Legume Symbiont Research Articles

Unearthing Optimal Symbiotic Rhizobia Partners from the Main Production Area of Phaseolus vulgaris in Yunnan.

Phaseolus vulgaris is a globally important legume cash crop, which can carry out symbiotic nitrogen fixation with rhizobia. The presence of suitable rhizobia in cultivating soils is crucial for legume cropping, especially in areas beyond the plant-host native range, where soils may lack efficient symbiotic partners. We analyzed the distribution patterns and traits of native rhizobia associated with P. vulgaris in soils of Yunnan, where the common bean experienced a recent expansion. A total of 608 rhizobial isolates were tracked from soils of fifteen sampling sites using two local varieties of P. vulgaris. The isolates were discriminated into 43 genotypes as defined by IGS PCR-RFLP. Multiple locus sequence analysis based on recA, atpD and rpoB of representative strains placed them into 11 rhizobial species of Rhizobium involving Rhizobium sophorae, Rhizobium acidisoli, Rhizobium ecuadorense, Rhizobium hidalgonense, Rhizobium vallis, Rhizobium sophoriradicis, Rhizobium croatiense, Rhizobium anhuiense, Rhizobium phaseoli, Rhizobium chutanense and Rhizobium etli, and five unknown Rhizobium species; Rhizobium genosp. I~V. R. phaseoli and R. anhuiense were the dominant species (28.0% and 28.8%) most widely distributed, followed by R. croatiense (14.8%). The other rhizobial species were less numerous or site-specific. Phylogenies of nodC and nifH markers, were divided into two specific symbiovars, sv. phaseoli regardless of the species affiliation and sv. viciae associated with R. vallis. Through symbiotic effect assessment, all the tested strains nodulated both P. vulgaris varieties, often resulting with a significant greenness index (91-98%). However, about half of them exhibited better plant biomass performance, at least on one common bean variety, and two isolates (CYAH-6 and BLYH-15) showed a better symbiotic efficiency score. Representative strains revealed diverse abiotic stress tolerance to NaCl, acidity, alkalinity, temperature, drought and glyphosate. One strain efficient on both varieties and exhibiting stress abiotic tolerance (BLYH-15) belonged to R. genosp. IV sv. phaseoli, a species first found as a legume symbiont.

A double-negative feedback loop between NtrBC and a small RNA rewires nitrogen metabolism in legume symbionts.

Root nodule endosymbioses between diazotrophic rhizobia and legumes provide the largest input of combined N to the biosphere, thus representing an alternative to harmful chemical fertilizers for sustainable crop production. Rhizobia have evolved intricate strategies to coordinate N assimilation for their own benefit with N2 fixation to sustain plant growth. The rhizobial N status is transduced by the NtrBC two-component system, the seemingly ubiquitous form of N signal transduction in Proteobacteria. Here, we show that the regulatory sRNA NfeR1 (nodule formation efficiency RNA) of the alfalfa symbiont Sinorhizobium meliloti is transcribed from a complex promoter repressed by NtrC in a N-dependent manner and feedback silences ntrBC by complementary base-pairing. These findings unveil a more prominent role of NtrC as a transcriptional repressor than hitherto anticipated and a novel RNA-based mechanism for NtrBC regulation. The NtrBC-NfeR1 double-negative feedback loop accurately rewires symbiotic S. meliloti N metabolism and is likely conserved in α-rhizobia.

A Selective Bottleneck During Host Entry Drives the Evolution of New Legume Symbionts.

