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.

The Bradyrhizobium japonicum fsrB gene is essential for utilization of structurally diverse ferric siderophores to fulfill its nutritional iron requirement.

In Bradyrhizobium japonicum, iron uptake from ferric siderophores involves selective outer membrane proteins and non-selective periplasmic and cytoplasmic membrane components that accommodate numerous structurally diverse siderophores. Free iron traverses the cytoplasmic membrane through the ferrous (Fe2+ ) transporter system FeoAB, but the other non-selective components have not been described. Here, we identify fsrB as an iron-regulated gene required for growth on iron chelates of catecholate- and hydroxymate-type siderophores, but not on inorganic iron. Utilization of the non-physiological iron chelator EDDHA as an iron source was also dependent on fsrB. Uptake activities of 55 Fe3+ bound to ferrioxamine B, ferrichrome or enterobactin were severely diminished in the fsrB mutant compared with the wild type. Growth of the fsrB or feoB strains on ferrichrome were rescued with plasmid-borne E. coli fhuCDB ferrichrome transport genes, suggesting that FsrB activity occurs in the periplasm rather than the cytoplasm. Whole cells of an fsrB mutant are defective in ferric reductase activity. Both whole cells and spheroplasts catalyzed the demetallation of ferric siderophores that were defective in an fsrB mutant. Collectively, the data support a model whereby FsrB is required for reduction of iron and its dissociation from the siderophore in the periplasm, followed by transport of the ferrous ion into the cytoplasm by FeoAB.

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 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.

The native distribution of a common legume shrub is limited by the range of its nitrogen-fixing mutualist.

Plant-microbe mutualisms, such as the legume-rhizobium symbiosis, are influenced by the geographical distributions of both partners. However, limitations on the native range of legumes, resulting from the absence of a compatible mutualist, have rarely been explored. We used a combination of a large-scale field survey and controlled experiments to determine the realized niche of Calicotome villosa, an abundant and widespread legume shrub. Soil type was a major factor affecting the distribution and abundance of C. villosa. In addition, we found a large region within its range in which neither C. villosa nor Bradyrhizobium, the bacterial genus that associates with it, were present. Seedlings grown in soil from this region failed to nodulate and were deficient in nitrogen. Inoculation of this soil with Bradyrhizobium isolated from root nodules of C. villosa resulted in the formation of nodules and higher growth rate, leaf N and shoot biomass compared with un-inoculated plants. We present evidence for the exclusion of a legume from parts of its native range by the absence of a compatible mutualist. This result highlights the importance of the co-distribution of both the host plant and its mutualist when attempting to understand present and future geographical distributions of legumes.

Bacillus suppresses nitrogen efficiency of soybean-rhizobium symbiosis through regulation of nitrogen-related transcriptional and microbial patterns.

The regulation of legume-rhizobia symbiosis by microorganisms has obtained considerable interest in recent research, particularly in the common rhizobacteria Bacillus. However, few studies have provided detailed explanations regarding the regulatory mechanisms involved. Here, we investigated the effects of Bacillus (Bac.B) on Bradyrhizobium-soybean (Glycine max) symbiosis and elucidated the underlying ecological mechanisms. We found that two Bradyrhizobium strains (i.e. Bra.Q2 and Bra.D) isolated from nodules significantly promoted nitrogen (N) efficiency of soybean via facilitating nodule formation, thereby enhanced plant growth and yield. However, the intrusion of Bac.B caused a reverse shift in the synergistic efficiency of N2 fixation in the soybean-Bradyrhizobium symbiosis. Biofilm formation and naringenin may be importantin suppression of Bra.Q2 growth regulated by Bac.B. In addition, transcriptome and microbiome analyses revealed that Bra.Q2 and Bac.B might interact to regulateN transport and assimilation, thus influence the bacterial composition related to plant N nutrition in nodules. Also, the metabolisms of secondary metabolites and hormones associated with plant-microbe interaction and growth regulation were modulated by Bra.Q2 and Bac.B coinoculation. Collectively, we demonstrate that Bacillus negatively affects Bradyrhizobium-soybean symbiosis and modulate microbial interactions in the nodule. Our findings highlight a novel Bacillus-based regulation to improve N efficiency and sustainable agricultural development.

