Symbiotic Nitrogen-fixing Bacteria Research Articles

Soil Bradyrhizobium population response to invasion of a natural Quercus suber forest by the introduced nitrogen-fixing tree Acacia mearnsii in El Kala National Park, Algeria

We analyzed the diversity and identity of the rhizobial populations nodulating an invasive Australian legume tree Acacia mearnsii in a natural Quercus suber forest in the El Kala National Park, Algeria. Soils from three different forest plots corresponding to non invaded original Q. suber stand, partially invaded by A. mearnsii, and totally invaded (monodominant) A. mearnsii stand were used to trap nodulating bacteria with the same tree species. Symbiotic nitrogen-fixing bacteria were isolated from root nodules and characterized by sequencing of the internal transcribed spacer region, then submitted to phylogenetic analyses. A total of 67 isolates was obtained, representing the 3 different forest plots, all renodulating A. mearnsii in monoxenic conditions. Phylogenetic analyses showed that all isolates belong to different Bradyrhizobium lineages, according to each of the three locations with little intermixing between forest plots. These results illustrate the adaptation of nodulating Bradyrhizobium populations to the new soil conditions induced by invasion. This symbiotic adaptability is presumed to be a key factor of the invasive character of this tree species.

RNA-Seq analysis of the multipartite genome of Rhizobium etli CE3 shows different replicon contributions under heat and saline shock.

BackgroundRegulation of transcription is essential for any organism and Rhizobium etli (a multi-replicon, nitrogen-fixing symbiotic bacterium) is no exception. This bacterium is commonly found in the rhizosphere (free-living) or inside of root-nodules of the common bean (Phaseolus vulgaris) in a symbiotic relationship. Abiotic stresses, such as high soil temperatures and salinity, compromise the genetic stability of R. etli and therefore its symbiotic interaction with P. vulgaris. However, it is still unclear which genes are up- or down-regulated to cope with these stress conditions. The aim of this study was to identify the genes and non-coding RNAs (ncRNAs) that are differentially expressed under heat and saline shock, as well as the promoter regions of the up-regulated loci.ResultsAnalysing the heat and saline shock responses of R. etli CE3 through RNA-Seq, we identified 756 and 392 differentially expressed genes, respectively, and 106 were up-regulated under both conditions. Notably, the set of genes over-expressed under either condition was preferentially encoded on plasmids, although this observation was more significant for the heat shock response. In contrast, during either saline shock or heat shock, the down-regulated genes were principally chromosomally encoded. Our functional analysis shows that genes encoding chaperone proteins were up-regulated during the heat shock response, whereas genes involved in the metabolism of compatible solutes were up-regulated following saline shock. Furthermore, we identified thirteen and nine ncRNAs that were differentially expressed under heat and saline shock, respectively, as well as eleven ncRNAs that had not been previously identified. Finally, using an in silico analysis, we studied the promoter motifs in all of the non-coding regions associated with the genes and ncRNAs up-regulated under both conditions.ConclusionsOur data suggest that the replicon contribution is different for different stress responses and that the heat shock response is more complex than the saline shock response. In general, this work exemplifies how strategies that not only consider differentially regulated genes but also regulatory elements of the stress response provide a more comprehensive view of bacterial gene regulation.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-770) contains supplementary material, which is available to authorized users.

Mutation in the pssA gene involved in exopolysaccharide synthesis leads to several physiological and symbiotic defects in Rhizobium leguminosarum bv. trifolii.

The symbiotic nitrogen-fixing bacterium Rhizobium leguminosarum bv. trifolii 24.2 secretes large amounts of acidic exopolysaccharide (EPS), which plays a crucial role in establishment of effective symbiosis with clover. The biosynthesis of this heteropolymer is conducted by a multi-enzymatic complex located in the bacterial inner membrane. PssA protein, responsible for the addition of glucose-1-phosphate to a polyprenyl phosphate carrier, is involved in the first step of EPS synthesis. In this work, we characterize R. leguminosarum bv. trifolii strain Rt270 containing a mini-Tn5 transposon insertion located in the 3′-end of the pssA gene. It has been established that a mutation in this gene causes a pleiotropic effect in rhizobial cells. This is confirmed by the phenotype of the mutant strain Rt270, which exhibits several physiological and symbiotic defects such as a deficiency in EPS synthesis, decreased motility and utilization of some nutrients, decreased sensitivity to several antibiotics, an altered extracellular protein profile, and failed host plant infection. The data of this study indicate that the protein product of the pssA gene is not only involved in EPS synthesis, but also required for proper functioning of Rhizobium leguminosarum bv. trifolii cells.

