In vitro interactions between Bradyrhizobium spp. and Tuber magnatum mycelium.

Tuber magnatum is the most expensive truffle, but its large-scale cultivation is still a challenge compared to other valuable Tuber species. T. magnatum mycelium has never been grown profitably until now, which has led to difficulties to studying it in vitro. This study describes beneficial interactions between T. magnatum mycelium and never before described bradyrhizobia, which allows the in vitro growth of T. magnatum mycelium. Three T. magnatum strains were co-isolated on modified Woody Plant Medium (mWPM) with aerobic bacteria and characterised through microscopic observations. The difficulties of growing alone both partners, bacteria and T. magnatum mycelium, on mWPM demonstrated the reciprocal dependency. Three bacterial isolates for each T. magnatum strain were obtained and molecularly characterised by sequencing the 16S rRNA, glnII, recA and nifH genes. Phylogenetic analyses showed that all nine bacterial strains were distributed among five subclades included in a new monophyletic lineage belonging to the Bradyrhizobium genus within the Bradyrhizobium jicamae supergroup. The nifH genes were detected in all bacterial isolates, suggesting nitrogen-fixing capacities. This is the first report of consistent T. magnatum mycelium growth in vitro conditions. It has important implications for the development of new technologies in white truffle cultivation and for further studies on T. magnatum biology and genetics.

The distinct cell physiology of Bradyrhizobium at the population and cellular level.

The α-Proteobacteria belonging to Bradyrhizobium genus are microorganisms of extreme slow growth. Despite their extended use as inoculants in soybean production, their physiology remains poorly characterized. In this work, we produced quantitative data on four different isolates: B. diazoefficens USDA110, B. diazoefficiens USDA122, B. japonicum E109 and B. japonicum USDA6 which are representative of specific genomic profiles. Notably, we found conserved physiological traits conserved in all the studied isolates: (i) the lag and initial exponential growth phases display cell aggregation; (ii) the increase in specific nutrient concentration such as yeast extract and gluconate hinders growth; (iii) cell size does not correlate with culture age; and (iv) cell cycle presents polar growth. Meanwhile, fitness, cell size and in vitro growth widely vary across isolates correlating to ribosomal RNA operon number. In summary, this study provides novel empirical data that enriches the comprehension of the Bradyrhizobium (slow) growth dynamics and cell cycle.

Bradyrhizobium ontarionense sp. nov., a novel bacterial symbiont isolated from Aeschynomene indica (Indian jointvetch), harbours photosynthesis, nitrogen fixation and nitrous oxide (N2O) reductase genes.

A novel bacterial symbiont, strain A19T, was previously isolated from a root-nodule of Aeschynomene indica and assigned to a new lineage in the photosynthetic clade of the genus Bradyrhizobium. Here data are presented for the detailed genomic and taxonomic analyses of novel strain A19T. Emphasis is placed on the analysis of genes of practical or ecological significance (photosynthesis, nitrous oxide reductase and nitrogen fixation genes). Phylogenomic analysis of whole genome sequences as well as 50 single-copy core gene sequences placed A19T in a highly supported lineage distinct from described Bradyrhizobium species with B. oligotrophicum as the closest relative. The digital DNA-DNA hybridization and average nucleotide identity values for A19T in pair-wise comparisons with close relatives were far lower than the respective threshold values of 70% and ~ 96% for definition of species boundaries. The complete genome of A19T consists of a single 8.44 Mbp chromosome and contains a photosynthesis gene cluster, nitrogen-fixation genes and genes encoding a complete denitrifying enzyme system including nitrous oxide reductase implicated in the reduction of N2O, a potent greenhouse gas, to inert dinitrogen. Nodulation and type III secretion system genes, needed for nodulation by most rhizobia, were not detected. Data for multiple phenotypic tests complemented the sequence-based analyses. Strain A19T elicits nitrogen-fixing nodules on stems and roots of A. indica plants but not on soybeans or Macroptilium atropurpureum. Based on the data presented, a new species named Bradyrhizobium ontarionense sp. nov. is proposed with strain A19T (= LMG 32638T = HAMBI 3761T) as the type strain.

Hopanoid lipids promote soybean-Bradyrhizobium symbiosis.