The radiation of nodulated Chamaecrista species from the rainforest into more diverse habitats has been accompanied by a reduction in growth form and a shift from fixation threads to symbiosomes.

All non-Mimosoid nodulated genera in the legume subfamily Caesalpinioideae confine their rhizobial symbionts within cell wall-bound 'fixation threads' (FTs). The exception is the large genus Chamaecrista in which shrubs and subshrubs house their rhizobial bacteroids more intimately within symbiosomes, whereas large trees have FTs. This study aimed to unravel the evolutionary relationships between Chamaecrista growth habit, habitat, nodule bacteroid type, and rhizobial genotype. The growth habit, bacteroid anatomy, and rhizobial symbionts of 30 nodulated Chamaecrista species native to different biomes in the Brazilian state of Bahia, a major centre of diversity for the genus, was plotted onto an ITS-trnL-F-derived phylogeny of Chamaecrista. The bacteroids from most of the Chamaecrista species examined were enclosed in symbiosomes (SYM-type nodules), but those in arborescent species in the section Apoucouita, at the base of the genus, were enclosed in cell wall material containing homogalacturonan (HG) and cellulose (FT-type nodules). Most symbionts were Bradyrhizobium genotypes grouped according to the growth habits of their hosts, but the tree, C. eitenorum, was nodulated by Paraburkholderia. Chamaecrista has a range of growth habits that allow it to occupy several different biomes and to co-evolve with a wide range of (mainly) bradyrhizobial symbionts. FTs represent a less intimate symbiosis linked with nodulation losses, so the evolution of SYM-type nodules by most Chamaecrista species may have (i) aided the genus-wide retention of nodulation, and (ii) assisted in its rapid speciation and radiation out of the rainforest into more diverse and challenging habitats.

Endophytic diazotrophic communities from rice roots are diverse and weakly associated with soil diazotrophic community composition and soil properties.

AIM: Bacteria that promote plant growth, such as diazotrophs, are valuable tools for achieving a more sustainable production of important non-legume crops like rice. Different strategies have been used to discover new bacteria capable of promoting plant growth. This work evaluated the contribution of soil diazotrophs to the endophytic communities established in the roots of rice seedlings cultivated on seven representative soils from Uruguay. METHODS AND RESULTS: The soils were classified into two groups according to the C and clay content. qPCR, terminal restriction fragment length polymorphism (T-RFLP), and 454-pyrosequencing of the nifH gene were used for analyzing diazotrophs in soil and plantlets' roots grown from seeds of the same genotype for 25 days under controlled conditions. A similar nifH abundance was found among the seven soils, roots, or leaves. The distribution of diazotrophs was more uneven in roots than in soils, with dominance indices significantly higher than in soils (nifH T-RFLP). Dominant soils' diazotrophs were mainly affiliated to Alphaproteobacteria and Planctomycetota. Conversely, Alpha, Beta, Gammaproteobacteria, and Bacillota were predominant in different roots, though undetectable in soils. Almost no nifH sequences were shared between soils and roots. CONCLUSIONS: Root endophytic diazotrophs comprised a broader taxonomic range of microorganisms than diazotrophs found in soils from which the plantlets were grown and showed strong colonization patterns.

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.

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.

Inter-species interaction of bradyrhizobia affects their colonization and plant growth promotion in Arachis hypogaea.