The Effect of pH Stress on the Survival of Rhizobium Trifolii and Sinorhizobium Meliloti in Vitro

Nitrogen-fixing symbiotic bacteria known as rhizobia can exist in different soils and adapt to different environmental conditions. The aim of this study was to determine the impact of pH on the growth of Rhizobium trifolii and Sinorhizobium meliloti. Rhizobial species were isolated using yeast extract mannitol agar medium) in which the pH values were adjusted to 5.0, 6.0, 8.0 and 9.0 by adding HCl and NaOH. The optimum pH for rhizobia is neutral or slightly alkaline (pH 8) and they are more sensitive to acidity. Sinorhizobium meliloti developed better in an acid medium compared to Rhizobium trifolii.

Legume Genomics: From Genomic Resources to Molecular Breeding

With an explosive growth rate, especially in developing countries, the world population of 7.2 billion is expected to reach 9.6 billion by 2050. There is a need to produce about 70% more food to feed this predicted population. Legumes form important constituents of a vegetarian diet and are rich sources of dietary protein (Duranti and Gius, 1997). Legumes comprise the third largest family of flowering plants and provide important sources of food, fodder, oil, and fiber products. Legume seeds typically contain 20 to 25% protein and are also a rich source of dietary fiber. In addition, legumes have the capability to fix atmospheric N2 with the help of symbiotic nitrogen fixing bacteria in root nodules, thereby reducing fertilizer use in agriculture, and the cost of nitrogen inputs by smallholder farmers in developing countries. Due to their higher protein content and other nutrients, legumes are considered important to confront malnutrition among resource-poor people in developing countries. In brief, legumes including beans (Phaseolus vulgaris), chickpea (Cicer arietinum), cowpea (Vigna unguiculata), lentils (Lens culinaris), pea (Pisum sativum), peanut (Arachis hypogaea), and soybean (Glycine max), etc. play an important role in ensuring food security, reducing poverty, improving human health and nutrition, and enhancing ecosystem resilience, especially in developing countries....

Microvirga vignae sp. nov., a root nodule symbiotic bacterium isolated from cowpea grown in semi-arid Brazil

16S rRNA gene sequence analysis of eight strains (BR 3299(T), BR 3296, BR 10192, BR 10193, BR 10194, BR 10195, BR 10196 and BR 10197) isolated from nodules of cowpea collected from a semi-arid region of Brazil showed 97 % similarity to sequences of recently described rhizobial species of the genus Microvirga. Phylogenetic analyses of four housekeeping genes (gyrB, recA, dnaK and rpoB), DNA-DNA relatedness and AFLP further indicated that these strains belong to a novel species within the genus Microvirga. Our data support the hypothesis that genes related to nitrogen fixation were obtained via horizontal gene transfer, as sequences of nifH genes were very similar to those found in members of the genera Rhizobium and Mesorhizobium, which are not immediate relatives of the genus Microvirga, as shown by 16S rRNA gene sequence analysis. Phenotypic traits, such as host range and carbon utilization, differentiate the novel strains from the most closely related species, Microvirga lotononidis, Microvirga zambiensis and Microvirga lupini. Therefore, these symbiotic nitrogen-fixing bacteria are proposed to be representatives of a novel species, for which the name Microvirga vignae sp. nov. is suggested. The type strain is BR3299(T) ( = HAMBI 3457(T)).

Draft genome sequence of Sinorhizobium meliloti RU11/001, a model organism for flagellum structure, motility and chemotaxis

Sinorhizobium meliloti of the order Rhizobiales is a symbiotic nitrogen-fixing bacterium nodulating plants of the genera Medicago, Trigonella and Melilotus and hence is of great agricultural importance. In its free-living state it is motile and capable of modulating its movement patterns in response to chemical attractants. Here, the draft genome consisting of a circular chromosome, the megaplasmids pSymA and pSymB and three accessory plasmids of Sinorhizobium meliloti RU11/001, a model organism for flagellum structure, motility and chemotaxis, is reported.