The symbioses between leguminous plants and nitrogen-fixing bacteria known as rhizobia are well known for promoting plant growth and sustainably increasing soil nitrogen. Recent evidence indicates that hopanoids, a family of steroid-like lipids, promote Bradyrhizobium symbioses with tropical legumes. To characterize hopanoids in Bradyrhizobium symbiosis with soybean, we validated a recently published cumate-inducible hopanoid mutant of Bradyrhizobium diazoefficiens USDA110, Pcu-shc::∆shc. GC-MS analysis showed that this strain does not produce hopanoids without cumate induction, and under this condition, is impaired in growth in rich medium and under osmotic, temperature, and pH stress. In planta, Pcu-shc::∆shc is an inefficient soybean symbiont with significantly lower rates of nitrogen fixation and low survival within the host tissue. RNA-seq revealed that hopanoid loss reduces the expression of flagellar motility and chemotaxis-related genes, further confirmed by swim plate assays, and enhances the expression of genes related to nitrogen metabolism and protein secretion. These results suggest that hopanoids provide a significant fitness advantage to B. diazoefficiens in legume hosts and provide a foundation for future mechanistic studies of hopanoid function in protein secretion and motility.A major problem for global sustainability is feeding our exponentially growing human population while available arable land decreases. Harnessing the power of plant-beneficial microbes is a potential solution, including increasing our reliance on the symbioses of leguminous plants and nitrogen-fixing rhizobia. This study examines the role of hopanoid lipids in the symbiosis between Bradyrhizobium diazoefficiens USDA110, an important commercial inoculant strain, and its economically significant host soybean. Our research extends our knowledge of the functions of bacterial lipids in symbiosis to an agricultural context, which may one day help improve the practical applications of plant-beneficial microbes in agriculture.

Pleiotropic Effects of PhaR Regulator in Bradyrhizobium diazoefficiens Microaerobic Metabolism.

Bradyrhizobium diazoefficiens can live inside soybean root nodules and in free-living conditions. In both states, when oxygen levels decrease, cells adjust their protein pools by gene transcription modulation. PhaR is a transcription factor involved in polyhydroxyalkanoate (PHA) metabolism but also plays a role in the microaerobic network of this bacterium. To deeply uncover the function of PhaR, we applied a multipronged approach, including the expression profile of a phaR mutant at the transcriptional and protein levels under microaerobic conditions, and the identification of direct targets and of proteins associated with PHA granules. Our results confirmed a pleiotropic function of PhaR, affecting several phenotypes, in addition to PHA cycle control. These include growth deficiency, regulation of carbon and nitrogen allocation, and bacterial motility. Interestingly, PhaR may also modulate the microoxic-responsive regulatory network by activating the expression of fixK2 and repressing nifA, both encoding two transcription factors relevant for microaerobic regulation. At the molecular level, two PhaR-binding motifs were predicted and direct control mediated by PhaR determined by protein-interaction assays revealed seven new direct targets for PhaR. Finally, among the proteins associated with PHA granules, we found PhaR, phasins, and other proteins, confirming a dual function of PhaR in microoxia.

Soybean-mediated suppression of BjaI/BjaR1 quorum sensing in Bradyrhizobium diazoefficiens impacts symbiotic nitrogen fixation.