Bradyrhizobia are the principal symbiotic partner of the leguminous plant and take active part in biological nitrogen-fixation. The present investigation explores the underlying competition among different strains during colonization in host roots. Six distinct GFP and RFP-tagged Bradyrhizobium strains were engineered to track them inside the peanut roots either independently or in combination. The Bradyrhizobium strains require different time-spans ranging from 4 to 21 days post-infection (dpi) for successful colonization which further varies in presence of another strain. While most of the individual strains enhanced the shoot and root dry weight, number of nodules, and nitrogen fixation capabilities of the host plants, no significant enhancement of plant growth and nodulation efficiency was observed when they were allowed to colonize in combinations. However, if among the combinations one strains is SEMIA 6144, the co-infection results in higher growth and nodulation efficiency of the hosts. From the competition experiments it has been found that Bradyrhizobium japonicum SEMIA 6144 was found to be the most dominant strain for effective nodulation in peanut. The extent of biofilm and exopolysaccharide (EPS) production by these isolates, individually or in combinations, were envisaged to correlate whether these parameters have any impact on the symbiotic association. But the extent of colonization, growth-promotion and nitrogen-fixation ability drastically lowered when a strain present together with other Bradyrhizobium strain. Therefore, it is imperative to understand the interaction between two co-inoculating Bradyrhizobium species for nodulation followed by plant growth promotion to develop suitable consortia for enhancing BNF in peanut and possibly for other legumes.

Denitrification by Bradyrhizobia under Feast and Famine and the Role of the bc1 Complex in Securing Electrons for N2O Reduction.

Rhizobia living as microsymbionts inside nodules have stable access to carbon substrates, but also must survive as free-living bacteria in soil where they are starved for carbon and energy most of the time. Many rhizobia can denitrify, thus switch to anaerobic respiration under low O2 tension using N-oxides as electron acceptors. The cellular machinery regulating this transition is relatively well known from studies under optimal laboratory conditions, while little is known about this regulation in starved organisms. It is, for example, not known if the strong preference for N2O- over NO3- reduction in bradyrhizobia is retained under carbon limitation. Here, we show that starved cultures of a Bradyrhizobium strain with respiration rates 1 to 18% of well-fed cultures reduced all available N2O before touching provided NO3-. These organisms, which carry out complete denitrification, have the periplasmic nitrate reductase NapA but lack the membrane-bound nitrate reductase NarG. Proteomics showed similar levels of NapA and NosZ (N2O reductase), excluding that the lack of NO3- reduction was due to low NapA abundance. Instead, this points to a metabolic-level phenomenon where the bc1 complex, which channels electrons to NosZ via cytochromes, is a much stronger competitor for electrons from the quinol pool than the NapC enzyme, which provides electrons to NapA via NapB. The results contrast the general notion that NosZ activity diminishes under carbon limitation and suggest that bradyrhizobia carrying NosZ can act as strong sinks for N2O under natural conditions, implying that this criterion should be considered in the development of biofertilizers. IMPORTANCE Legume cropped farmlands account for substantial N2O emissions globally. Legumes are commonly inoculated with N2-fixing bacteria, rhizobia, to improve crop yields. Rhizobia belonging to Bradyrhizobium, the microsymbionts of several economically important legumes, are generally capable of denitrification but many lack genes encoding N2O reductase and will be N2O sources. Bradyrhizobia with complete denitrification will instead act as sinks since N2O-reduction efficiently competes for electrons over nitrate reduction in these organisms. This phenomenon has only been demonstrated under optimal conditions and it is not known how carbon substrate limitation, which is the common situation in most soils, affects the denitrification phenotype. Here, we demonstrate that bradyrhizobia retain their strong preference for N2O under carbon starvation. The findings add basic knowledge about mechanisms controlling denitrification and support the potential for developing novel methods for greenhouse gas mitigation based on legume inoculants with the dual capacity to optimize N2 fixation and minimize N2O emission.

Bradyrhizobium commune sp. nov., isolated from nodules of a wide range of native legumes across the Australian continent.