Functional Conservation of the Capacity for ent-Kaurene Biosynthesis and an Associated Operon in Certain Rhizobia

Bacterial interactions with plants are accompanied by complex signal exchange processes. Previously, the nitrogen-fixing symbiotic (rhizo)bacterium Bradyrhizobium japonicum was found to carry adjacent genes encoding two sequentially acting diterpene cyclases that together transform geranylgeranyl diphosphate to ent-kaurene, the olefin precursor to the gibberellin plant hormones. Species from the three other major genera of rhizobia were found to have homologous terpene synthase genes. Cloning and functional characterization of a representative set of these enzymes confirmed the capacity of each genus to produce ent-kaurene. Moreover, comparison of their genomic context revealed that these diterpene synthases are found in a conserved operon which includes an adjacent isoprenyl diphosphate synthase, shown here to produce the geranylgeranyl diphosphate precursor, providing a critical link to central metabolism. In addition, the rest of the operon consists of enzymatic genes that presumably lead to a more elaborated diterpenoid, although the production of gibberellins was not observed. Nevertheless, it has previously been shown that the operon is selectively expressed during nodulation, and the scattered distribution of the operon via independent horizontal gene transfer within the symbiotic plasmid or genomic island shown here suggests that such diterpenoid production may modulate the interaction of these particular symbionts with their host plants.

The Sinorhizobium meliloti essential porin RopA1 is a target for numerous bacteriophages.

The symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti harbors a gene, SMc02396, which encodes a predicted outer membrane porin that is conserved in many symbiotic and pathogenic bacteria in the order Rhizobiales. Here, this gene (renamed ropA1) is shown to be required for infection by two commonly utilized transducing bacteriophages (ΦM12 and N3). Mapping of S. meliloti mutations conferring resistance to ΦM12, N3, or both phages simultaneously revealed diverse mutations mapping within the ropA1 open reading frame. Subsequent tests determined that RopA1, lipopolysaccharide, or both are required for infection by all of a larger collection of Sinorhizobium-specific phages. Failed attempts to disrupt or delete ropA1 suggest that this gene is essential for viability. Phylogenetic analysis reveals that ropA1 homologs in many Rhizobiales species are often found as two genetically linked copies and that the intraspecies duplicates are always more closely related to each other than to homologs in other species, suggesting multiple independent duplication events.

Flavodoxin overexpression confers tolerance to oxidative stress in beneficial soil bacteria and improves survival in the presence of the herbicides paraquat and atrazine

To determine whether expression of a cyanobacterial flavodoxin in soil bacteria of agronomic interest confers protection against the widely used herbicides paraquat and atrazine. The model bacterium Escherichia coli, the symbiotic nitrogen-fixing bacterium Ensifer meliloti and the plant growth-promoting rhizobacterium Pseudomonas fluorescens Aur6 were transformed with expression vectors containing the flavodoxin gene of Anabaena variabilis. Expression of the cyanobacterial protein was confirmed by Western blot. Bacterial tolerance to oxidative stress was tested in solid medium supplemented with hydrogen peroxide, paraquat or atrazine. In all three bacterial strains, flavodoxin expression enhanced tolerance to the oxidative stress provoked by hydrogen peroxide and by the reactive oxygen species-inducing herbicides, witnessed by the enhanced survival of the transformed bacteria in the presence of these oxidizing agents. Flavodoxin overexpression in beneficial soil bacteria confers tolerance to oxidative stress and improves their survival in the presence of the herbicides paraquat and atrazine. Flavodoxin could be considered as a general antioxidant resource to face oxidative challenges in different micro-organisms. The use of plant growth-promoting rhizobacteria or nitrogen-fixing bacteria with enhanced tolerance to oxidative stress in contaminated soils is of significant agronomic interest. The enhanced tolerance of flavodoxin-expressing bacteria to atrazine and paraquat points to potential applications in herbicide-treated soils.

