation, two-component system, DNA replication and conjugation, but only one gene for nodulation was identified.     

Strains of Mesorhizobium amorphae and Mesorhizobium tianshanense, carrying symbiotic genes of common chickpea endosymbiotic species, constitute a novel biovar (ciceri) capable of nodulating Cicer arietinum

AIMS: To identify several strains of Mesorhizobium amorphae and Mesorhizobium tianshanense nodulating Cicer arietinum in Spain and Portugal, and to study the symbiotic genes carried by these strains. METHODS AND RESULTS: The sequences of 16S-23S intergenic spacer (ITS), 16S rRNA gene and symbiotic genes nodC and nifH were analysed. According to their 16S rRNA gene and ITS sequences, the strains from this study were identified as M. amorphae and M. tianshanense. The type strains of these species were isolated in China from Glycyrrhiza pallidiflora and Amorpha fruticosa nodules, respectively, and are not capable of nodulating chickpea. These strains carry symbiotic genes, phylogenetically divergent from those of the chickpea isolates, whose nodC and nifH genes showed more than 99% similarity with respect to those from Mesorhizobium ciceri and Mesorhizobium mediterraneum, the two common chickpea nodulating species in Spain and Portugal. CONCLUSIONS: The results from this study showed that different symbiotic genes have been acquired by strains from the same species during their coevolution with different legumes in distinct geographical locations. SIGNIFICANCE AND IMPACT OF THE STUDY: A new infrasubspecific division named biovar ciceri is proposed within M. amorphae and M. tianshanense to include the strains able to effectively nodulate Cicer arietinum.     

Control of the expression of bacterial genes involved in symbiotic nitrogen fixation

Several genera of N₂-fixing bacteria establish symbiotic associations with plants. Among these, the genus Rhizobium has the most significant contribution, in terms of yield, in many important crop plants. The establishment of the Rhizobium-legume symbiosis is a very complex process involving many genes which need to be co-ordinately regulated. In the first instance, plant signal molecules, known to be flavonoids, trigger the expression of host-specific genes in the bacterial partner through the action of the regulatory NodD protein. In response to these signals, Rhizobium bacteria synthesize lipo-oligosaccharide molecules which in turn cause cell differentiation and nodule development. Once the nodule has formed, Rhizobium cells differentiate into bacteroids and another set of genes is activated. These genes, designated nif and fix, are responsible for N₂ fixation. In this system, several regulatory proteins are involved in a complex manner, the most important being NifA and a two component (FixK and FixL) regulatory system. Our knowledge about the establishment of these symbioses has advanced recently, although there are many questions yet to be solved.     

Phylogenetic analyses of symbiotic genes and characterization of functional traits of Mesorhizobium spp. strains associated with the promiscuous species Acacia seyal Del.

Aims: To assess the phenotypic, symbiotic and genotypic diversity scope of Mesorhizobium spp. strains associated with Acacia seyal (Del.) isolated from different agro‐ecological zones in Senegal, and uses of susceptible microbial inoculum in a reafforestation process. Methods and Results: A polyphasic approach including phenotypic and genotypic techniques was used to study the diversity and their relationships with other biovars and species of rhizobia. The geographical origins of the strains have limited effect on their phylogenetic and phenotypic classification. Nodulation tests indicated promiscuity of the strains studied, because they were capable of nodulating six woody legume species (Acacia auriculiformis, Acacia senegal, A. seyal, Acacia tortilis ssp. raddiana, Leucaena leucocephala and Prosopis juliflora). Sequencing and phylogenetic analyses of nodA, nodC and nifH genes pointed out that in contrast to nodA gene, the phylogenies of nodC and nifH genes were not consistent with that of 16S rRNA, indicating that these genes of the A. seyal‐nodulating rhizobia might have different origins. Microbial inoculation on nonsterile soil had significant effect on the nodules number and the growth of the seedlings, indicating that these strains of rhizobia might be used as inoculum. Conclusions: The results indicated that A. seyal is a nonselective host that can establish effective symbiosis with Mesorhizobium spp. strains from diverse genomic backgrounds and that the selected A. seyal‐nodulating rhizobia could enhance plant growth. Significance and Impact of the Study: These results showed the important role that A. seyal could play in the improvement of reafforestation process as a promiscuous host, which can establish effective symbiosis with rhizobia from diverse genomic backgrounds.     