The acyl-homoserine lactones (AHLs)-mediated LuxI/LuxR quorum sensing (QS) system orchestrates diverse bacterial behaviors in response to changes in population density. The role of the BjaI/BjaR1 QS system in Bradyrhizobium diazoefficiens USDA 110, which shares homology with LuxI/LuxR, remains elusive during symbiotic interaction with soybean. Here this genetic system in wild-type (WT) bacteria residing inside nodules exhibited significantly reduced activity compared to free-living cells, potentially attributed to soybean-mediated suppression. The deletion mutant strain ΔbjaR1 showed significantly enhanced nodulation induction and nitrogen fixation ability. Nevertheless, its ultimate symbiotic outcome (plant dry weight) in soybeans was compromised. Furthermore, comparative analysis of the transcriptome, proteome, and promoter activity revealed that the inactivation of BjaR1 systematically activated and inhibited genomic modules associated with nodulation and nitrogen metabolism. The former appeared to be linked to a significant decrease in the expression of NodD2, a key cell-density-dependent repressor of nodulation genes, while the latter conferred bacterial growth and nitrogen fixation insensitivity to environmental nitrogen. In addition, BjaR1 exerted a positive influence on the transcription of multiple genes involved in a so-called central intermediate metabolism within the nodule. In conclusion, our findings highlight the crucial role of the BjaI/BjaR1 QS circuit in positively regulating bacterial nitrogen metabolism and emphasize the significance of the soybean-mediated suppression of this genetic system for promoting efficient symbiotic nitrogen fixation by B. diazoefficiens.IMPORTANCEThe present study demonstrates, for the first time, that the BjaI/BjaR1 QS system of Bradyrhizobium diazoefficiens has a significant impact on its nodulation and nitrogen fixation capability in soybean by positively regulating NodD2 expression and bacterial nitrogen metabolism. Moreover, it provides novel insights into the importance of suppressing the activity of this QS circuit by the soybean host plant in establishing an efficient mutual relationship between the two symbiotic partners. This research expands our understanding of legumes' role in modulating symbiotic nitrogen fixation through rhizobial QS-mediated metabolic functioning, thereby deepening our comprehension of symbiotic coevolution theory. In addition, these findings may hold great promise for developing quorum quenching technology in agriculture.

The divalent metal ion exporter IhpABC is required to maintain iron homeostasis under low to moderate environmental iron conditions in the bacterium Bradyrhizobium japonicum.

Bacterial iron export mitigates high iron stress, but a role for it under lower iron conditions has not been established. MbfA is the high iron stress exporter in Bradyrhizobium japonicum. Here, we identify the ihpABC genes in a selection for secondary site mutations that suppress the poor growth phenotype of feoAB mutants defective in iron acquisition. IhpABC belongs to the RND tripartite efflux pump family. High iron conditions that derepress the mbfA gene partially rescued the growth of an ihpC mutant but reverted the feoB ihpC mutant to the feoB growth phenotype. The ihpA mutant grown under low iron conditions accumulated higher levels of iron compared to the wild type, and it displayed aberrant iron-responsive gene expression. The mbfA mutant was more sensitive than the wild type to H2 O2 , but the ihpA mutant was not sensitive. The ihpA mutant accumulated more Zn, Co and Cd than was found in the wild type, and growth of the mutant was more sensitive to inhibition by ZnCl2 , CoCl2 and CdCl2 . The findings suggest that IhpABC is a divalent metal ion exporter that helps maintain iron homeostasis under low to moderate environmental iron levels. Thus, iron export is not limited to managing high iron stress.

Convergent gene pair dSH3 and irr regulate Pi and Fe homeostasis in Bradyrhizobium diazoefficiens USDA110 and symbiotic nitrogen fixation efficiency.

The nitrogen-fixing bacteroids inhabit inside legume root nodules must manage finely the utilization of P and Fe, the two most critical elements, due to their antagonistic interactions. While the balance mechanism for them remains unclear. A double SH3 domain-containing protein (dSH3) in the Bradyrhizobium diazoefficiens USDA110 was found to inhibit the alkaline phosphatase activity, thereby reducing P supply from organophosphates. The dSH3 gene is adjacent to the irr gene, which encodes the iron response repressor and regulates Fe homeostasis under Fe-limited conditions. Their transcription directions converge to a common intergenic sequence (IGS) region, forming a convergent transcription. Extending the IGS region through Tn5 transposon or pVO155 plasmid insertion significantly down-regulated expression of this gene pair, leading to a remarkable accumulation of P and an inability to grow under Fe-limited conditions. Inoculation of soybean with either of the insertion mutants resulted in N2-fixing failure. However, the IGS-deleted mutant showed no visible changes in N2-fixing efficiency on soybean compared to that inoculated with wild type. These findings reveal a novel regulative strategy in the IGS region and its flanking convergent gene pair for antagonistic utilization of P and Fe in rhizobia and coordination of N2-fixing efficiency.

Bradyrhizobium ottawaense efficiently reduces nitrous oxide through high nosZ gene expression.

