Abstract
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.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Introduction
Bacteria belonging to the genus Bradyrhizobium represent one of the most abundant taxa in soils globally and are considered a priority group for research on furthering the understanding of the contribution of soil microbes to ecosystem functioning (Delgado-Baquerizo et al. 2018). Moreover, the genus Bradyrhizobium includes economically important species that fix nitrogen in symbiotic association with agricultural plants such as soybean (Glycine max) and peanut (Arachis hypogaea) (Lindström and Mousavi 2019), species that are capable of photosynthesis (Giraud et al. 2007), and, species that possess genes for nitrous oxide (N2O) reductase and are able to reduce N2O, a potent greenhouse gas, to dinitrogen (N2) (Minamisawa 2023).
Bacterial species possessing photosynthesis genes are distributed across the genus Bradyrhizobium (Avontuur et al. 2023). These include apparently free-living (non-symbiotic) species such as B. betae (Rivas et al. 2004; Cloutier and Bromfield 2019), B. amphicarpaeae (Bromfield et al. 2019) and B. cosmicum (Wasai-Hara et al. 2020b). Others are represented by the species, B. oligotrophicum (Okubo et al. 2013; Ramirez-Bahena et al. 2013), ‘B. aeschynomenes’ (Sun et al. 2022), and, B. denitrificans (van Berkum et al. 2006) that are symbionts of tropical plant species of the genus Aeschynomene, and based on core gene analyses, are placed exclusively in the so called “photosynthetic clade” (Avontuur et al. 2023) of the genus Bradyrhizobium. These bacterial species lack the Type III Secretion System (T3SS) and nodulation (nod) genes needed for symbiosis by most rhizobia and yet are still capable of eliciting root- and stem-nodules on Aeschynomene species in a nodulation (nod) factor -T3SS independent manner (Giraud et al. 2007; Camuel et al. 2023).
In previous work (unpublished) we grew plants of Aeschynomene indica (Indian jointvetch) in pots in the glasshouse to produce seed for our research. After several weeks of plant growth in the glasshouse, a few sporadic nodules were observed on plant roots that were apparently due to “volunteer” bacteria in the rooting medium. Analysis of recA house-keeping (core) gene sequences placed bacteria isolated from these root-nodules in several novel lineages in the genus Bradyrhizobium.
The objective of the current work was the detailed genomic, phylogenetic and phenotypic description of one of these lineages represented by strain A19T. The novel strain, placed in the “photosynthetic clade” of the genus Bradyrhizobium possesses photosynthesis, nitrogen fixation and nitrous oxide (N2O) reductase genes and is capable of eliciting nitrogen-fixing nodules on the stems and roots of A. indica plants. Based on the data presented, a new species is proposed with the name Bradyrhizobium ontarionense sp. nov.
Materials and methods
Bacterial strains
Bacterial strain A19T was isolated from a nodule that formed on the root of an Aeschynomene indica plant raised from surface sterilized seed and grown in a pot in a glasshouse at Agriculture and Agri-Food Canada, Ottawa for six weeks (16 h, 25 − 28 °C (day); 8 h, 16 − 18 °C, (night)). The rooting medium consisted of a mixture of Canadian peat moss and locally sourced (Ottawa, Ontario) black-earth that had been sterilized by steaming for 8 h and then stored in bulk without aseptic precautions until use.
Bacteria employed in this work are listed in Table S1 or in the Tables and Figures of the main text and Appendix. Bacterial strains were grown on modified yeast extract-mannitol (YEM) agar medium having the following composition (g/l−1): yeast-extract (Thermo Scientific™ Oxoid™), 1.5; mannitol, 1.0; NaCl, 0.1; K2HPO4, 0.5; MgSO4· 7H2O, 0.2; Bacteriological agar (Thermo Scientific™ Oxoid™), 18.0. Bacterial cultures were maintained in 20% w/v glycerol at − 80 °C.
Genomic DNA sequencing and phylogenetic analysis
Genomic DNAs were extracted and purified from bacterial cells grown for 7 days at 28 °C on YEM agar medium as detailed by Bromfield et al. (2023).
Sequencing of the genome of strain A19T was done at the Genome Quebec Innovation Centre, Canada, employing Pacific Biosciences (PacBio) Sequel Single-Molecule Real-Time (SMRT) technology (Ardui et al. 2018). Flye software (version 2.9) (Kolmogorov et al. 2019) was used for genome sequence assembly.
