Does Rhizobial Inoculation Change the Microbial Community in Field Soils? A| |Comparison with Agricultural Land-use Changes.

Although microbial inoculation may be effective for sustainable crop production, detrimental aspects have been argued because of the potential of inoculated microorganisms to behave as invaders and negatively affect the microbial ecosystem. We herein compared the impact of rhizobial inoculation on the soil bacterial community with that of agricultural land-use changes using a 16S rRNA amplicon ana-lysis. Soybean plants were cultivated with and without five types of bradyrhizobial inoculants (Bradyrhizobium diazoefficiens or Bradyrhizobium ottawaense) in experimental fields of Andosol, and the high nodule occupancy (35-72%) of bradyrhizobial inoculants was confirmed by nosZ PCR. However, bradyrhizobial inoculants did not significantly affect Shannon's diversity index (α-diversity) or shifts (ß-diversity) in the bacterial community in the soils. Moreover, the soil bacterial community was significantly affected by land-use types (conventional cropping, organic cropping, and original forest), where ß-diversity correlated with soil chemical properties (pH, carbon, and nitrogen contents). Therefore, the effects of bradyrhizobial inoculation on bacterial communities in bulk soil were minor, regardless of high nodule occupancy. We also observed a correlation between the relative abundance of bacterial classes (Alphaproteobacteria, Gammaproteobacteria, and Gemmatimonadetes) and land-use types or soil chemical properties. The impact of microbial inoculation on soil microbial ecosystems has been exami-ned to a limited extent, such as rhizosphere communities and viability. In the present study, we found that bacterial community shifts in soil were more strongly affected by land usage than by rhizobial inoculation. Therefore, the results obtained herein highlight the importance of assessing microbial inoculants in consideration of the entire land management system.

Diversity of soybean rhizobia in Northeast China and their application.

Biological nitrogen fixation is the main source of nitrogen in ecosystems. The diversity of soil rhizobia and their effects on soybeans need further research. In this study, we collected soybean rhizosphere samples from eight sites in the black soil soybean planting area in Northeast China. A total of 94 strains of bacteria were isolated and identified using the 16S rRNA and symbiotic genes (nodC, nifH) analysis, of which 70 strains were identified as rhizobia belonging to the genus Bradyrhizobium. To further validate the application effects of rhizobia, we selec-ted seven representative indigenous rhizobia based on the results of phylogenetic analysis, and conducted laboratory experiments to determine their nodulation and the impacts on soybeans. The results showed that, compared to the control without rhizobial inoculation, all the seven indigenous rhizobia exhibited good promoting and nodulation abilities. Among them, strains H7-L22 and H34-L6 performed the best, with the former significantly increasing plant height by 25.7% and the latter increasing root nodule dry weight by 20.9% to 67.1% compared to other indi-genous rhizobia treatments. We tested these two efficient rhizobia strains as soybean rhizobial inoculants in field experiments. The promoting effect of mixed rhizobial inoculants was significantly better than single ones. Compared to the control without inoculation, soybean yield increased by 8.4% with the strain H7-L22 treatment and by 17.9% with the mixed inoculant treatment. Additionally, there was a significant increase in the number of four-seed pods in soybeans. In conclusion, the application of rhizobial inoculants can significantly increase soybean yield, thereby reducing dependence on nitrogen fertilizer during soybean production, improving soil health, and promoting green development in agriculture in the black soil region of Northeast China.

Propane Monooxygenases in Soil Associated Metagenomes Align Most Closely to those in the Genera Kribbella, Amycolatopsis, Bradyrhizobium, Paraburkholderia and Burkholderia.