Evolutionary trend of phylogenetic diversity of nitrogen fixation genes in the gut community of wood-feeding termites

Nitrogen fixation by gut microorganisms is one of the crucial aspects of symbiosis in wood-feeding termites since these termites thrive on a nitrogen-poor diet. In order to understand the evolution of this symbiosis, we analysed the nitrogenase structural gene nifH in the gut microbial communities. In conjunction with the published sequences, we compared approximately 320 putatively functional NifH protein sequences obtained from a total of 19 termite samples that represent all the major branches of their currently proposed phylogeny, and from one species of the cockroach Cryptocercus that shares a common ancestor with termites. Using multivariate techniques for clustering and ordination, a phylogeny of NifH protein sequences was created and plotted variously with host termite families, genera, and species. Close concordance was observed between NifH communities and the host termites at genus level, but family level relationships were not always congruent with accepted termite clade structure. Host groups examined included basal families (Mastotermitidae, Termopsidae, Kalotermitidae, as well as Cryptocercus), the most derived lower termite family Rhinotermitidae, and subfamilies representing the advanced and highly diverse apical family Termitidae (Macrotermitinae, Termitinae, and Nasutitermitinae). This selection encompassed the major nesting and feeding styles recognized in termites, and it was evident that NifH phylogenetic divergence, as well as the occurrence of alternative nitrogenase-type NifH, was to some extent dependent on host lifestyle as well as phylogenetic position.     

Interaction of nitrogen monoxide with hemoglobin and the artefactual production of S-nitroso-hemoglobin

Hemoglobin (Hb) is probably the most thoroughly studied protein in the human body. However, it has recently been proposed that in addition to the well known function of dioxygen and carbon dioxide transporter, one of the main roles of hemoglobin is to store and transport nitrogen monoxide. This hypothesis is highly disputed and is in contrast to the proposal that hemoglobin serves as an NO. scavenger in the blood. In this short review, I have presented the current status of research on the much-debated mechanism of the reaction between circulating hemoglobin and NO.. Despite the fact that oxyHb is extremely rapidly oxidized by NO., under basal physiological conditions the biological activity of NO. in the blood vessels is not completely lost. It has been shown that three factors reduce the efficiency of hemoglobin to scavenge NO.: a so-called red blood cell-free zone created close to the vessel wall by intravascular flow, an undisturbed layer around the red blood cells--where the NO. concentration is much smaller than the bulk concentration--and/or the red blood cell membrane. Alternatively, it has been proposed that NO. binds to Cys beta 93 of oxyHb, is liberated after deoxygenation of Hb, and consequently allows for a more effective delivery of O2 to peripheral tissues. However, because of the extremely fast rate of the reaction between NO. and oxyHb, experiments in vitro lead to artefactual production of large amounts of S-nitroso-hemoglobin. These results, together with other data, which challenge most steps of the NO.-transporter hypothesis, are discussed.     

Seasonal flooding regimes influence survival,nitrogen fixation,and the partitioning of nitrogen and biomass in Alnus incana ssp. rugosa

