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Leguminous tree root nodule nitrogen-fixing bacteria are critical for recuperation of soil C and N cycle processes after disturbance in tropical forests, while other nodule-associated bacteria (NAB) may enhance nodule development and activity, and plant growth. However, little is known of these root nodule microbiomes. Through DNA analysis, we evaluated the bacterial taxa associated with the root nodules of the 1-year-old, 2-year-old, 13-year-old, and old growth Inga punctata trees in a cloud forest. Bradyrhizobium diazoefficiens was the dominant taxon found in all nodules at 63.16% to 85.71% mean percent sequences (MPS) of the total nodule bacterial DNA and was found in the youngest nodules examined (1 year old), suggesting that it is the primary nodular bacteria. There were 26 other NAB genera with collective MPS levels between 7.4% to 12.2%, while 15 of these genera were found in the Bulk Forest soils at collective MPS levels of 4.6%. These bacterial community compositions were different between the NAB and Bulk Forest soils, suggesting the NAB became concentrated within the root nodules, resulting in communities with different compositions from the Bulk Forest soils. Twenty-three of the 26 NAB genera were previously identified with the potential to perform 9 plant growth promoting (PGP) activities, suggesting their importance in root nodule development and plant growth. These NAB communities appeared to successionally develop over time into more complex taxonomic communities, which is consistent with the outcome of advanced microbial communities following succession. The presence of both B. diazoefficiens and the NAB communities in the nodules across all ages of tree roots, and the potential for PGP activities linked with most of the NAB genera, suggest the importance of B. diazoefficiens and the NAB community for nodule development and enhanced development and growth of I. punctata throughout its lifespan, and most critically in the younger plants., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 the Author(s), licensee AIMS Press.)

Members of the genus Mesorhizobium, which are core components of the rhizosphere and specific symbionts of legume plants, possess genes for acyl-homoserine lactone (AHL) quorum sensing (QS). Here we show Mesorhizobium japonicum MAFF 303099 (formerly M. loti ) synthesizes and responds to N -[(2 E , 4 E )-2,4-dodecadienoyl] homoserine lactone (2 E, 4 E- C 12:2 -HSL). We show that the 2 E, 4 E- C 12:2 -HSL QS circuit involves one of four luxR-luxI -type genes found in the sequenced genome of MAFF 303099. We refer to this circuit, which appears to be conserved among Mesorhizobium species, as R1-I1. We show that two other Mesorhizobium strains also produce 2 E, 4 E- C 12:2 -HSL. The 2 E, 4 E- C 12:2 -HSL is unique among known AHLs in its arrangement of two trans double bonds. The R1 response to 2 E, 4 E- C 12:2 -HSL is extremely selective in comparison with other LuxR homologs, and the trans double bonds appear critical for R1 signal recognition. Most well-studied LuxI-like proteins use S -adenosylmethionine and an acyl-acyl carrier protein as substrates for synthesis of AHLs. Others that form a subgroup of LuxI-type proteins use acyl-coenzyme A substrates rather than acyl-acyl carrier proteins. I1 clusters with the acyl-coenzyme A-type AHL synthases. We show that a gene linked to the I1 AHL synthase is involved in the production of the QS signal. The discovery of the unique I1 product enforces the view that further study of acyl-coenzyme A-dependent LuxI homologs will expand our knowledge of AHL diversity. The involvement of an additional enzyme in AHL generation leads us to consider this system a three-component QS circuit. IMPORTANCE We report a Mesorhizobium japonicum quorum sensing (QS) system involving a novel acyl-homoserine lactone (AHL) signal. This system is known to be involved in root nodule symbiosis with host plants. The chemistry of the newly described QS signal indicated that there may be a dedicated cellular enzyme involved in its synthesis in addition to the types known for production of other AHLs. Indeed, we report that an additional gene is required for synthesis of the unique signal, and we propose that this is a three-component QS circuit as opposed to the canonical two-component AHL QS circuits. The signaling system is exquisitely selective. The selectivity may be important when this species resides in the complex microbial communities around host plants and may make this system useful in various synthetic biology applications of QS circuits., Competing Interests: The authors declare no conflict of interest.