N2O is an important greenhouse gas influencing global warming, and agricultural land is the predominant (anthropogenic) source of N2O emissions. Here, we report the high N2O-reducing activity of Bradyrhizobium ottawaense, suggesting the potential for efficiently mitigating N2O emission from agricultural lands. Among the 15 B. ottawaense isolates examined, the N2O-reducing activities of most (13) strains were approximately five-fold higher than that of Bradyrhizobium diazoefficiens USDA110T under anaerobic conditions. This robust N2O-reducing activity of B. ottawaense was confirmed by N2O reductase (NosZ) protein levels and by mitigation of N2O emitted by nodule decomposition in laboratory system. While the NosZ of B. ottawaense and B. diazoefficiens showed high homology, nosZ gene expression in B. ottawaense was over 150-fold higher than that in B. diazoefficiens USDA110T, suggesting the high N2O-reducing activity of B. ottawaense is achieved by high nos expression. Furthermore, we examined the nos operon transcription start sites and found that, unlike B. diazoefficiens, B. ottawaense has two transcription start sites under N2O-respiring conditions, which may contribute to the high nosZ expression. Our study indicates the potential of B. ottawaense for effective N2O reduction and unique regulation of nos gene expression towards the high performance of N2O mitigation in the soil.

Exploring potential soybean bradyrhizobia from high trehalose-accumulating soybean genotypes for improved symbiotic effectiveness in soybean / Exploración de bradirizobios potenciales de la soja a partir de genotipos de soja con alto contenido de trehalosa para mejorar la eficacia simbiótica en la soja

Drought is the most important factor limiting the activity of rhizobia during N-fixation and plant growth. In the present study, we isolated Bradyrhizobium spp. from root nodules of higher trehalose-accumulating soybean genotypes and examined for moisture stress tolerance on a gradient of polyethylene glycol (PEG 6000) amended in yeast extract mannitol (YEM) broth. In addition, the bradyrhizobial strains were also evaluated for symbiotic effectiveness on soybean. Based on 16S rDNA gene sequences, four bradyrhizobial species were recovered from high trehalose-accumulating genotypes, i.e., two Bradyrhizobium liaoningense strains (accession number KX230053, KX230054) from EC 538828 and PK-472, respectively, one Bradyrhizobium daqingense (accession number KX230052) from PK-472, and one Bradyrhizobium kavangense (accession number MN197775) from Valder genotype having low trehalose. These strains, along with two native strains, viz., Bradyrhizobium japonicum (JF792425), Bradyrhizobium liaoningense (JF792426), and one commercial rhizobium, were studied for nodulation, leghaemoglobin, and N-fixation abilities on soybean under sterilized sand microcosm conditions in a completely randomized design. Among all the strains, D-4A (B. daqingense) followed by D-4B (B. liaoningense) was found to have significantly higher nodulation traits and acetylene reduction assay (ARA) activity when compared to other strains and commercial rhizobia. The bradyrhizobia isolates showed plant growth promotion traits such as indole acetic acid (IAA), exopolysaccharide (EPS), and siderophore production, phosphate-solubilizing potential, and proline accumulation. The novel species B. daqingense was reported for the first time from Indian soil and observed to be a potential candidate strain and should be evaluated for conferring drought tolerance in soybean under simulated stress conditions.(AU)

Legume-nodulating rhizobia are widespread in soils and plants across the island of O'ahu, Hawai'i.

Legumes and their interaction with rhizobia represent one of the most well-characterized symbioses that are widespread across both natural and agricultural environments. However, larger distribution patterns and host associations on isolated Pacific islands with many native and introduced hosts have not been well-documented. Here, we used molecular and culturing techniques to characterize rhizobia from soils and 24 native and introduced legume species on the island of O'ahu, Hawai'i. We chose two of these isolates to inoculate an endemic legume tree, Erythina sandwicensis to measure nodulation potentials and host benefits. We found that all rhizobia genera can be found in the soil, where only Cupriavidus was found at all sites, although at lower abundance relative to other more common genera such as Rhizobium (and close relatives), Bradyzhizobium, and Devosia. Bradyrhizobium was the most common nodulator of legumes, where the strain Bradyrhizobium sp. strain JA1 is a generalist capable of forming nodules on nine different host species, including two native species. In greenhouse nursery inoculations, the two different Bradyrhizobium strains successfully nodulate the endemic E. sandwicensis; both strains equally and significantly increased seedling biomass in nursery inoculations. Overall, this work provides a molecular-based framework in which to study potential native and introduced rhizobia on one of the most isolated archipelagos on the planet.