Analysis of the core gene sequences (atpD, glnII, gyrB, recA, rpoB and 16S rRNA) of strain A19T together with type strains of Bradyrhizobium species and widely studied members of the “photosynthetic clade” (Bradyrhizobium spp. strains BTAi1, ORS278 and ORS285 (Giraud et al 2007; Renier et al 2011)) were done using sequences retrieved from whole genome sequences (where available). Analysis of photosynthesis reaction center (pufLM) genes, nitrogen fixation (nifHDK) genes and the nitrous oxide reductase (nosZ) gene were done using full-length gene sequences retrieved from genome sequences. Alignment of nucleotide sequences was performed using MUSCLE (Edgar 2004). Sequence accession numbers are listed in Table S1.
Further analysis was carried out using 50 single-copy core gene sequences encoding ribosome protein subunits (rps) of novel strain A19T and 77 Bradyrhizobium reference strains (Jolley et al. 2012). Aligned and concatenated sequences of rps genes of A19T and reference strains were obtained from genome sequences using the Genome Comparator tool implemented in the domain genome database of the BIGSdb software platform (Jolley and Maiden 2010). To avoid confounding the phylogenetic analysis, the sequences rpsU, rpmH and rpmJ were excluded because they were either incomplete or paralogous in several Bradyrhizobium reference strains.
The ModelTest-NG tool (Darriba et al. 2020) in the web-based CIPRES Science Gateway version 3.3 (Miller et al. 2010) was used to select best fit substitution models. Phylogenetic analyses using MrBayes (software version 3.2.1) were done employing default priors as described previously (Yu et al. 2014). Maximum-likelihood analyses were performed using 1,000 non-parametric bootstrap replications (Guindon et al. 2010). Bayesian trees are only presented in this work as trees reconstructed from Bayesian and Maximum-likelihood methods exhibited similar topologies (results not shown).
A whole genome sequence based phylogenetic tree was reconstructed using the online Type Strain Genome Server (TYGS) (Lefort et al. 2015; Meier-Kolthoff and Göker 2019).
Genomic analyses
The overall genome relatedness indices of digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) are routinely employed in bacterial taxonomic studies to facilitate species circumscription (Chun et al. 2018; Meier-Kolthoff and Göker 2019). Algorithms implemented in the TYGS were used to calculate dDDH values and associated confidence intervals (Holland et al. 2002; Meier-Kolthoff et al. 2013; Meier-Kolthoff and Göker 2019). The accepted dDDH threshold of 70% was used to define species boundaries (Meier-Kolthoff and Göker 2019). ANI values were calculated utilizing FastANI (Jain et al. 2018) performed in the K base web server (Arkin et al. 2018). The ANI threshold ~ 96% was used for delineation of bacterial species boundaries (Richter and Rosselló-Móra 2009; Lee et al. 2016; Ciufo et al. 2018).
Genome sequence comparisons were facilitated by utilizing the software, Geneious Prime 2023.0.4 (https://www.geneious.com) and GenomeMatcher (Ohtsubo et al. 2008).
Phenotypic characterisation
The Gram-stain reactivity of bacterial cells was assessed using the protocol outlined by Buck (1982).
Analysis of fatty acids, was done using bacteria grown at 28 °C on YEM agar medium for 7 days. Fatty acids were extracted as detailed by Sasser (1990). Identification of fatty acids was performed using the Sherlock Microbial Identification System (MIDI) version 6.0 and RTSBA6 database.
Tests of chemical sensitivity and carbon source utilization were done using BIOLOG GEN III MicroPlates (Biolog™, United States) as detailed in the manufacturer’s instructions.
Cells of strain A19T were examined using a scanning electron microscope (model, Hitachi SU7000 FESEM) and a transmission electron microscope (model, H-7000; Hitachi). For microscopic examination of cells, strain A19T was grown for 96 h in YEM broth at 28 °C as outlined previously (Bromfield et al. 2023).
Tests of acid or alkali production by A19T and reference strains grown for 21 days on YEM agar medium at 28 °C were performed as detailed by Bromfield et al. (2010).