Soybean is one of the most important legumes in the world, and its advantages and disadvantages are well known. As a result of symbiosis with the bacterium Bradyrhizobium japonicum , soybean can assimilate nitrogen from the air and is therefore not fertilized with this element, or if it is, only at small doses. In soybean agriculture practice, an important treatment is the inoculation of seeds with symbiotic bacteria and optimal fertilization with selected nutrients. Therefore, a three-year (2019-2021) field experiment was carried out to investigate the effects of soybean in the field to a seed Rhizobium inoculation or coating and molybdenum foliar fertilization. There were no significant interactions between the tested treatments over the years. It was demonstrated that the best variant was seed inoculation before sowing in combination with foliar molybdenum application. As a result of this treatment, a significant increase in nodulation, soil plant analysis development (SPAD) index, leaf area index (LAI) and seed yield (by 0.61 t·ha -1 ) was obtained compared to the control. In addition, the content of total protein in the seeds increased, while the content of crude fat decreased, which significantly modified the yield of both components. Sowing coated seeds in the Fix Fertig technology was less effective compared to inoculation, but it was significantly better than that in the control. Coating seeds with B. japonicum , in combination with foliar fertilization with molybdenum, could be recommended for agricultural practice, which was confirmed by economic calculations. Future experiments will assess the soybean's response to seed inoculation or coating and fertilization with other micronutrients.

Strain UY79 T was isolated from a root nodule of Arachis villosa , collected at the Esteros de Farrapos National Park, Río Negro, Uruguay. Cells were non-motile Gram-variable rods with central to subterminal oval to ellipsoidal endospores that swell the sporangia. Growth was observed in the range of 15-42 °C (optimum, 30 °C), pH 5.0-9.0 (optimum, pH 7.0-8.0) and with up to 3 % (w/v) NaCl (optimum, 1-2 %). Strain UY79 T was facultative anaerobic, catalase-positive and oxidase-negative. According to the results of 16S rRNA gene sequence analysis, UY79 T belongs to the genus Paenibacillus and is closely related to P. ottowii MS2379 T , P. peoriae BD-57 T , P. polymyxa ATCC 842 T and P. brasilensis PB172 T , exhibiting 99.4, 99.0, 99.0 and 98.9% sequence identity, respectively. Average nucleotide identity and digital DNA-DNA hybridization values with the most closely related type strains were 74.3-88.6% and 38.2-48.7 %, respectively. Major fatty acids (>10 %) were anteiso-C 15:0 , iso-C 15:0, and C 16 : 0 . Menaquinones MK-7 and MK-6 were the only isoprenoid quinones detected. Major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified glycolipid. Spermidine was the predominant polyamine. The DNA G+C content based on the draft genome sequence was 46.34 mol%. Based on the current polyphasic study, UY79 T represents a novel species of the genus Paenibacillus , for which the name Paenibacillus farraposensis sp. nov. is proposed. The type strain is UY79 T (=CCM 9147 T =CGMCC 1.19038 T ).

Two Gram-stain-negative strains, RCAM04680 T and RCAM04685, were isolated from root nodules of the relict legume Caragana jubata (Pall.) Poir. originating from the south-western shore of Lake Khuvsgul (Mongolia). The 16S rRNA gene ( rrs ) sequencing data showed that these novel isolates belong to the genus Bosea and are phylogenetically closest to the type strains Bosea lathyri LMG 26379 T , Bosea vaviloviae LMG 28367 T , Bosea massiliensis LMG 26221 T and Bosea lupini LMG 26383 T (the rrs -similarity levels were 98.7-98.8 %). The recA gene of strain RCAM04680 T showed the highest sequence similarity to the type strain B. lupini LMG 26383 T (95.4 %), while its atpD gene was closest to that of B. lathyri LMG 26379 T (94.4 %). The ITS, dnaK and gyrB sequences of this isolate were most similar to the B. vaviloviae LMG 28367 T (86.8 % for ITS, 90.4 % for the other genes). The most abundant fatty acid was C 18 : 1 ω7 c (40.8 %). The whole genomes of strains RCAM04680 T and RCAM04685 were identical (100 % average nucleotide identity). The highest average nucleotide identity value (82.8 %) was found between the genome of strain RCAM04680 T and B. vaviloviae LMG 28367 T . The common nod ABC genes required for legume nodulation were absent in both strains; however, some other symbiotic nol , nod , nif and fix genes were detected. Based on the genetic study, as well as analyses of the whole-cell fatty acid compositions and phenotypic properties, a new species, Boseacaraganae sp. nov. (type strain RCAM04680 T (=LMG 31125 T ), is proposed.