Independent Component Analysis Reveals the Transcriptional Regulatory Modules in Bradyrhizobium diazoefficiens USDA110.

The dynamic adaptation of bacteria to environmental changes is achieved through the coordinated expression of many genes, which constitutes a transcriptional regulatory network (TRN). Bradyrhizobium diazoefficiens USDA110 is an important model strain for the study of symbiotic nitrogen fixation (SNF), and its SNF ability largely depends on the TRN. In this study, independent component analysis was applied to 226 high-quality gene expression profiles of B. diazoefficiens USDA110 microarray datasets, from which 64 iModulons were identified. Using these iModulons and their condition-specific activity levels, we (1) provided new insights into the connection between the FixLJ-FixK2-FixK1 regulatory cascade and quorum sensing, (2) discovered the independence of the FixLJ-FixK2-FixK1 and NifA/RpoN regulatory cascades in response to oxygen, (3) identified the FixLJ-FixK2 cascade as a mediator connecting the FixK2-2 iModulon and the Phenylalanine iModulon, (4) described the differential activation of iModulons in B. diazoefficiens USDA110 under different environmental conditions, and (5) proposed a notion of active-TRN based on the changes in iModulon activity to better illustrate the relationship between gene regulation and environmental condition. In sum, this research offered an iModulon-based TRN for B. diazoefficiens USDA110, which formed a foundation for comprehensively understanding the intricate transcriptional regulation during SNF.

The copper-responsive regulator CsoR is indirectly involved in Bradyrhizobium diazoefficiens denitrification.

The soybean endosymbiont Bradyrhizobium diazoefficiens harbours the complete denitrification pathway that is catalysed by a periplasmic nitrate reductase (Nap), a copper (Cu)-containing nitrite reductase (NirK), a c-type nitric oxide reductase (cNor), and a nitrous oxide reductase (Nos), encoded by the napEDABC, nirK, norCBQD, and nosRZDFYLX genes, respectively. Induction of denitrification genes requires low oxygen and nitric oxide, both signals integrated into a complex regulatory network comprised by two interconnected cascades, FixLJ-FixK2-NnrR and RegSR-NifA. Copper is a cofactor of NirK and Nos, but it has also a role in denitrification gene expression and protein synthesis. In fact, Cu limitation triggers a substantial down-regulation of nirK, norCBQD, and nosRZDFYLX gene expression under denitrifying conditions. Bradyrhizobium diazoefficiens genome possesses a gene predicted to encode a Cu-responsive repressor of the CsoR family, which is located adjacent to copA, a gene encoding a putative Cu+-ATPase transporter. To investigate the role of CsoR in the control of denitrification gene expression in response to Cu, a csoR deletion mutant was constructed in this work. Mutation of csoR did not affect the capacity of B. diazoefficiens to grow under denitrifying conditions. However, by using qRT-PCR analyses, we showed that nirK and norCBQD expression was much lower in the csoR mutant compared to wild-type levels under Cu-limiting denitrifying conditions. On the contrary, copA expression was significantly increased in the csoR mutant. The results obtained suggest that CsoR acts as a repressor of copA. Under Cu limitation, CsoR has also an indirect role in the expression of nirK and norCBQD genes.

Competitive interference among rhizobia reduces benefits to hosts.

The capacity of beneficial microbes to compete for host infection-and the ability of hosts to discriminate among them-introduces evolutionary conflict that is predicted to destabilize mutualism. We investigated fitness outcomes in associations between legumes and their symbiotic rhizobia to characterize fitness impacts of microbial competition. Diverse Bradyrhizobium strains varying in their capacity to fix nitrogen symbiotically with a common host plant, Acmispon strigosus, were tested in full-factorial coinoculation experiments involving 28 pairwise strain combinations. We analyzed the effects of interstrain competition and host discrimination on symbiotic-interaction outcomes by relativizing fitness proxies to clonally infected and uninfected controls. More than one thousand root nodules of coinoculated plants were genotyped to quantify strain occupancy, and the Bradyrhizobium strain genome sequences were analyzed to uncover the genetic bases of interstrain competition outcomes. Strikingly, interstrain competition favored a fast-growing, minimally beneficial rhizobia strain. Host benefits were significantly diminished in coinoculation treatments relative to expectations from clonally inoculated controls, consistent with competitive interference among rhizobia that reduced both nodulation and plant growth. Competition traits appear polygenic, linked with inter-strain allelopathic interactions in the rhizosphere. This study confirms that competition among strains can destabilize mutualism by favoring microbes that are superior in colonizing host tissues but provide minimal benefits to host plants. Moreover, our findings help resolve the paradox that despite efficient host control post infection, legumes nonetheless encounter rhizobia that vary in their nitrogen fixation.