During the emergence of new host-microbe symbioses, microbial fitness results from the ability to complete the different steps of symbiotic life cycles, where each step imposes specific selective pressures. However, the relative contribution of these different selective pressures to the adaptive trajectories of microbial symbionts is still poorly known. Here, we characterized the dynamics of phenotypic adaptation to a simplified symbiotic life cycle during the experimental evolution of a plant pathogenic bacterium into a legume symbiont. We observed that fast adaptation was predominantly explained by improved competitiveness for host entry, which outweighed adaptation to within-host proliferation. Whole-population sequencing of bacteria at regular time intervals along this evolution experiment revealed the continuous accumulation of new mutations (fuelled by a transient hypermutagenesis phase occurring at each cycle before host entry, a phenomenon described in previous work) and sequential sweeps of cohorts of mutations with similar temporal trajectories. The identification of adaptive mutations within the fixed mutational cohorts showed that several adaptive mutations can co-occur in the same cohort. Moreover, all adaptive mutations improved competitiveness for host entry, while only a subset of those also improved within-host proliferation. Computer simulations predict that this effect emerges from the presence of a strong selective bottleneck at host entry occurring before within-host proliferation and just after the hypermutagenesis phase in the rhizosphere. Together, these results show how selective bottlenecks can alter the relative influence of selective pressures acting during bacterial adaptation to multistep infection processes.

Soil Protists Can Actively Redistribute Beneficial Bacteria along Medicago truncatula Roots.

The rhizosphere is the region of soil directly influenced by plant roots. The microbial community in the rhizosphere includes fungi, protists, and bacteria: all play significant roles in plant health. The beneficial bacterium Sinorhizobium meliloti infects growing root hairs on nitrogen-starved leguminous plants. Infection leads to the formation of a root nodule, where S. meliloti converts atmospheric nitrogen to ammonia, a bioavailable form. In soil, S. meliloti is often found in biofilms and travels slowly along the roots, leaving developing root hairs at the growing root tips uninfected. Soil protists are an important component of the rhizosphere system, able to travel quickly along roots and water films, who prey on soil bacteria and have been known to egest undigested phagosomes. We show that a soil protist, Colpoda sp., can transport S. meliloti down Medicago truncatula roots. Using model soil microcosms, we directly observed fluorescently labeled S. meliloti along M. truncatula roots and tracked the displacement of the fluorescence signal over time. Two weeks after co-inoculation, this signal extended 52 mm farther down plant roots when Colpoda sp. was also present versus treatments that contained bacteria but not protists. Direct counts also showed protists are required for viable bacteria to reach the deeper sections of our microcosms. Facilitating bacterial transport may be an important mechanism whereby soil protists promote plant health. IMPORTANCE Soil protists are an important part of the microbial community in the rhizosphere. Plants grown with protists fare better than plants grown without protists. Mechanisms through which protists support plant health include nutrient cycling, alteration of the bacterial community through selective feeding, and consumption of plant pathogens. Here, we provide data in support of an additional mechanism: protists act as transport vehicles for bacteria in soil. We show that protist-facilitated transport can deliver plant-beneficial bacteria to the growing tips of roots that may otherwise be sparsely inhabited with bacteria originating from a seed-associated inoculum. By co-inoculating Medicago truncatula roots with both S. meliloti, a nitrogen-fixing legume symbiont, and Colpoda sp., a ciliated protist, we show substantial and statistically significant transport with depth and breadth of bacteria-associated fluorescence as well as transport of viable bacteria. Co-inoculation with shelf-stable encysted soil protists may be employed as a sustainable agriculture biotechnology to better distribute beneficial bacteria and enhance the performance of inoculants.

RopB protein of Rhizobium leguminosarum bv. viciae adopts amyloid state during symbiotic interactions with pea (Pisum sativum L.).