Given that phosphate supplies may diminish and become uneconomic to mine after 2020, there is a compelling need to develop alternative industries to support the population on Christmas Island. Former mine sites could be turned into productive agricultural land, however, large-scale commercial agriculture has never been attempted, and, given the uniqueness of the island, the diversity of rhizobia prior to introducing legumes needed evaluation. Therefore, 84 rhizobia isolates were obtained from nine different hosts, both crop and introduced legumes, located at seven sites across the island. Based on 16S rRNA and recA gene sequence analysis, the isolates grouped into 13 clades clustering within the genus Bradyrhizobium, Ensifer, Cupriavidus and Rhizobium. According to the sequences of their symbiosis genes nodC and nifH, the isolates were classified into 12 and 11 clades, respectively, and clustered closest to tropical or crop legume isolates. Moreover, the symbiosis gene phylogeny and Multi Locus Sequence Analysis gene phylogeny suggested vertical transmission in the Alpha-rhizobia but horizontal transmission within the Beta-rhizobia. Furthermore, this study provides evidence of a large diversity of endemic rhizobia associated with both crop and introduced legumes, and highlights the necessity of inoculation for common bean, chickpea and soybean on the Island., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

The importance of root nodule bacteria in biotechnology is determined by their distinctive feature: symbiotic nitrogen fixation resulting in the production of organic nitrogen-containing compounds. While interacting with host legume plants, the cells of these bacteria undergo global changes at all levels of expression of genetic information leading to the formation in root nodules of so-called bacteroids functioning as nitrogen fixation factories. The molecular mechanisms underlying plant-microbial symbiosis are actively investigated, and one of the most interesting and poorly studied aspects of this problem is the species-specificity of interaction between root nodule bacteria and host plants. In this work we have performed the proteomic analysis of the Sinorhizobium meliloti bacteroids isolated from two legume species: alfalfa (Medicago sativa L.) and yellow sweet clover (Melilotus officinalis L.). It has been shown that the S. meliloti bacteroids produce a lot of proteins (many of them associated with symbiosis) in a host-specific manner, i.e., only in certain host plant species. It has been demonstrated for the first time that the levels of expression in bacteroids of the genes encoding the ExoZ and MscL proteins responsible for the synthesis of surface lipopolysaccha-rides and formation of a large conductance mechanosensitive channel, respectively, depend on a host plant species that confirms the results of proteomic analysis. Overall, our data show that the regulation of bacteroid development by the host plant has species-specific features.

Gram-negative strains Tri-36, Tri-38, Tri-48 T and Tri-53 were isolated from root nodules of the relict legume Oxytropis triphylla (Pall.) Pers. originating from Zunduk Cape (Baikal Lake region, Russia). 16S rRNA gene sequencing showed that the novel isolates were phylogenetically closest to the type strains Phyllobacterium sophorae LMG 27899 T , Phyllobacterium brassicacearum LMG 22836 T , Phyllobacterium endophyticum LMG 26470 T and Phyllobacterium bourgognense LMG 22837 T while similarity levels between the isolates and the most closely related strain P. endophyticum LMG 26470 T were 98.8-99.5 %. The recA and glnII genes of the isolates showed highest sequence similarities with P. sophorae LMG 27899 T (95.4 and 89.5 %, respectively) and P. brassicacearum LMG 22836 T (91.4 and 85.1 %, respectively). Comparative analysis of phenotypic properties between the novel isolates and the closest reference strains P. sophorae LMG 27899 T , P. brassicacearum LMG 22836 T and P. endophyticum LMG 26470 T was performed using a microassay system. Average nucleotide identities between the whole genome sequences of the isolates Tri-38 and Tri-48 T and P. sophorae LMG 27899 T , P. brassicacearum LMG 22836 T and P. endophyticum LMG 26470 T ranged from 79.23 % for P. endophyticum LMG 26470 T to 85.74 % for P. sophorae LMG 27899 T . The common nodABC genes required for legume nodulation were absent from strains Tri-38 and Tri-48 T , although some other symbiotic nod and fix genes were detected. On the basis of genotypic and phenotypic analysis, a novel species, Phyllobacterium zundukense sp. nov. (type strain Tri-48 T =LMG 30371 T =RCAM 03910 T ), is proposed.