RNA-Seq analysis of mung bean (Vigna radiata L.) roots shows differential gene expression and predicts regulatory pathways responding to taxonomically different rhizobia.

Symbiotic interaction among legume and rhizobia is a complex phenomenon which results in the formation of nitrogen-fixing nodules. Mung bean is promiscuous host however expression profile of this important legume plant in response to rhizobial infection was particularly lacking and urgently needed. We have demonstrated the pattern of gene expression of mung bean roots inoculated with two symbionts Bradyrhizobium yuanmingense Vr50 and Sinorhizobium (Ensifer) aridi Vr33 and non-inoculated control (CK). The RNA-Seq data analyzed at two growth stages i.e., 1-3 h and 10-16 days post inoculation revealed significantly higher number of differentially expressed genes (DEGs) at nodulation stage. The DEGs encoding receptor kinases identified at early stage might be involved in perception of Nod factors produced by different rhizobia. At nodulation stage important genes involved in plant hormone signal transduction, nitrogen and sulfur metabolism were identified. KEGG pathway enrichment analysis showed that metabolic pathways were most prominent in both groups (Group 1: Vr33 vs CK; Group 2: Vr50 vs CK), followed by biosynthesis of secondary metabolites, plant hormone signal transduction and biosynthesis of amino acids. Furthermore, DEGs involved in cell communication and plant hormone signal transduction were found to be different among two symbiotic systems while DEGs involved in carbon, nitrogen and sulfur metabolism were similar but their expression varied in response to two rhizobial strains. This study provides the first insight into the mechanisms underlying interactions of mung bean host with two taxonomically different symbionts (Bradyrhizobium and Sinorhizobium) and the candidate genes for better understanding the mechanisms of symbiotic host-specificity.

GFP labeling of a Bradyrhizobium strain and an attempt to track the crack entry process during symbiosis with peanuts.

Compared to the well-studied model legumes, where symbiosis is established via root hair entry, the peanut is infected by Bradyrhizobium through the crack entry, which is less common and not fully understood. Crack entry is, however, considered a primitive symbiotic infection pathway, which could be potentially utilized for engineering non-legume species with nitrogen fixation ability. We utilized a fluorescence-labeled Bradyrhizobium strain to help in understanding the crack entry process at the cellular level. A modified plasmid pRJPaph-bjGFP, harboring the codon-optimized GFP gene and tetracycline resistance gene, was created and conjugated into Bradyrhizobium strain Lb8, an isolate from peanut nodules, through tri-parental mating. Microscopic observation and peanut inoculation assays confirmed the successful GFP tagging of Lb8, which is capable of generating root nodules. A marking system for peanut root potential infection sites and an optimized sample preparation protocol for cryostat sectioning was developed. The feasibility of using the GFP-tagged Lb8 for observing crack entry was examined. GFP signal was detected at the nodule primordial stage and the following nodule developmental stages with robust GFP signals observed in infected cells in the mature nodules. Spherical bacteroids in the root tissue were visualized at the nodules' inner cortex under higher magnification, reflecting the trace along the rhizobial infection path. The GFP labeled Lb8 can serve as an essential tool for plant-microbe studies between the cultivated peanut and Bradyrhizobium, which could facilitate further study of the crack entry process during the legume-rhizobia symbiosis.

Widespread Bradyrhizobium distribution of diverse Type III effectors that trigger legume nodulation in the absence of Nod factor.