Amyloids represent protein aggregates with highly ordered fibrillar structure associated with the development of various disorders in humans and animals and involved in implementation of different vital functions in all three domains of life. In prokaryotes, amyloids perform a wide repertoire of functions mostly attributed to their interactions with other organisms including interspecies interactions within bacterial communities and host-pathogen interactions. Recently, we demonstrated that free-living cells of Rhizobium leguminosarum, a nitrogen-fixing symbiont of legumes, produce RopA and RopB which form amyloid fibrils at cell surface during the stationary growth phase thus connecting amyloid formation and host-symbiont interactions. Here we focused on a more detailed analysis of the RopB amyloid state in vitro and in vivo, during the symbiotic interaction between R. leguminosarum bv. viciae with its macrosymbiont, garden pea (Pisum sativum L.). We confirmed that RopB is the bona fide amyloid protein since its fibrils exhibit circular x-ray reflections indicating its cross-β structure specific for amyloids. We found that fibrils containing RopB and exhibiting amyloid properties are formed in vivo at the surface of bacteroids of R. leguminosarum extracted from pea nodules. Moreover, using pea sym31 mutant we demonstrated that formation of extracellular RopB amyloid state occurs at different stages of bacteroid development but is enhanced in juvenile symbiosomes. Proteomic screening of potentially amyloidogenic proteins in the nodules revealed the presence of detergent-resistant aggregates of different plant and bacterial proteins including pea amyloid vicilin. We demonstrated that preformed vicilin amyloids can cross-seed RopB amyloid formation suggesting for probable interaction between bacterial and plant amyloidogenic proteins in the nodules. Taken together, we demonstrate that R. leguminosarum bacteroids produce extracellular RopB amyloids in pea nodules in vivo and these nodules also contain aggregates of pea vicilin amyloid protein, which is able to cross-seed RopB fibrillogenesis in vitro. Thus, we hypothesize that plant nodules contain a complex amyloid network consisting of plant and bacterial amyloids and probably modulating host-symbiont interactions.

Effect of Rhizobium Inoculation on Growth of Common Bean in Low-Fertility Tropical Soil Amended with Phosphorus and Lime

The cultivation of grain legumes (e.g., common bean) in sub-Saharan Africa contributes to the provision of food for a growing population and delivers environmental benefits such as inputs of nitrogen (N) to crops and soil via symbiotic nitrogen fixation (SNF). However, the success of SNF is constrained by several factors such as the poor efficiency of native rhizobial strains to fix N, the low availability of phosphorus (P) and the acidity of soils. Two trials have been conducted in low-fertility tropical soils at the smallholder farm scale in Madagascar to assess the effects of Rhizobium inoculation together with inputs of P and lime on the growth of the common bean. We showed that inoculation with native strains of Rhizobium had significant effects on bean root nodulation, which was increased by up to 15-fold on plant growth, which increased by 78% and on bean yield, which increased by 126%. Moreover, we observed positive and significant relationships between inoculation with Rhizobium and P fertilization on nodulation, plant growth and yield. However, the addition of dolomite lime did not show any effect in our study. The addition of P decreased the mycorrhization rate of roots. Additional research is still needed to improve our understanding of soil fertility conditions (mainly on nutrient availability, including micronutrients) allowing better efficiency of legume symbionts (rhizobium and mycorrhiza) in such low-fertility soils.

An epigenetic switch activates bacterial quorum sensing and horizontal transfer of an integrative and conjugative element.

Horizontal transfer of the integrative and conjugative element ICEMlSymR7A converts non-symbiotic Mesorhizobium spp. into nitrogen-fixing legume symbionts. Here, we discover subpopulations of Mesorhizobium japonicum R7A become epigenetically primed for quorum-sensing (QS) and QS-activated horizontal transfer. Isolated populations in this state termed R7A* maintained these phenotypes in laboratory culture but did not transfer the R7A* state to recipients of ICEMlSymR7A following conjugation. We previously demonstrated ICEMlSymR7A transfer and QS are repressed by the antiactivator QseM in R7A populations and that the adjacently-coded DNA-binding protein QseC represses qseM transcription. Here RNA-sequencing revealed qseM expression was repressed in R7A* cells and that RNA antisense to qseC was abundant in R7A but not R7A*. Deletion of the antisense-qseC promoter converted cells into an R7A*-like state. An adjacently coded QseC2 protein bound two operator sites and repressed antisense-qseC transcription. Plasmid overexpression of QseC2 stimulated the R7A* state, which persisted following curing of this plasmid. The epigenetic maintenance of the R7A* state required ICEMlSymR7A-encoded copies of both qseC and qseC2. Therefore, QseC and QseC2, together with their DNA-binding sites and overlapping promoters, form a stable epigenetic switch that establishes binary control over qseM transcription and primes a subpopulation of R7A cells for QS and horizontal transfer.

Harmony but Not Uniformity: Role of Strigolactone in Plants.