Alteration of natural flooding regimes can expose lowlands to waterlogged soil conditions during any month of the year. The seasonality of flooding may have profound effects on the carbon and nitrogen budgets of N-fixing alders (Alnus spp.), and in turn, may impact the C and N economy of extensive alder-dominated, wetland ecosystems, including those dominated by speckled alder (Alnus incana ssp. rugosa). To better understand this process, two-year-old, nodulated seedlings of speckled alder were subjected to late spring (May 10 – July 10), summer (July 10 – September 8), and fall (September 8 – November 8) flooding treatments. Alders were root-flooded outdoors in tanks containing an N-free nutrient solution and compared with unflooded alders at the experimental site. Flooding arrested N fixation, photosynthesis, and growth of alders without recovery in all flooding treatments for the remainder of the growing season. Late spring and summer flooding resulted in complete mortality of alders while all seedlings survived flooding in the fall. Fall flooding increased foliar N resorption by 140% over unflooded seedlings. Eighty-seven percent of the total N fixed and 89% of biomass accumulation for the entire growing season occurred in unflooded alders after July 10. In unflooded alders, nitrogen fixation rates per unit mass declined by 63% for nodules, 28% for leaves, and 48% for whole seedlings during the fall, while total N fixed per plant in the fall was similar to that fixed in the summer. The majority of newly fixed N in unflooded alders was allocated to leaves before September 8 and to roots/nodules combined after September 8. In unflooded plants, the greatest proportion of new biomass was partitioned to leaves before July 10, to stems between July 10 and September 8, and equally to stems and roots/nodules after September 8. Fall-flooded alders did not increase root or nodule biomass. Proportional allocation of plant resources were such that the ratio of N fixed to seedling growth of unflooded alders decreased by 19% during summer before rebounding by 6% in fall. Seasonality of flooding alters seedling survival, growth, and resource allocation, and may be a critical determinant of speckled alder recruitment and occurrence in wetlands.     

Expression of a class 1 hemoglobin gene and production of nitric oxide in response to symbiotic and pathogenic bacteria in Lotus japonicus

Symbiotic nitrogen fixation by the collaboration between leguminous plants and rhizobia is an important system in the global nitrogen cycle, and some molecular aspects during the early stage of host-symbiont recognition have been revealed. To understand the responses of a host plant against various bacteria, we examined expression of hemoglobin (Hb) genes and production of nitric oxide (NO) in Lotus japonicus after inoculation with rhizobia or plant pathogens. When the symbiotic rhizobium Mesorhizobium loti was inoculated, expression of LjHb1 and NO production were induced transiently in the roots at 4 h after inoculation. In contrast, inoculation with the nonsymbiotic rhizobia Sinorhizobium meliloti and Bradyrhizobium japonicum induced neither expression of LjHb1 nor NO production. When L. japonicus was inoculated with plant pathogens (Ralstonia solanacearum or Pseudomonas syringae), continuous NO production was observed in roots but induction of LjHb1 did not occur. These results suggest that modulation of NO levels and expression of class 1 Hb are involved in the establishment of the symbiosis.     

木麻黄共生固氮菌Frankia的分离鉴定及固氮效应

从浙江省短枝木麻黄(Casuarina equisetifolia)和细枝木麻黄(C.curninghamiana的根瘤中共分离获得14株共生菌株.形态观察表明,菌株具有分枝状菌丝、多腔孢囊、泡囊等典型的Frankia结构、16S rDNA测序结果表明,供试菌株均为Frankia,其中4株属于生理类群A,7株属于生理类群B,3株属于生理类群AB.固氮效应研究表明,菌株均具有固氨酶生物学活性,但菌株之间存在显著差异,其中菌株ZCN192固氨酶活性最强,可达2.897 μmol·mg-1·h-1,菌株ZCN199最低,固氮酶活性为0.056 μmol·mg-1·h-1.活体固氮试验显示,与阴性对照相比,供试菌株能显著提高苗高、地径和干重,且一般情况下,离体固氮酶活性强的菌株在活体接种时能获得更明显的固氮效应.     