An overview of the metabolic differences betweenBradyrhizobium japonicum110 bacteria and differentiated bacteroids from soybean (Glycine max) root nodules: anin vitro13C- and31P-nuclear magnetic resonance spectroscopy study

Bradyrhizobium japonicum is a symbiotic nitrogen-fixing soil bacteria that induce root nodules formation in legume soybean (Glycine max.). Using (13)C- and (31)P-nuclear magnetic resonance (NMR) spectroscopy, we have analysed the metabolite profiles of cultivated B. japonicum cells and bacteroids isolated from soybean nodules. Our results revealed some quantitative and qualitative differences between the metabolite profiles of bacteroids and their vegetative state. This includes in bacteroids a huge accumulation of soluble carbohydrates such as trehalose, glutamate, myo-inositol and homospermidine as well as Pi, nucleotide pools and intermediates of the primary carbon metabolism. Using this novel approach, these data show that most of the compounds detected in bacteroids reflect the metabolic adaptation of rhizobia to the surrounding microenvironment with its host plant cells.

NODULE INCEPTION Directly Targets NF-Y Subunit Genes to Regulate Essential Processes of Root Nodule Development in Lotus japonicus

The interactions of legumes with symbiotic nitrogen-fixing bacteria cause the formation of specialized lateral root organs called root nodules. It has been postulated that this root nodule symbiosis system has recruited factors that act in early signaling pathways (common SYM genes) partly from the ancestral mycorrhizal symbiosis. However, the origins of factors needed for root nodule organogenesis are largely unknown. NODULE INCEPTION (NIN) is a nodulation-specific gene that encodes a putative transcription factor and acts downstream of the common SYM genes. Here, we identified two Nuclear Factor-Y (NF-Y) subunit genes, LjNF-YA1 and LjNF-YB1, as transcriptional targets of NIN in Lotus japonicus. These genes are expressed in root nodule primordia and their translational products interact in plant cells, indicating that they form an NF-Y complex in root nodule primordia. The knockdown of LjNF-YA1 inhibited root nodule organogenesis, as did the loss of function of NIN. Furthermore, we found that NIN overexpression induced root nodule primordium-like structures that originated from cortical cells in the absence of bacterial symbionts. Thus, NIN is a crucial factor responsible for initiating nodulation-specific symbiotic processes. In addition, ectopic expression of either NIN or the NF-Y subunit genes caused abnormal cell division during lateral root development. This indicated that the Lotus NF-Y subunits can function to stimulate cell division. Thus, transcriptional regulation by NIN, including the activation of the NF-Y subunit genes, induces cortical cell division, which is an initial step in root nodule organogenesis. Unlike the legume-specific NIN protein, NF-Y is a major CCAAT box binding protein complex that is widespread among eukaryotes. We propose that the evolution of root nodules in legume plants was associated with changes in the function of NIN. NIN has acquired functions that allow it to divert pathways involved in the regulation of cell division to root nodule organogenesis.

Selection/ Isolation of High Temperature Tolerant Strains of Rhizobium for Management of High Temperature Stress on Rhizobium- Legume Symbiosis

Biological nitrogen fixation (BNF) is carried out by free-living, non-symbiotic, associative and symbiotic nitrogen fixing bacteria, symbiotic fixers being the major contributors. Legume- Rhizobium association being most effectively and widely studied is the major area of concern with regard to BNF technology. The symbiotic association between legumes and rhizobia is highly influenced by various environmental factors, temperature being the most important one. The growth and survival of rhizobia in soils and their symbiotic association with leguminous plants are adversely affected by high soil temperatures. Therefore, the potential benefit of legume - Rhizobium association is not fully exploited when soil temperatures become limiting, as is generally seen in tropical and subtropical regions, where the temperatures rise beyond 45 o C in the uppermost layers of the soil during the summer season. Isolating/ selecting temperature- resistant/ tolerant strains of rhizobia is, therefore, necessary before establishing inoculant production for these regions. The present review is aimed at understanding the effect of high temperature on Rhizobium-legume symbiosis; and at exploring the on-going efforts being employed for the management of this adverse effect.

Mitigation of nitrous oxide emissions from soils by Bradyrhizobium japonicum inoculation

Soybean hosts the symbiotic nitrogen-fixing soil bacterium Bradyrhizobium japonicum, that can produce the greenhouse gas nitrous oxide. This study shows that nitrous oxide emissions from soybean ecosystems can be biologically mitigated at a field scale by inoculation with strains of B. japonicum that have increased nitrous oxide reductase activity.