Bradyrhizobia are particularly abundant in Australia, where they nodulate native legumes growing in the acidic and seasonally dry soils that predominate in these environments. They are essential to Australian ecosystems by helping legumes to compensate for nutrient deficiencies and the low fertility of Australian soils. During a survey of Australian native rhizobial communities in 1994-1995, several Bradyrhizobium genospecies were identified, among which genospecies B appeared to be present in various edaphic and climatic conditions and associate with a large range of leguminous hosts across the whole continent. We took advantage of the recent sequencing of the genome of strain BDV5040T, representative of Bradyrhizobium genospecies B, to re-evaluate the taxonomic status of this lineage. We further characterized strain BDV5040T based on morpho-physiological traits and determined its phylogenetic relationships with the type strains of all currently described Bradyrhizobium species using both small subunit (SSU) rRNA gene and complete genome sequences. The digital DNA-DNA hybridization relatedness with any type strain was less than 35 % and both SSU rRNA gene and genome phylogenies confirmed the initial observation that this strain does not belong to any formerly described species within the genus Bradyrhizobium. All data thus support the description of the novel species Bradyrhizobium commune sp. nov. for which the type strain is BDV5040T (=CFBP 9110T=LMG 32898T), isolated from a nodule of Bossiaea ensata in Ben Boyd National Park in New South Wales, Australia.

Exploring potential soybean bradyrhizobia from high trehalose-accumulating soybean genotypes for improved symbiotic effectiveness in soybean.

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.

Not just passengers, but co-pilots! Non-rhizobial nodule-associated bacteria promote cowpea growth and symbiosis with (brady)rhizobia.

AIMS: To isolate and characterize non-rhizobial nodule-associated bacteria (NAB) from cowpea root-nodules regarding their performance of plant-growth-promoting mechanisms and their ability to enhance cowpea growth and symbiosis when co-inoculated with bradyrhizobia. METHODS AND RESULTS: Sixteen NAB were isolated, identified, and in vitro evaluated for plant growth promotion traits. The ability to promote cowpea growth was analyzed when co-inoculated with Bradyrhizobium pachyrhizi BR 3262 in sterile and non-sterile substrates. The 16S rRNA gene sequences analysis revealed that NAB belonged to the genera Chryseobacterium (4), Bacillus (3), Microbacterium (3), Agrobacterium (1), Escherichia (1), Delftia (1), Pelomonas (1), Sphingomonas (1), and Staphylococcus (1). All strains produced different amounts of auxin siderophores and formed biofilms. Twelve out of the 16 strains carried the nifH, a gene associated with nitrogen fixation. Co-inoculation of NAB (ESA 424 and ESA 29) with Bradyrhizobium pachyrhizi BR 3262 significantly promoted cowpea growth, especially after simultaneous inoculation with the three strains. CONCLUSIONS: NAB are efficient cowpea growth promoters and can improve the efficiency of the symbiosis between cowpea and the N2-fixing microsymbiont B. pachyrhizi BR 3262, mainly under a specific triple microbial association.

Bacteria isolated from explosive contaminated environments transform pentaerythritol tetranitrate (PETN) under aerobic and anaerobic conditions.

Pentaerythritol tetranitrate (PETN) is a nitrate ester explosive that may be persistent with scarce reports on its environmental fate and impacts. Our main objective was to isolate and characterize bacteria that transform PETN under aerobic and anaerobic conditions. Biotransformation of PETN (100 mg L-1) was evaluated using mineral medium with (M + C) and without (M - C) additional carbon sources under aerobic conditions and with additional carbon sources under anaerobic conditions. Here, we report on the isolation of 12 PETN-transforming cultures (4 pure and 8 co-cultures) from environmental samples collected at an explosive manufacturing plant. The highest transformation of PETN was observed for cultures in M + C under aerobic conditions, reaching up to 91% ± 2% in 2 d. Under this condition, PETN biotransformation was observed in conjunction with the release of nitrites and bacterial growth. No substantial transformation of PETN (<45%) was observed during 21 d in M - C under aerobic conditions. Under anaerobic conditions, five cultures could transform PETN (up to 52% ± 13%) as the sole nitrogen source, concurrent with the formation of two unidentified metabolites. PETN-transforming cultures belonged to Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Actinobacteria. In conclusion, we isolated 12 PETN-transforming cultures belonging to diverse taxa, suggesting that PETN transformation is phylogenetically widespread.

