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Bradyrhizobium septentrionale sp. nov. (sv. septentrionale) and Bradyrhizobium quebecense sp. nov. (sv. septentrionale) associated with legumes native to Canada possess rearranged symbiosis genes and numerous insertion sequences.

ABSTRACT: Six bacterial strains isolated from root nodules of soybean plants that had been inoculated with root-zone soil of legumes native to Canada were previously characterized and 1) placed in two novel lineages within the genus Bradyrhizobium and 2) assigned to symbiovar septentrionale. Here we verified the taxonomic status of these strains using genomic and phenotypic analyses. Phylogenetic analyses of five protein encoding partial gene sequences as well as 52 full length ribosome protein subunit gene sequences confirmed placement of the novel strains in two highly supported lineages distinct from named Bradyrhizobium species. The highest average nucleotide identity values of strains representing these two lineages relative to type strains of closest relatives were 90.7 and 92.3% which is well below the threshold value for bacterial species circumscription. The genomes of representative strains 1S1^T, 162S2 and 66S1MB^T have sizes of 10598256, 10733150 and 9032145 bp with DNA G+C contents of 63.5, 63.4 and 63.8 mol%, respectively. These strains possess between one and three plasmids based on copy number of plasmid replication and segregation (repABC) genes. Novel strains also possess numerous insertion sequences, and, relative to reference strain Bradyrhizobium diazoefficiens USDA110^T, exhibit inversion and fragmentation of nodulation (nod) and nitrogen-fixation (nif) gene clusters. Phylogenetic analyses of nodC and nifH gene sequences confirmed placement of novel strains in a distinct lineage corresponding to symbiovar septentrionale. Data for morphological, physiological and symbiotic characteristics complement the sequence-based results. The data presented here support the description of two new species for which the names Bradyrhizobium septentrionale sp. nov. (sv. septentrionale) and Bradyrhizobium quebecense sp. nov. (sv. septentrionale) are proposed, with 1S1^T (=LMG 29930^T=HAMBI 3676^T) and 66S1MB^T (=LMG 31547^T=HAMBI 3720^T) as type strains, respectively.

SUBMITTER: Bromfield ESP

PROVIDER: S-EPMC8374602 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Strains of the genus Bradyrhizobium associated with agronomically important crops such as soybean (Glycine max) are increasingly studied; however, information about symbionts of wild Glycine species is scarce. Australia is a genetic centre of wild Glycine species and we performed a polyphasic analysis of three Bradyrhizobium strains-CNPSo 4010T, CNPSo 4016T, and CNPSo 4019T-trapped from Western Australian soils with Glycine clandestina, Glycine tabacina and Glycine max, respectively. The phylogenetic tree of the 16S rRNA gene clustered all strains into the Bradyrhizobium japonicum superclade; strains CNPSo 4010T and CNPSo 4016T had Bradyrhizobium yuanmingense CCBAU 10071T as the closest species, whereas strain CNPSo 4019T was closer to Bradyrhizobium liaoningense LMG 18230T. The multilocus sequence analysis (MLSA) with five housekeeping genes-dnaK, glnII, gyrB, recA and rpoB-confirmed the same clusters as the 16S rRNA phylogeny, but indicated low similarity to described species, with nucleotide identities ranging from 93.6 to 97.6% of similarity. Considering the genomes of the three strains, the average nucleotide identity and digital DNA-DNA hybridization values were lower than 94.97 and 59.80 %, respectively, with the closest species. In the nodC phylogeny, strains CNPSo 4010T and CNPSo 4019T grouped with Bradyrhizobium zhanjiangense and Bradyrhizobium ganzhouense, respectively, while strain CNPSo 4016T was positioned separately from the all symbiotic Bradyrhizobium species. Other genomic (BOX-PCR), phenotypic and symbiotic properties were evaluated and corroborated with the description of three new lineages of Bradyrhizobium. We propose the names of Bradyrhizobium agreste sp. nov. for CNPSo 4010T (=WSM 4802T=LMG 31645T) isolated from Glycine clandestina, Bradyrhizobium glycinis sp. nov. for CNPSo 4016T (=WSM 4801T=LMG 31649T) isolated from Glycine tabacina and Bradyrhizobium diversitatis sp. nov. for CNPSo 4019T (=WSM 4799T=LMG 31650T) isolated from G. max.