Strigolactones (SLs) represent an important new plant hormone class marked by their multifunctional roles in plants and rhizosphere interactions, which stimulate hyphal branching in arbuscular mycorrhizal fungi (AMF) and seed germination of root parasitic plants. SLs have been broadly implicated in regulating root growth, shoot architecture, leaf senescence, nodulation, and legume–symbionts interaction, as well as a response to various external stimuli, such as abiotic and biotic stresses. These functional properties of SLs enable the genetic engineering of crop plants to improve crop yield and productivity. In this review, the conservation and divergence of SL pathways and its biological processes in multiple plant species have been extensively discussed with a particular emphasis on its interactions with other different phytohormones. These interactions may shed further light on the regulatory networks underlying plant growth, development, and stress responses, ultimately providing certain strategies for promoting crop yield and productivity with the challenges of global climate and environmental changes.

Bradyrhizobium altum sp. nov., Bradyrhizobium oropedii sp. nov. and Bradyrhizobium acaciae sp. nov. from South Africa show locally restricted and pantropical nodA phylogeographic patterns

Africa is known for its rich legume diversity with a significant number of endemic species originating in South Africa. Many of these legumes associate with rhizobial symbionts of the genus Bradyrhizobium, of which most represent new species. Yet, none of the Bradyrhizobium species from South Africa have been described. In this study, phylogenetic analysis of 16S rRNA gene sequences of fourteen strains isolated in southern Africa from root nodules of diverse legumes (i.e., from the tribes Crotalarieae, Acacieae, Genisteae, Phaseoleae and Cassieae) revealed that they belong to the Bradyrhizobium elkanii supergroup. The taxonomic position and possible novelty of these strains were further interrogated using genealogical concordance of five housekeeping genes (atpD, dnaK, glnII, gyrB and rpoB). These phylogenies consistently recovered four monophyletic groups and one singleton within Bradyrhizobium. Of these groups, two were conspecific with Bradyrhizobium brasilense UFLA 03-321T and Bradyrhizobium ivorense CI-1BT, while the remaining three represented novel taxa. Their existence was further supported with genome data, as well as metabolic and physiological traits. Analysis of nodA gene sequences further showed that the evolution of these bacteria likely involved adapting to local legume hosts and environmental conditions through the acquisition, via horizontal gene transfer, of optimal symbiotic loci. We accordingly propose the following names Bradyrhizobium acaciae sp. nov. 10BBT (SARCC 730T = LMG 31409T), Bradyrhizobium oropedii sp. nov. Pear76T (SARCC 731T = LMG 31408T), and Bradyrhizobium altum sp. nov. Pear77T (SARCC 754T = LMG 31407T) to accommodate three novel species, all of which are symbionts of legumes in South Africa.

Structure of Lipopolysaccharide from Liberibacter crescens Is Low Molecular Weight and Offers Insight into Candidatus Liberibacter Biology.

Huanglongbing (HLB) disease, also known as citrus greening disease, was first reported in the US in 2005. Since then, the disease has decimated the citrus industry in Florida, resulting in billions of dollars in crop losses and the destruction of thousands of acres of citrus groves. The causative agent of citrus greening disease is the phloem limited pathogen Candidatus Liberibacter asiaticus. As it has not been cultured, very little is known about the structural biology of the organism. Liberibacter are part of the Rhizobiaceae family, which includes nitrogen-fixing symbionts of legumes as well as the Agrobacterium plant pathogens. To better understand the Liberibacter genus, a closely related culturable bacterium (Liberibacter crescens or Lcr) has attracted attention as a model organism for structural and functional genomics of Liberibacters. Given that the structure of lipopolysaccharides (LPS) from Gram-negative bacteria plays a crucial role in mediating host-pathogen interactions, we sought to characterize the LPS from Lcr. We found that the major lipid A component of the LPS consisted of a pentaacylated molecule with a β-6-GlcN disaccharide backbone lacking phosphate. The polysaccharide portion of the LPS was unusual compared to previously described members of the Rhizobiaceae family in that it contained ribofuranosyl residues. The LPS structure presented here allows us to extrapolate known LPS structure/function relationships to members of the Liberibacter genus which cannot yet be cultured. It also offers insights into the biology of the organism and how they manage to effectively attack citrus trees.