Propanotrophs are a focus of interest because of their ability to degrade numerous environmental contaminants. To explore the phylogeny of microorganisms containing the propane monooxygenase gene cluster (prmABCD), NCBI bacterial genomes and publicly available soil associated metagenomes (from soils, rhizospheres, tree roots) were both examined. Nucleic acid sequences were collected only if all four subunits were located together, were of the expected length and were annotated as propane monooxygenase subunits. In the bacterial genomes, this resulted in data collection only from the phyla Actinomycetota and Pseudomonadota. For the soil associated metagenomes, reads from four studies were subject to quality control, assembly and annotation. Following this, the propane monooxygenase subunit nucleic acid sequences were collected and aligned to the collected bacterial sequences. In total, forty-two propane monooxygenase gene clusters were annotated from the soil associated metagenomes. The majority aligned closely to those from the Actinomycetota, followed by the Alphaproteobacteria, then the Betaproteobacteria. Actinomycetota aligning propane monooxygenase sequences were obtained from all four datasets and most closely aligned to the genera Kribbella and Amycolatopsis. Alphaproteobacteria aligning sequences largely originated from metagenomes associated with miscanthus and switchgrass rhizospheres and primarily aligned with the genera Bradyrhizobium, Acidiphilium and unclassified Rhizobiales. Betaproteobacteria aligning sequences were obtained from only the Red Oak root metagenomes and primarily aligned with the genera Paraburkholderia, Burkholderia and Caballeronia. Interestingly, sequences from the environmental metagenomes were not closely aligned to those from well-studied propanotrophs, such as Mycobacterium and Rhodococcus. Overall, the study highlights the previously unreported diversity of putative propanotrophs in environmental samples. The common occurrence of propane monooxygenase gene clusters has implications for their potential use for contaminant biodegradation.

Developing a genomic-based strategy to confirm microbial identity in bio-inputs containing multiple strains: an easy, fast, and low-cost multiplex PCR applied to inoculants carrying soybean Bradyrhizobium.

Brazil stands out in research, industrial development, and farmers' use of microbial inoculants, with an emphasis on getting benefits from the biological nitrogen fixation process with the soybean crop. Nowadays, about 140 million doses of inoculants are commercialized annually for the soybean in the country, and strain identification is achieved by rep-PCR, an effective but time-consuming method. Aiming to develop an easy, low-cost, and low-time-consuming method, we used a complete genome-based approach based on the unequivocal identification of unique genes present in the genomes of each of the four Bradyrhizobium strains used in commercial inoculants: Bradyrhizobium elkanii strains SEMIA 587 and SEMIA 5019, Bradyrhizobium japonicum SEMIA 5079, and Bradyrhizobium diazoefficiens SEMIA 5080. The unique pairs of primers able to amplify genomic regions of different sizes allowed the identification of the four strains in a simple multiplex polymerase chain reaction (PCR). Validation was confirmed by using single colonies, multiple cultures, and commercial inoculants. The number of labor hours of a technician was 3.08 times higher, and the final cost was 3.25 times higher in the rep-PCR than in the multiplex PCR. Most importantly, the results for multiplex PCR were obtained on the same day, in contrast with 15 days in the traditional methodology. The genomic approach developed can be easily applied to a variety of microbial inoculants worldwide, in addition to studies of ecology and evaluation of the competitiveness of the strains.

Comparative genomic analysis of Bradyrhizobium strains with natural variability in the efficiency of nitrogen fixation, competitiveness, and adaptation to stressful edaphoclimatic conditions.