马桑结瘤固氮与光合作用的关系

马桑(Coriaria sinica)植株的结瘤量、根瘤固氮活性和固氮能力均与植株叶面积和光合能力呈显著的直线相关关系,叶面积大、光合能力强的植株结瘤量大,根瘤固氮活性高,固氮能力强。马桑根瘤固氮活性呈白天升高夜间降低的昼夜变化特点,昼夜变幅为10～20μmol C2H2/g.h,光合作用是引起固氮活性昼夜变化的主要因素,同时受土壤温湿度的影响,遮阴或光照不足将引起马桑结瘤固氮能力的大幅度降低。     

Fertilizers and biological nitrogen fixation as sources of plant nutrients: Perspectives for future agriculture

Biological nitrogen fixation (BNF) has an assured place in agriculture, mainly as a source of nitrogen for legumes. Legumes are currently grown mostly as a source of vegetable oil and as food for humans and animals, but not as nitrogen source.Other crops with BNF capability may be eventually be developed eventually. Such crops will also need mineral fertilizers to maintain a good status of soil nutrients, but their possible effects to the environment is also a concern. Fertilizers, however, will remain a necessary and sustainable input to agriculture to feed the present and increasing human population. It is not a case of whether BNF is better or worse than mineral fertilizers because both plays an important role in agriculture.     

The contribution of associative and symbiotic nitrogen fixation to the nitrogen nutrition of non-legumes

During the past 10 years estimates of N₂ fixation associated with sugar cane, forage grasses, cereals and actinorhizal plants grown in soil with and without addition of inoculum have been obtained using the ¹⁵N isotope dilution technique. These experiments are reviewed in this paper with the aim of determining the proportional and absolute contribution of N₂ fixation to the N nutrition of non-legumes, and its role as a source of N in agriculture. The review also identifies deficiencies in both the totality of data which are currently available and the experimental approaches used to quantify N₂ fixation associated with non-legumes.Field data indicate that associative N₂ fixation can potentially contribute agronomically-significant amounts of N (>30–40 kg N ha^-1 y^-1) to the N nutrition of plants of importance in tropical agriculture, including sugar cane (Saccharum sp.) and forage grasses (Panicum maximum, Brachiaria sp. and Leptochloa fusca) when grown in uninoculated, N-deficient soils. Marked variations in proportions of plant N derived from the atmosphere have been measured between species or cultivars within species.Limited pot-culture data indicate that rice can benefit naturally from associative N₂ fixation, and that inoculation responses due to N₂ fixation can occur. Wheat can also respond to inoculation but responses do not appear to be due to associative N₂ fixation. ¹⁵N dilution studies confirm that substantial amounts of N₂ can be fixed by actinorhizal plants.     

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.     

Loss‐of‐function of ASPARTIC PEPTIDASE NODULE‐INDUCED 1 (APN1) in Lotus japonicus restricts efficient nitrogen‐fixing symbiosis with specific Mesorhizobium loti strains

The nitrogen‐fixing symbiosis of legumes and Rhizobium bacteria is established by complex interactions between the two symbiotic partners. Legume Fix^– mutants form apparently normal nodules with endosymbiotic rhizobia but fail to induce rhizobial nitrogen fixation. These mutants are useful for identifying the legume genes involved in the interactions essential for symbiotic nitrogen fixation. We describe here a Fix^– mutant of Lotus japonicus, apn1, which showed a very specific symbiotic phenotype. It formed ineffective nodules when inoculated with the Mesorhizobium loti strain TONO. In these nodules, infected cells disintegrated and successively became necrotic, indicating premature senescence typical of Fix^– mutants. However, it formed effective nodules when inoculated with the M. loti strain MAFF303099. Among nine different M. loti strains tested, four formed ineffective nodules and five formed effective nodules on apn1 roots. The identified causal gene, ASPARTIC PEPTIDASE NODULE‐INDUCED 1 (LjAPN1), encodes a nepenthesin‐type aspartic peptidase. The well characterized Arabidopsis aspartic peptidase CDR1 could complement the strain‐specific Fix^– phenotype of apn1. LjAPN1 is a typical late nodulin; its gene expression was exclusively induced during nodule development. LjAPN1 was most abundantly expressed in the infected cells in the nodules. Our findings indicate that LjAPN1 is required for the development and persistence of functional (nitrogen‐fixing) symbiosis in a rhizobial strain‐dependent manner, and thus determines compatibility between M. loti and L. japonicus at the level of nitrogen fixation.