The history and distribution of nodulating Paraburkholderia, a potential inoculum for Fynbos forage species

AbstractLegumes in the Fynbos vegetation of the Western Cape of South Africa have emerged as candidates for domestication, particularly for their adaptation to acidic and infertile soils. However, South African rhizobia have been shown to be very diverse and unique, and a detailed understanding of them is essential to success in forage breeding programs that seek to exploit these “new” legumes. Symbionts of legumes in South Africa that belong to traditional rhizobial genera have been shown to have a unique origin for their symbiotic loci in comparison to members sampled from other regions of the world. Some of the legume tribes in the Fynbos have also been shown to associate predominantly with unique species in the Betaproteobacterial genus Paraburkholderia. The rhizobial members of this genus have two main centres of diversity, of which South Africa is one. In this centre, the legume hosts are principally from the Papilionoideae subfamily while hosts from the mimosoid clade (now in the Caesalpinioideae) are abundant in the South American centre. Not only do these rhizobia differ in terms of host, but their symbiotic loci also show separate origins. The dominance and uniqueness of the Paraburkholderia symbionts, in the context of indigenous South African legumes, makes understanding the history and factors that affect the distribution of this genus essential if successful adaptation and effective nodulation of these legumes in Agriculture are to be achieved globally.

Similarities and differences between 6S RNAs from Bradyrhizobium japonicum and Sinorhizobium meliloti.

6S RNA, a conserved and abundant small non-coding RNA found in most bacteria, regulates gene expression by inhibiting RNA polymerase (RNAP) holoenzyme. 6S RNAs from α-proteobacteria have been studied poorly so far. Here, we present a first in-depth analysis of 6S RNAs from two α-proteobacteria species, Bradyrhizobium japonicum and Sinorhizobium meliloti. Although both belong to the order Rhizobiales and are typical nitrogen-fixing symbionts of legumes, their 6S RNA expression profiles were found to differ: B. japonicum 6S RNA accumulated in the stationary phase, thus being reminiscent of Escherichia coli 6S RNA, whereas S. meliloti 6S RNA level peaked at the transition to the stationary phase, similarly to Rhodobacter sphaeroides 6S RNA. We demonstrated in vitro that both RNAs have hallmarks of 6S RNAs: they bind to the σ70-type RNAP holoenzyme and serve as templates for de novo transcription of so-called product RNAs (pRNAs) ranging in length from ∼13 to 24 nucleotides, with further evidence of the synthesis of even longer pRNAs. Likewise, stably bound pRNAs were found to rearrange the 6S RNA structure to induce its dissociation from RNAP. Compared with B. japonicum 6S RNA, considerable conformational heterogeneity was observed for S. meliloti 6S RNA and its complexes with pRNAs, even though the two 6S RNAs share ∼75% sequence identity. Overall, our findings suggest that the two rhizobial 6S RNAs have diverged with respect to their regulatory impact on gene expression throughout the bacterial life cycle.

Phylogenetic distribution and evolutionary dynamics of nod and T3SS genes in the genus Bradyrhizobium.

Bradyrhizobium are abundant soil bacteria and the major symbiont of legumes. The recent availability of Bradyrhizobium genome sequences provides a large source of information for analysis of symbiotic traits. In this study, we investigated the evolutionary dynamics of the nodulation genes (nod) and their relationship with the genes encoding type III secretion systems (T3SS) and their effectors among bradyrhizobia. Based on the comparative analysis of 146 Bradyrhizobium genome sequences, we identified six different types of T3SS gene clusters. The two predominant cluster types are designated RhcIa and RhcIb and both belong to the RhcI-T3SS family previously described in other rhizobia. They are found in 92/146 strains, most of them also containing nod genes. RhcIa and RhcIb gene clusters differ in the genes they carry: while the translocon-encoding gene nopX is systematically found in strains containing RhcIb, the nopE and nopH genes are specifically conserved in strains containing RhcIa, suggesting that these last two genes might functionally substitute nopX and play a role related to effector translocation. Phylogenetic analysis suggests that bradyrhizobia simultaneously gained nod and RhcI-T3SS gene clusters via horizontal transfer or subsequent vertical inheritance of a symbiotic island containing both. Sequence similarity searches for known Nop effector proteins in bradyrhizobial proteomes revealed the absence of a so-called core effectome, i.e. that no effector is conserved among all Bradyrhizobium strains. However, NopM and SUMO proteases were found to be the main effector families, being represented in the majority of the genus. This study indicates that bradyrhizobial T3SSs might play a more significant symbiotic role than previously thought and provides new candidates among T3SS structural proteins and effectors for future functional investigations.