Bradyrhizobium is known for fixing atmospheric nitrogen in symbiosis with agronomically important crops. This study focused on two groups of strains, each containing eight natural variants of the parental strains, Bradyrhizobium japonicum SEMIA 586 (=CNPSo 17) or Bradyrhizobium diazoefficiens SEMIA 566 (=CNPSo 10). CNPSo 17 and CNPSo 10 were used as commercial inoculants for soybean crops in Brazil at the beginning of the crop expansion in the southern region in the 1960s-1970s. Variants derived from these parental strains were obtained in the late 1980s through a strain selection program aimed at identifying elite strains adapted to a new cropping frontier in the central-western Cerrado region, with a higher capacity of biological nitrogen fixation (BNF) and competitiveness. Here, we aimed to detect genetic variations possibly related to BNF, competitiveness for nodule occupancy, and adaptation to the stressful conditions of the Brazilian Cerrado soils. High-quality genome assemblies were produced for all strains. The core genome phylogeny revealed that strains of each group are closely related, as confirmed by high average nucleotide identity values. However, variants accumulated divergences resulting from horizontal gene transfer, genomic rearrangements, and nucleotide polymorphisms. The B. japonicum group presented a larger pangenome and a higher number of nucleotide polymorphisms than the B. diazoefficiens group, possibly due to its longer adaptation time to the Cerrado soil. Interestingly, five strains of the B. japonicum group carry two plasmids. The genetic variability found in both groups is discussed considering the observed differences in their BNF capacity, competitiveness for nodule occupancy, and environmental adaptation.IMPORTANCEToday, Brazil is a global leader in the study and use of biological nitrogen fixation with soybean crops. As Brazilian soils are naturally void of soybean-compatible bradyrhizobia, strain selection programs were established, starting with foreign isolates. Selection searched for adaptation to the local edaphoclimatic conditions, higher efficiency of nitrogen fixation, and strong competitiveness for nodule occupancy. We analyzed the genomes of two parental strains of Bradyrhizobium japonicum and Bradyrhizobium diazoefficiens and eight variant strains derived from each parental strain. We detected two plasmids in five strains and several genetic differences that might be related to adaptation to the stressful conditions of the soils of the Brazilian Cerrado biome. We also detected genetic variations in specific regions that may impact symbiotic nitrogen fixation. Our analysis contributes to new insights into the evolution of Bradyrhizobium, and some of the identified differences may be applied as genetic markers to assist strain selection programs.

Single-cell transcriptome atlases of soybean root and mature nodule reveal new regulatory programs that control the nodulation process.

The soybean root system is complex. In addition to being composed of various cell types, the soybean root system includes the primary root, the lateral roots, and the nodule, an organ in which mutualistic symbiosis with N-fixing rhizobia occurs. A mature soybean root nodule is characterized by a central infection zone where atmospheric nitrogen is fixed and assimilated by the symbiont, resulting from the close cooperation between the plant cell and the bacteria. To date, the transcriptome of individual cells isolated from developing soybean nodules has been established, but the transcriptomic signatures of cells from the mature soybean nodule have not yet been characterized. Using single-nucleus RNA-seq and Molecular Cartography technologies, we precisely characterized the transcriptomic signature of soybean root and mature nodule cell types and revealed the co-existence of different sub-populations of B. diazoefficiens-infected cells in the mature soybean nodule, including those actively involved in nitrogen fixation and those engaged in senescence. Mining of the single-cell-resolution nodule transcriptome atlas and the associated gene co-expression network confirmed the role of known nodulation-related genes and identified new genes that control the nodulation process. For instance, we functionally characterized the role of GmFWL3, a plasma membrane microdomain-associated protein that controls rhizobial infection. Our study reveals the unique cellular complexity of the mature soybean nodule and helps redefine the concept of cell types when considering the infection zone of the soybean nodule.

Broad-spectrum ubiquitin/ubiquitin-like deconjugation activity of the rhizobial effector NopD from Bradyrhizobium (sp. XS1150).

The post-translational modification of proteins by ubiquitin-like modifiers (UbLs), such as SUMO, ubiquitin, and Nedd8, regulates a vast array of cellular processes. Dedicated UbL deconjugating proteases families reverse these modifications. During bacterial infection, effector proteins, including deconjugating proteases, are released to disrupt host cell defenses and promote bacterial survival. NopD, an effector protein from rhizobia involved in legume nodule symbiosis, exhibits deSUMOylation activity and, unexpectedly, also deubiquitination and deNeddylation activities. Here, we present two crystal structures of Bradyrhizobium (sp. XS1150) NopD complexed with either Arabidopsis SUMO2 or ubiquitin at 1.50 Å and 1.94 Å resolution, respectively. Despite their low sequence similarity, SUMO and ubiquitin bind to a similar NopD interface, employing a unique loop insertion in the NopD sequence. In vitro binding and activity assays reveal specific residues that distinguish between deubiquitination and deSUMOylation. These unique multifaceted deconjugating activities against SUMO, ubiquitin, and Nedd8 exemplify an optimized bacterial protease that disrupts distinct UbL post-translational modifications during host cell infection.

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.