Effects of single and co-inoculation with native phosphate solubilising strain Pantoea sp J49 and the symbiotic nitrogen fixing bacterium Bradyrhizobium sp SEMIA 6144 on peanut (Arachis hypogaea L.) growth

In the present study, attempts were made to analyze the effect of co-inoculation with an efficient phosphate solubilising native isolate Pantoea sp J49 and the symbiotic nitrogen fixing Bradyrhizobium sp SEMIA 6144 strain on Arachis hypogaea L. plants growth. Single and co-inoculation of peanut plants growing in plastic pots containing soil with low P content were developed. Plants were harvested at R1 and R4 growth stages and were analyzed in different growth parameters. Survival of strain Pantoea sp J49 was analyzed in soil samples and in root tissues. Plants inoculated only with Pantoea sp J49 showed the highest shoot and root weight in both reproductive growth stages evaluated. Plants co-inoculated with this strain and Bradyrhizobium sp SEMIA 6144 showed increase in aerial dry weight at R1 stage. Survival assays demonstrated that Pantoea sp J49 survives not only in the peanut rhizosphere but also inside plant tissues, including nodules formed when it was co-inoculated with Bradyrhizobium sp SEMIA 6144. Results obtained in this study confirm the great potential of the native Pantoea sp J49 isolate in the promotion of peanut plant growth, probably related with its capacity to solubilise phosphate.

Arbuscular mycorrhizal symbiosis and alleviation of salinity stress

Several environmental factors adversely affect plant growth and development and final yield performance of a crop. Drought, salinity, nutrient imbalances (including mineral toxicities and deficiencies) and extremes of temperature are among the major environmental constraints to crop productivity worldwide. Development of crop plants with stress tolerance, however, requires, among others, knowledge of the physiological mechanisms and genetic controls of the contributing traits at different plant developmental stages. In the past two decades,biotechnology research has provided considerable insights into the mechanism of biotic stress tolerance in plants at the molecular level. Furthermore, different abiotic stress factors may provoke osmotic stress, oxidative stress and protein denaturation in plants, which lead to similar cellular adaptive responses such as accumulation of compatible solutes, induction of stress proteins, and acceleration of reactive oxygen species scavenging systems. Recently, various methods are adapted to improve plant tolerance to salinity injury through either chemical treatments (plant hormones, minerals, amino acids, quaternary ammonium compounds, polyamines and vitamins) or biofertilizers treatments (Asymbiotic nitrogen-fixing bacteria, symbiotic nitrogen-fixing bacteria) or enhanced a process used naturally by plants (mycorrhiza) to minimise the movement of Na+ to the shoot. Proper management of Arbuscular Mycorrhizal Fungi (AMF) has the potential to improve the profitability and sustainability of salt tolerance. In this review article, the discussion is restricted to the mycorrhizal symbiosis and alleviation of salinity stress.

Structure of Bradavidin – C-Terminal Residues Act as Intrinsic Ligands

Bradavidin is a homotetrameric biotin-binding protein from Bradyrhizobium japonicum, a nitrogen fixing and root nodule-forming symbiotic bacterium of the soybean. Wild-type (wt) bradavidin has 138 amino acid residues, whereas the C-terminally truncated core-bradavidin has only 118 residues. We have solved the X-ray structure of wt bradavidin and found that the C-terminal amino acids of each subunit were uniquely bound to the biotin-binding pocket of an adjacent subunit. The biotin-binding pocket occupying peptide (SEKLSNTK) was named “Brad-tag” and it serves as an intrinsic stabilizing ligand in wt bradavidin. The binding of Brad-tag to core-bradavidin was analysed by isothermal titration calorimetry and a binding affinity of ∼25 µM was measured. In order to study the potential of Brad-tag, a green fluorescent protein tagged with Brad-tag was prepared and successfully concentrated from a bacterial cell lysate using core-bradavidin-functionalized Sepharose resin.