High-Throughput Fingerprinting of Rhizobial Free Fatty Acids by Chemical Thin-Film Deposition and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.

Fatty acids (FAs) represent an important class of metabolites, impacting on membrane building blocks and signaling compounds in cellular regulatory networks. In nature, prokaryotes are characterized with the most impressing FA structural diversity and the highest relative content of free fatty acids (FFAs). In this context, nitrogen-fixing bacteria (order Rhizobiales), the symbionts of legumes, are particularly interesting. Indeed, the FA profiles influence the structure of rhizobial nodulation factors, required for successful infection of plant root. Although FA patterns can be assessed by gas chromatography—(GC-) and liquid chromatography—mass spectrometry (LC-MS), sample preparation for these methods is time-consuming and quantification suffers from compromised sensitivity, low stability of derivatives and artifacts. In contrast, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) represents an excellent platform for high-efficient metabolite fingerprinting, also applicable to FFAs. Therefore, here we propose a simple and straightforward protocol for high-throughput relative quantification of FFAs in rhizobia by combination of Langmuir technology and MALDI-TOF-MS featuring a high sensitivity, accuracy and precision of quantification. We describe a step-by-step procedure comprising rhizobia culturing, pre-cleaning, extraction, sample preparation, mass spectrometric analysis, data processing and post-processing. As a case study, a comparison of the FFA metabolomes of two rhizobia species—Rhizobium leguminosarum and Sinorhizobium meliloti, demonstrates the analytical potential of the protocol.

Experimental Evolution of Legume Symbionts: What Have We Learnt?

Rhizobia, the nitrogen-fixing symbionts of legumes, are polyphyletic bacteria distributed in many alpha- and beta-proteobacterial genera. They likely emerged and diversified through independent horizontal transfers of key symbiotic genes. To replay the evolution of a new rhizobium genus under laboratory conditions, the symbiotic plasmid of Cupriavidus taiwanensis was introduced in the plant pathogen Ralstonia solanacearum, and the generated proto-rhizobium was submitted to repeated inoculations to the C. taiwanensis host, Mimosa pudica L. This experiment validated a two-step evolutionary scenario of key symbiotic gene acquisition followed by genome remodeling under plant selection. Nodulation and nodule cell infection were obtained and optimized mainly via the rewiring of regulatory circuits of the recipient bacterium. Symbiotic adaptation was shown to be accelerated by the activity of a mutagenesis cassette conserved in most rhizobia. Investigating mutated genes led us to identify new components of R. solanacearum virulence and C. taiwanensis symbiosis. Nitrogen fixation was not acquired in our short experiment. However, we showed that post-infection sanctions allowed the increase in frequency of nitrogen-fixing variants among a non-fixing population in the M. pudica–C. taiwanensis system and likely allowed the spread of this trait in natura. Experimental evolution thus provided new insights into rhizobium biology and evolution.

Riboregulation in Nitrogen-Fixing Endosymbiotic Bacteria.

Small non-coding RNAs (sRNAs) are ubiquitous components of bacterial adaptive regulatory networks underlying stress responses and chronic intracellular infection of eukaryotic hosts. Thus, sRNA-mediated regulation of gene expression is expected to play a major role in the establishment of mutualistic root nodule endosymbiosis between nitrogen-fixing rhizobia and legume plants. However, knowledge about this level of genetic regulation in this group of plant-interacting bacteria is still rather scarce. Here, we review insights into the rhizobial non-coding transcriptome and sRNA-mediated post-transcriptional regulation of symbiotic relevant traits such as nutrient uptake, cell cycle, quorum sensing, or nodule development. We provide details about the transcriptional control and protein-assisted activity mechanisms of the functionally characterized sRNAs involved in these processes. Finally, we discuss the forthcoming research on riboregulation in legume symbionts.