Amyloids are protein fibrils with a highly ordered spatial structure called cross-β. To date, amyloids were shown to be implicated in a wide range of biological processes, both pathogenic and functional. In bacteria, functional amyloids are involved in forming biofilms, storing toxins, overcoming the surface tension, and other functions. Rhizobiales represent an economically important group of Alphaproteobacteria , various species of which are not only capable of fixing nitrogen in the symbiosis with leguminous plants but also act as the causative agents of infectious diseases in animals and plants. Here, we implemented bioinformatic screening for potentially amyloidogenic proteins in the proteomes of more than 80 species belonging to the order Rhizobiales . Using SARP ( S equence A nalysis based on the R anking of P robabilities) and Waltz bioinformatic algorithms, we identified the biological processes, where potentially amyloidogenic proteins are overrepresented. We detected protein domains and regions associated with amyloidogenic sequences in the proteomes of various Rhizobiales species. We demonstrated that amyloidogenic regions tend to occur in the membrane or extracellular proteins, many of which are involved in pathogenesis-related processes, including adhesion, assembly of flagellum, and transport of siderophores and lipopolysaccharides, and contain domains typical of the virulence factors (hemolysin, RTX, YadA, LptD); some of them (rhizobiocins, LptD) are also related to symbiosis., Competing Interests: Declaration of Conflicting Interests:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Phylogenomic analyses showed two major superclades within the family Rhizobiaceae that corresponded to the Rhizobium/Agrobacterium and Shinella/Ensifer groups. Within the Rhizobium/Agrobacterium group, four highly supported clades were evident that could correspond to distinct genera. The Shinella/Ensifer group encompassed not only the genera Shinella and Ensifer but also a separate clade containing the type strain of Rhizobium giardinii. Ensifer adhaerens (Casida A(T)) was an outlier within its group, separated from the rest of the Ensifer strains. The phylogenomic analysis presented provided support for the revival of Allorhizobium as a bona fide genus within the Rhizobiaceae, the distinctiveness of Agrobacterium and the recently proposed Neorhizobium genus, and suggested that R. giardinii may be transferred to a novel genus. Genomics has provided data for defining bacterial-species limits from estimates of average nucleotide identity (ANI) and in silico DNA-DNA hybridization (DDH). ANI reference values are becoming the gold standard in rhizobial taxonomy and are being used to recognize novel rhizobial lineages and species that seem to be biologically coherent, as shown in this study., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Root nodule bacteria are free-living soil bacteria, belonging to diverse genera within the Alphaproteobacteria and Betaproteobacteria, that have the capacity to form nitrogen-fixing symbioses with legumes. The symbiosis is specific and is governed by signaling molecules produced from both host and bacteria. Sequencing of several model RNB genomes has provided valuable insights into the genetic basis of symbiosis. However, the small number of sequenced RNB genomes available does not currently reflect the phylogenetic diversity of RNB, or the variety of mechanisms that lead to symbiosis in different legume hosts. This prevents a broad understanding of symbiotic interactions and the factors that govern the biogeography of host-microbe symbioses. Here, we outline a proposal to expand the number of sequenced RNB strains, which aims to capture this phylogenetic and biogeographic diversity. Through the Vavilov centers of diversity (Proposal ID: 231) and GEBA-RNB (Proposal ID: 882) projects we will sequence 107 RNB strains, isolated from diverse legume hosts in various geographic locations around the world. The nominated strains belong to nine of the 16 currently validly described RNB genera. They include 13 type strains, as well as elite inoculant strains of high commercial importance. These projects will strongly support systematic sequence-based studies of RNB and contribute to our understanding of the effects of biogeography on the evolution of different species of RNB, as well as the mechanisms that determine the specificity and effectiveness of nodulation and symbiotic nitrogen fixation by RNB with diverse legume hosts.

It was found that both bacteroids and free-living cells of Rhizobium leguminosarum bv. phaseoli were highly susceptible to hydroxylysine (Hyl) and the inhibition of RNA and/or protein synthesis caused by the hydroxyl residue of Hyl appeared to be responsible for the growth inhibition. The size of free-living cells was enlarged by the addition of Hyl and some cells reached around 5 μm, which were close to the length of bacteroids. Under the same condition, the polyhydroxybutyrate (PHB) content in the cells was conspicuously increased. These results suggest that Hyl is not only a notable growth inhibitor of Rhizobium bacteria but also plays a role in a differentiation to bacteroids.