Bradyrhizobium japonicum HmuP is an RNA-binding protein that positively controls hmuR operon expression by suppression of a negative regulatory RNA element in the 5' untranslated region.

The hmuR operon encodes proteins for the uptake and utilization of heme as a nutritional iron source in Bradyrhizobium japonicum. The hmuR operon is transcriptionally activated by the Irr protein and is also positively controlled by HmuP by an unknown mechanism. An hmuP mutant does not express the hmuR operon genes nor does it grow on heme. Here, we show that hmuR expression from a heterologous promoter still requires hmuP, suggesting that HmuP does not regulate at the transcriptional level. Replacement of the 5' untranslated region (5'UTR) of an HmuP-independent gene with the hmuR 5'UTR conferred HmuP-dependent expression on that gene. Recombinant HmuP bound an HmuP-responsive RNA element (HPRE) within the hmuR 5'UTR. A 2 nt substitution predicted to destabilize the secondary structure of the HPRE abolished both HmuP binding activity in vitro and hmuR expression in cells. However, deletion of the HPRE partially restored hmuR expression in an hmuP mutant, and it rescued growth of the hmuP mutant on heme. These findings suggest that the HPRE is a negative regulatory RNA element that is suppressed when bound by HmuP to express the hmuR operon.

Identifying functional multi-host shuttle plasmids to advance synthetic biology applications in Mesorhizobium and Bradyrhizobium.

Ammonia availability has a crucial role in agriculture as it ensures healthy plant growth and increased crop yields. Since diazotrophs are the only organisms capable of reducing dinitrogen to ammonia, they have great ecological importance and potential to mitigate the environmental and economic costs of synthetic fertilizer use. Rhizobia are especially valuable being that they can engage in nitrogen-fixing symbiotic relationships with legumes, and they demonstrate great diversity and plasticity in genomic and phenotypic traits. However, few rhizobial species have sufficient genetic tractability for synthetic biology applications. This study established a basic genetic toolbox with antibiotic resistance markers, multi-host shuttle plasmids and a streamlined protocol for biparental conjugation with Mesorhizobium and Bradyrhizobium species. We identified two repABC origins of replication from Sinorhizobium meliloti (pSymB) and Rhizobium etli (p42d) that were stable across all three strains of interest. Furthermore, the NZP2235 genome was sequenced and phylogenetic analysis determined its reclassification to Mesorhizobium huakuii. These tools will enable the use of plasmid-based strategies for more advanced genetic engineering projects and ultimately contribute towards the development of more sustainable agriculture practices by means of novel nitrogen-fixing organelles, elite bioinoculants, or symbiotic association with nonlegumes.

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.

The elite strain INPA03-11B approved as a cowpea inoculant in Brazil represents a new Bradyrhizobium species and it has high adaptability to stressful soil conditions.

The strain INPA03-11BT, isolated in the 1980s from nodules of Centrosema sp. collected in Manaus, Amazonas, Brazil, was approved by the Brazilian Ministry of Agriculture as a cowpea inoculant in 2004. Since then, several studies have been conducted regarding its phenotypic, genetic, and symbiotic characteristics under axenic and field conditions. Phenotypic features demonstrate its high adaptability to stressful soil conditions, such as tolerance to acidity, high temperatures, and 13 antibiotics, and, especially, its high symbiotic efficiency with cowpea and soybean, proven in the field. The nodC and nifH phylogenies placed the INPA strain in the same clade as the species B. macuxiense BR 10303T which was also isolated from the Amazon region. The sequencing of the 16S rRNA ribosomal gene and housekeeping genes, as well as BOX-PCR profiles, showed its potential as a new species, which was confirmed by a similarity percentage of 94.7% and 92.6% in Average Nucleotide Identity with the closest phylogenetically related species Bradyrhizobium tropiciagri CNPSo1112T and B. viridifuturi SEMIA690T, respectively. dDDH values between INPA03-11BT and both CNPSo 1112T and SEMIA690T were respectively 58.5% and 48.1%, which are much lower than the limit for species boundary (70%). Therefore, we propose the name Bradyrhizobium amazonense for INPA03-11BT (= BR3301 = SEMIA6463).

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.