A Positive Correlation between Bacterial Autoaggregation and Biofilm Formation in Native Sinorhizobium meliloti Isolates from Argentina

Sinorhizobium meliloti is a symbiotic nitrogen-fixing bacterium that elicits nodule formation on roots of alfalfa plants. S. meliloti produces two exopolysaccharides (EPSs), termed EPS I and EPS II, that are both able to promote symbiosis. EPS I and EPS II are secreted in two major fractions that reflect differing degrees of subunit polymerization, designated high- and low-molecular-weight fractions. We reported previously that EPSs are crucial for autoaggregation and biofilm formation in S. meliloti reference strains and isogenic mutants. However, the previous observations were obtained by use of "domesticated" laboratory strains, with mutations resulting from successive passages under unnatural conditions, as has been documented for reference strain Rm1021. In the present study, we analyzed the autoaggregation and biofilm formation abilities of native S. meliloti strains isolated from root nodules of alfalfa plants grown in four regions of Argentina. 16S rRNA gene analysis of all the native isolates revealed a high degree of identity with reference S. meliloti strains. PCR analysis of the expR gene of all the isolates showed that, as in the case of reference strain Rm8530, this gene is not interrupted by an insertion sequence (IS) element. A positive correlation was found between autoaggregation and biofilm formation abilities in these rhizobia, indicating that both processes depend on the same physical adhesive forces. Extracellular complementation experiments using mutants of the native strains showed that autoaggregation was dependent on EPS II production. Our results indicate that a functional EPS II synthetic pathway and its proper regulation are essential for cell-cell interactions and surface attachment of S. meliloti.

Diversity and stress-tolerance of symbiotic nitrogen-fixing bacteria from legumes in the dry-hot valleys of southwest China

The Dry-Hot valleys in Southwest China are hostile environments for living organisms. The dominant plants in the valleys are drought-resistant legumes. Here, we examined the diversity and physiology of the nitrogen-fixing bacteria associated with these legumes. Thirty-three strains were isolated from the nodules of six legume species located in five valleys. Analyses of their 16S rRNA gene sequences indicated that each strain had a unique 16S rRNA gene sequence and none of the strains had a sequence identical to those reported in the GenBank. The 16S rRNA gene sequence showed little clustering based on host tree species or geographic locations. PCR fingerprinting confirmed the genetic uniqueness of these strains. Physiological tests showed that these strains were all capable of growing at 35°C or above and at hypertonic environments. All strains formed root nodules on Acacia richii, the most common legume in these valleys. The bacterial strains obtained here could help future reforestation efforts in these valleys and other environments with similar conditions. Key words: Rhizobium spp., Bradyrhizobium spp., Mesorhizobium spp., 16S rRNA, high temperature resistance, drought resistanc

Corals Form Characteristic Associations with Symbiotic Nitrogen-Fixing Bacteria

The complex symbiotic relationship between corals and their dinoflagellate partner Symbiodinium is believed to be sustained through close associations with mutualistic bacterial communities, though little is known about coral associations with bacterial groups able to fix nitrogen (diazotrophs). In this study, we investigated the diversity of diazotrophic bacterial communities associated with three common coral species (Acropora millepora, Acropora muricata, and Pocillopora damicormis) from three midshelf locations of the Great Barrier Reef (GBR) by profiling the conserved subunit of the nifH gene, which encodes the dinitrogenase iron protein. Comparisons of diazotrophic community diversity among coral tissue and mucus microenvironments and the surrounding seawater revealed that corals harbor diverse nifH phylotypes that differ between tissue and mucus microhabitats. Coral mucus nifH sequences displayed high heterogeneity, and many bacterial groups overlapped with those found in seawater. Moreover, coral mucus diazotrophs were specific neither to coral species nor to reef location, reflecting the ephemeral nature of coral mucus. In contrast, the dominant diazotrophic bacteria in tissue samples differed among coral species, with differences remaining consistent at all three reefs, indicating that coral-diazotroph associations are species specific. Notably, dominant diazotrophs for all coral species were closely related to the bacterial group rhizobia, which represented 71% of the total sequences retrieved from tissue samples. The species specificity of coral-diazotroph associations further supports the coral holobiont model that bacterial groups associated with corals are conserved. Our results suggest that, as in terrestrial plants, rhizobia have developed a mutualistic relationship with corals and may contribute fixed nitrogen to Symbiodinium.