Project description:The type strain of the prospective 10.1601/nm.30737 sp. nov. ERR11T, was isolated from a nodule of the leguminous tree Erythrina brucei native to Ethiopia. The type strain 10.1601/nm.1463 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071 T, was isolated from the nodules of Lespedeza cuneata in Beijing, China. The genomes of ERR11T and 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071 T were sequenced by DOE-JGI and deposited at the DOE-JGI genome portal as well as at the European Nucleotide Archive. The genome of ERR11T is 9,163,226 bp in length and has 102 scaffolds, containing 8548 protein-coding and 86 RNA genes. The 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071 T genome is arranged in 108 scaffolds and consists of 8,201,522 bp long and 7776 protein-coding and 85 RNA genes. Both genomes contain symbiotic genes, which are homologous to the genes found in the complete genome sequence of 10.1601/nm.24498 10.1601/strainfinder?urlappend=%3Fid%3DUSDA+110 T. The genes encoding for nodulation and nitrogen fixation in ERR11T showed high sequence similarity with homologous genes found in the draft genome of peanut-nodulating 10.1601/nm.27386 10.1601/strainfinder?urlappend=%3Fid%3DLMG+26795 T. The nodulation genes nolYA-nodD2D1YABCSUIJ-nolO-nodZ of ERR11T and 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071 T are organized in a similar way to the homologous genes identified in the genomes of 10.1601/strainfinder?urlappend=%3Fid%3DUSDA+110 T, 10.1601/nm.25806 10.1601/strainfinder?urlappend=%3Fid%3DUSDA+4 and 10.1601/nm.1462 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+05525. The genomes harbor hupSLCFHK and hypBFDE genes that code the expression of hydrogenase, an enzyme that helps rhizobia to uptake hydrogen released by the N2-fixation process and genes encoding denitrification functions napEDABC and norCBQD for nitrate and nitric oxide reduction, respectively. The genome of ERR11T also contains nosRZDFYLX genes encoding nitrous oxide reductase. Based on multilocus sequence analysis of housekeeping genes, the novel species, which contains eight strains formed a unique group close to the 10.1601/nm.25806 branch. Genome Average Nucleotide Identity (ANI) calculated between the genome sequences of ERR11T and closely related sequences revealed that strains belonging to 10.1601/nm.25806 branch (10.1601/strainfinder?urlappend=%3Fid%3DUSDA+4 and 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+15615), were the closest strains to the strain ERR11T with 95.2% ANI. Type strain ERR11T showed the highest DDH predicted value with 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+15615 (58.5%), followed by 10.1601/strainfinder?urlappend=%3Fid%3DUSDA+4 (53.1%). Nevertheless, the ANI and DDH values obtained between ERR11T and 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+15615 or 10.1601/strainfinder?urlappend=%3Fid%3DUSDA+4 were below the cutoff values (ANI???96.5%; DDH???70%) for strains belonging to the same species, suggesting that ERR11T is a new species. Therefore, based on the phylogenetic analysis, ANI and DDH values, we formally propose the creation of 10.1601/nm.30737 sp. nov. with strain ERR11T (10.1601/strainfinder?urlappend=%3Fid%3DHAMBI+3532 T=10.1601/strainfinder?urlappend=%3Fid%3DLMG+30162 T) as the type strain.

Project description:Rhizobia are nitrogen-fixing bacteria that establish endosymbiotic associations with legumes. Nodule formation depends on various bacterial carbohydrates, including lipopolysaccharides, K-antigens, and exopolysaccharides (EPS). An acidic EPS from Rhizobium sp. strain NGR234 consists of glucosyl (Glc), galactosyl (Gal), glucuronosyl (GlcA), and 4,6-pyruvylated galactosyl (PvGal) residues with beta-1,3, beta-1,4, beta-1,6, alpha-1,3, and alpha-1,4 glycoside linkages. Here we examined the role of NGR234 genes in the synthesis of EPS. Deletions within the exoF, exoL, exoP, exoQ, and exoY genes suppressed accumulation of EPS in bacterial supernatants, a finding that was confirmed by chemical analyses. The data suggest that the repeating subunits of EPS are assembled by an ExoQ/ExoP/ExoF-dependent mechanism, which is related to the Wzy polymerization system of group 1 capsular polysaccharides in Escherichia coli. Mutation of exoK (NGROmegaexoK), which encodes a putative glycanase, resulted in the absence of low-molecular-weight forms of EPS. Analysis of the extracellular carbohydrates revealed that NGROmegaexoK is unable to accumulate exo-oligosaccharides (EOSs), which are O-acetylated nonasaccharide subunits of EPS having the formula Gal(Glc)5(GlcA)2PvGal. When used as inoculants, both the exo-deficient mutants and NGROmegaexoK were unable to form nitrogen-fixing nodules on some hosts (e.g., Albizia lebbeck and Leucaena leucocephala), but they were able to form nitrogen-fixing nodules on other hosts (e.g., Vigna unguiculata). EOSs of the parent strain were biologically active at very low levels (yield in culture supernatants, approximately 50 microg per liter). Thus, NGR234 produces symbiotically active EOSs by enzymatic degradation of EPS, using the extracellular endo-beta-1,4-glycanase encoded by exoK (glycoside hydrolase family 16). We propose that the derived EOSs (and not EPS) are bacterial components that play a crucial role in nodule formation in various legumes.

Dataset Information

Bradyrhizobium septentrionale sp. nov. (sv. septentrionale) and Bradyrhizobium quebecense sp. nov. (sv. septentrionale) associated with legumes native to Canada possess rearranged symbiosis genes and numerous insertion sequences.

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