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.

Lupin, a Unique Legume That Is Nodulated by Multiple Microsymbionts: The Role of Horizontal Gene Transfer.

Lupin is a high-protein legume crop that grows in a wide range of edaphoclimatic conditions where other crops are not viable. Its unique seed nutrient profile can promote health benefits, and it has been proposed as a phytoremediation plant. Most rhizobia nodulating Lupinus species belong to the genus Bradyrhizobium, comprising strains that are phylogenetically related to B. cytisi, B. hipponenese, B. rifense, B. iriomotense/B. stylosanthis, B. diazoefficiens, B. japonicum, B. canariense/B. lupini, and B. retamae/B. valentinum. Lupins are also nodulated by fast-growing bacteria within the genera Microvirga, Ochrobactrum, Devosia, Phyllobacterium, Agrobacterium, Rhizobium, and Neorhizobium. Phylogenetic analyses of the nod and nif genes, involved in microbial colonization and symbiotic nitrogen fixation, respectively, suggest that fast-growing lupin-nodulating bacteria have acquired their symbiotic genes from rhizobial genera other than Bradyrhizobium. Horizontal transfer represents a key mechanism allowing lupin to form symbioses with bacteria that were previously considered as non-symbiotic or unable to nodulate lupin, which might favor lupin's adaptation to specific habitats. The characterization of yet-unstudied Lupinus species, including microsymbiont whole genome analyses, will most likely expand and modify the current lupin microsymbiont taxonomy, and provide additional knowledge that might help to further increase lupin's adaptability to marginal soils and climates.

Response of peanut (Arachis hypogaea L.) plant to bio-fertilizer and plant residues in sandy soil.

Nitrogen (N) fertilizer has been intensively used to improve peanut productivity. However, the high cost of N fertilizer, and the need for sustainable alternative fertilizer sources have increased the strategic importance of nitrogen fixation (NF). Thus, field experiments were conducted in an experimental farm with a drip irrigation system, at the Atomic Energy Authority, Inshas, Egypt, in order to measure the impact of efficiency symbiotic Bradyrhizobium sp. and asymbiotic Azotobacter sp. on NF, from air and soil, in the presence or absence of plant residues on the growth and yield of peanut plant. All treatments received nitrogen fertilizer at a rate of 72 kg N per hectare. Nitrogen dose was applied using ammonium sulphate 15N labeled of 10% atom excess from the peanut. Results indicated that the application of Bradyrhizobium sp. with plant residues significantly increased fresh and dry weight/m2, pod and seed weight/plant-1,100- seed weight, and biological yield kg ha-1, where the highest mean values of seed yield (4648 and 4529 kg ha-1), oil % (52.29 and 52.21%), seed protein percentage (16.09 and 15.89%), as well as nitrogen derived from air (63.14 and 66.20%) in the first and second seasons were recorded under the application of Bradyrhizobium sp, respectively. Bradyrhizobium sp. inoculation showed nearly close portions of Ndfa to those recorded with Azotobacter sp., in both the presence and absence of plant residue application through the two seasons. The investigated yield signs and their properties were significantly enhanced by bacterial inoculation with plant residue application. The present study shows that both possibility of NF of peanut, and nitrogen uptake in the soil are enhanced by field inoculation with effective Bradyrhizobium sp. with plant residue application. In practice, inoculation is a great strategy to improve soil fertility for subsequent planting, since it helps boost the import of nitrogen from plant biomass into the soil.

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.