Emergence of β-rhizobia as new root nodulating bacteria in legumes and current status of the legume–rhizobium host specificity dogma

Recent developments in the legume rhizobium symbiotic interaction particularly those related to the emergence of novel strains of bacteria that nodulate and fix nitrogen in legumes is gaining momentum. These novel strains of bacteria were mostly isolated from the root nodules of indigenous and invasive legumes belonging to the sub families Papilionoideae and Mimosoideae in South Africa, South America and South East China. These rhizobia are phylogenetically and taxonomically different from the traditional 'alpha rhizobia' and are termed 'β-rhizobia' as they belong to the β-sub class of Proteobacteria. There are also new reports of novel species of root nodulating bacteria from the α-Proteobacteria, not known for several decades since the discovery of rhizobia. However, in this review focus is given to the emerging β-rhizobia isolated from the indigenous Papilionoid legumes in the Cape Floristic regions in South Africa and the indigenous and invasive Mimosoid legumes in South America and South East Asia respectively. The nodulation of the indigenous South African Papilionoid legumes including that of Aspalathus linearis (rooibos) is discussed in a bit detail. Previous reports indicated that A. linearis is very specific in its rhizobium requirement and was reported to be nodulated by the slow growing Bradyrhizobium spp. This review however summarizes that the bacteria associated with the root nodules of A. linearis belong to members of both the alpha (α) Proteobacteria that include Mesorhizobium, Rhizobium and Bradyrhizobium spp. and the beta (β) Proteobacteria represented by the genus Burkholderia (now reclassified as Paraburkholderia). In addition, the occurrence of Paraburkholderia as the newly emerging root nodule symbionts of various other legumes has been discussed. In doing so, the review highlights that nodulation is no longer restricted to the traditional 'rhizobia' group following the emergence of the new beta rhizobia as potential nodulators of various indigenous legumes. It thus provides some insights on the status of the legume-rhizobium host specificity concept and the loss of this specificity in several symbiotic associations that puts the long held dogma of host specificity of the legume rhizobium symbiosis in a dilemma.

Modulation of Quorum Sensing as an Adaptation to Nodule Cell Infection during Experimental Evolution of Legume Symbionts.

Over millions of years, changes have occurred in regulatory circuitries in response to genome reorganization and/or persistent changes in environmental conditions. How bacteria optimize regulatory circuitries is crucial to understand bacterial adaptation. Here, we analyzed the experimental evolution of the plant pathogen Ralstonia solanacearum into legume symbionts after the transfer of a natural plasmid encoding the essential mutualistic genes. We showed that the Phc quorum sensing system required for the virulence of the ancestral bacterium was reconfigured to improve intracellular infection of root nodules induced by evolved Ralstonia A single mutation in either the PhcB autoinducer synthase or the PhcQ regulator of the sensory cascade tuned the kinetics of activation of the central regulator PhcA in response to cell density so that the minimal stimulatory concentration of autoinducers needed for a given response was increased. Yet, a change in the expression of a PhcA target gene was observed in infection threads progressing in root hairs, suggesting early programming for the late accommodation of bacteria in nodule cells. Moreover, this delayed switch to the quorum sensing mode decreased the pathogenicity of the ancestral strain, illustrating the functional plasticity of regulatory systems and showing how a small modulation in signal response can produce drastic changes in bacterial lifestyle.IMPORTANCE Rhizobia are soil bacteria from unrelated genera able to form a mutualistic relationship with legumes. Bacteria induce the formation of root nodules, invade nodule cells, and fix nitrogen to the benefit of the plant. Rhizobial lineages emerged from the horizontal transfer of essential symbiotic genes followed by genome remodeling to activate and/or optimize the acquired symbiotic potential. This evolutionary scenario was replayed in a laboratory evolution experiment in which the plant pathogen Ralstonia solanacearum successively evolved the capacities to nodulate Mimosa pudica and poorly invade, then massively invade, nodule cells. In some lines, the improvement of intracellular infection was achieved by mutations modulating a quorum sensing regulatory system of the ancestral strain. This modulation that affects the activity of a central regulator during the earliest stages of symbiosis has a huge impact on late stages of symbiosis. This work showed that regulatory rewiring is the main driver of this pathogeny-symbiosis transition.