The establishment of the rhizobium-legume symbiosis is generally based on plant perception of Nod factors (NFs) synthesized by the bacteria. However, some Bradyrhizobium strains can nodulate certain legume species, such as Aeschynomene spp. or Glycine max, independently of NFs, and via two different processes that are distinguished by the necessity or not of a type III secretion system (T3SS). ErnA is the first known type III effector (T3E) triggering nodulation in Aeschynomene indica. In this study, a collection of 196 sequenced Bradyrhizobium strains was tested on A. indica. Only strains belonging to the photosynthetic supergroup can develop a NF-T3SS-independent symbiosis, while the ability to use a T3SS-dependent process is found in multiple supergroups. Of these, 14 strains lacking ernA were tested by mutagenesis to identify new T3Es triggering nodulation. We discovered a novel T3E, Sup3, a putative SUMO-protease without similarity to ErnA. Its mutation in Bradyrhizobium strains NAS96.2 and WSM1744 abolishes nodulation and its introduction in an ernA mutant of strain ORS3257 restores nodulation. Moreover, ectopic expression of sup3 in A. indica roots led to the formation of spontaneous nodules. We also report three other new T3Es, Ubi1, Ubi2 and Ubi3, which each contribute to the nodulation capacity of strain LMTR13. These T3Es have no homology to known proteins but share with ErnA three motifs necessary for ErnA activity. Together, our results highlight an unsuspected distribution and diversity of T3Es within the Bradyrhizobium genus that may contribute to their symbiotic efficiency by participating in triggering legume nodulation.

The Type IV Secretion System (T4SS) Mediates Symbiosis between Bradyrhizobium sp. SUTN9-2 and Legumes.

There has been little study of the type IV secretion system (T4SS) of bradyrhizobia and its role in legume symbiosis. Therefore, broad host range Bradyrhizobium sp. SUTN9-2 was selected for study. The chromosome of Bradyrhizobium sp. SUTN9-2 contains two copies of the T4SS gene, homologous with the tra/trb operons. A phylogenetic tree of the T4SS gene traG was constructed, which exemplified its horizontal transfer among Bradyrhizobium and Mesorhizobium genera. They also showed similar gene arrangements for the tra/trb operons. However, the virD2 gene was not observed in Mesorhizobium, except M. oppotunistum WSM2075. Interestingly, the orientation of copG, traG, and virD2 cluster was unique to the Bradyrhizobium genus. The phylogenetic tree of copG, traG, and virD2 demonstrated that copies 1 and 2 of these genes were grouped in different clades. In addition, the derived mutant and complementation strains of T4SS were investigated in representative legumes Genistoids, Dalbergioids, and Millettiods. When T4SS copy 1 (T4SS1) was deleted, the nodule number and nitrogenase activity decreased. This supports a positive effect of T4SS1 on symbiosis. In addition, delayed nodulation was observed 7 dpi, which was restored by the complementation of T4SS1. Therefore, T4SS plays an important role in the symbiotic interaction between Bradyrhizobium sp. SUTN9-2 and its leguminous hosts. IMPORTANCE SUTN9-2 is a broad host range strain capable of symbiosis with several legumes. Two copies of T4SS clusters belonging to the tra/trb operon are observed on chromosomes with different gene arrangements. We use phylogenetic tree and gene annotation analysis to predict the evolution of the tra/trb operon of rhizobia. Our finding suggests that the gene encoding the T4SS gene among Bradyrhizobium and Mesorhizobium may have coevolution. In addition, Bradyrhizobium has a uniquely arranged copG, traG, and virD2 gene cluster. The results of T4SS1 gene deletion and complementation revealed its positive effect on nodulation. Therefore, T4SS seems to be another determinant for symbiosis. This is the first report on the role of T4SS in Bradyrhizobium symbiosis.

Novel symbiovars ingae, lysilomae and lysilomaefficiens in bradyrhizobia from tree-legume nodules.

Inga vera and Lysiloma tree legumes form nodules with Bradyrhizobium spp. from the japonicum group that represent novel genomospecies, for which we describe here using genome data, symbiovars lysilomae, lysilomaefficiens and ingae. Genes encoding Type three secretion system (TTSS) that could affect host specificity were found in ingae but not in lysilomae nor in lysilomaefficiens symbiovars and uptake hydrogenase hup genes (that affect nitrogen fixation) were observed in bradyrhizobia from the symbiovars ingae and lysilomaefficiens. nolA gene was found in the symbiovar lysilomaefficiens but not in strains from lysilomae. We discuss that multiple genes may dictate symbiosis specificity. Besides, toxin-antitoxin genes were found in the symbiosis islands in bradyrhizobia from symbiovars ingae and lysilomaefficiens. A limit (95%) to define symbiovars with nifH gene sequences was proposed here.