Reduction of Organoarsenical Herbicides and Antimicrobial Growth Promoters by the Legume Symbiont Sinorhizobium meliloti.

Massive amounts of methyl [e.g., methylarsenate, MAs(V)] and aromatic arsenicals [e.g., roxarsone (4-hydroxy-3-nitrophenylarsonate, Rox(V)] have been utilized as herbicides for weed control and growth promotors for poultry and swine, respectively. The majority of these organoarsenicals degrade into more toxic inorganic species. Here, we demonstrate that the legume symbiont Sinorhizobium meliloti both reduces MAs(V) to MAs(III) and catalyzes sequential two-step reduction of nitro and arsenate groups in Rox(V), producing the highly toxic trivalent amino aromatic derivative 4-hydroxy-3-aminophenylarsenite (HAPA(III)). The existence of this process suggests that S. meliloti possesses the ability to transform pentavalent methyl and aromatic arsenicals into antibiotics to provide a competitive advantage over other microbes, which would be a critical process for the synthetic aromatic arsenicals to function as antimicrobial growth promoters. The activated trivalent aromatic arsenicals are degraded into less-toxic inorganic species by an MAs(III)-demethylating aerobe, suggesting that environmental aromatic arsenicals also undergo a multiple-step degradation pathway, in analogy with the previously reported demethylation pathway of the methylarsenate herbicide. We further show that an FAD-NADPH-dependent nitroreductase encoded by mdaB gene catalyzes nitroreduction of roxarsone both in vivo and in vitro. Our results demonstrate that environmental organoarsenicals trigger competition between members of microbial communities, resulting in gradual degradation of organoarsenicals and contamination by inorganic arsenic.

Bradyrhizobium niftali sp. nov., an effective nitrogen-fixing symbiont of partridge pea [Chamaecrista fasciculata (Michx.) Greene], a native caesalpinioid legume broadly distributed in the USA.

Information about the symbionts of legumes of the Caesalpinioideae subfamily is still limited, and we performed a polyphasic approach with three Bradyrhizobium strains-CNPSo 3448T, CNPSo 3394 and CNPSo 3442-isolated from Chamaecrista fasciculata, a native legume broadly distributed in the USA. In the phylogenetic analysis of both the 16S rRNA gene and the intergenic transcribed spacer, the CNPSo strains were clustered within the Bradyrhizobium japonicumsuperclade. Multilocus sequence analysis with six housekeeping genes-glnII, gyrB, recA, rpoB, atpD and dnaK-indicated that Bradyrhizobium diazoefficiens is the closest species, with 83 % of nucleotide identity. In the genome analyses of CNPSo 3448T, average nucleotide identity and digital DNA-DNA hybridization results confirmed higher similarity with B. diazoefficiens, with values estimated of 93.35 and 51.50 %, respectively, both below the threshold of the same species, confirming that the CNPSo strains represent a new lineage. BOX-PCR profiles indicated high intraspecific genetic diversity between the CNPSo strains. In the analyses of the symbiotic genes nodC and nifH the CNPSo strains were clustered with Bradyrhizobium arachidis, Bradyrhizobium forestalis, Bradyrhizobium cajani, Bradyrhizobium kavangense and Bradyrhizobium vignae, indicating a different phylogenetic history compared to the conserved core genes. Other physiological (C utilization, tolerance to antibiotics and abiotic stresses), chemical (fatty acid profile) and symbiotic (nodulation host range) properties were evaluated and are described. The data from our study support the description of the CNPSo strains as the novel species Bradyrhizobiumniftali sp. nov., with CNPSo 3448T (=USDA 10051T=U687T=CL 40T) designated as the type strain.