Plant tests were carried out with modified Leonard jars (Vincent 1970) (two plants per jar with three replicate jars for each inoculation treatment) using nitrogen-free nutrient solution as detailed by Bromfield et al. (2010). Seeds of A. indica were vernalized by immersion in liquid nitrogen (− 196 °C) for 20 s and surface sterilized by serial immersion in 70% ethanol (30 s), 10.5% sodium hypochlorite solution (90 s) followed by multiple washes in water over two hours. The seeds were left in sterile water overnight at 4 °C to facilitate imbibition and then transferred to water agar plates (Bacteriological agar (Thermo Scientific™ Oxoid™) 15 g/l) at 25 °C to germinate. For tests of A. indica stem nodulation, cell suspensions of Bradyrhizobium test strains (ca. 109 cells/ ml in sterile water) were applied to stems with cotton-wool swabs. The inoculated portions of stems were kept moist for 48 h using wrappings of moistened tissue paper covered with plastic cling film.
Nodulation and symbiotic nitrogen fixation were assessed by visual comparison of shoots and roots of plants inoculated with A19T (as test strain) relative to negative control plants (uninoculated) and positive control plants inoculated with an effective strain: Bradyrhizobium sp. BTAi1 (for A. indica plants) and B. diazoefficiens USDA110 (for Glycine max (soybeans) and Macroptilium atropurpureum ‘siratro’). Symbiotic nitrogen fixation was considered to be ‘effective’ based on the size and colour of shoots (i.e., large, green and healthy) and size and number of root-nodules possessing pink pigmented interiors (characteristic of leghaemoglobin, a phytoglobin necessary for symbiotic nitrogen fixation).
Results and discussion
Genomic and phylogenetic characterisation
A complete genome sequence of novel strain A19T was generated in this work; genome coverage was 634-fold with 60,448 polymerase reads and 91,671 bp average read length. The genome of strain A19T consists of a single chromosome of size 8,435,845 bp and has a DNA G+C content of 64.9 mol% (Table 1).
The analysis of 16S rRNA gene sequences has traditionally been used as a taxonomic tool in species descriptions. However, this gene is highly conserved and different bacterial species may possess identical 16S rRNA sequences (Richter and Rosselló-Móra 2009; de Lajudie et al. 2019). Nevertheless, the analysis of 16S rRNA gene sequences is considered to be useful for verifying the genus level identity of bacteria (Young et al. 2023).
The phylogenetic tree of 16S rRNA gene sequences (Fig. S1) of strain A19T and 86 Bradyrhizobium species (type strains) confirms placement of A19T in the genus Bradyrhizobium. The tree also shows that strain A19T is placed in a novel lineage with B. oligotrophicum as the most closely related species.
Multiple Locus Sequence Analysis (MLSA) of single copy, protein encoding gene sequences is a widely used tool for species differentiation (Jolley et al. 2012; de Lajudie et al. 2019).
The Bayesian tree of concatenated core gene sequences (atpD-glnII-gyrB-recA-rpoB; alignment length, 2679 positions) of strain A19T and reference strains (Fig. S2), corroborates the placement of A19T in a new Bradyrhizobium lineage with B. oligotrophicum S58T as the closest relative. Fig. S2 also shows that strain A19T is placed in the “photosynthetic clade” (represented by B. oligotrophicum) containing photosynthetic symbionts of the tropical legume A. indica. It should be noted that Bradyrhizobium sp. BTAi1 shares a lineage with the type strain of B. denitrificans and therefore represents a potential member of this species. Moreover, strains ORS278 and ORS285 are placed in distinct lineages and represent potential genospecies.
The taxonomic status of strain A19T was further investigated by MLSA of 50 single-copy core gene sequences consisting of concatenated full-length bacterial ribosome protein subunit (rps) gene sequences (Jolley et al. 2012) as well as a phylogenomic analysis (based on whole genome sequences) implemented in the TYGS (Meier-Kolthoff and Göker 2019). A genome sequence of reference strain, B. denitrificans IFAM 1005T, is not available in public databases. As our phylogenetic analysis of five concatenated core gene sequences (Fig. S2) showed that the widely studied strain, Bradyrhizobium sp. BTAi1, is a potential member of the species, B. denitrificans, we used BTAi1 as a proxy for IFAM 1005T in subsequent phylogenetic and genomic analyses.
The phylogenetic tree of 50 core (rps) gene sequences (Fig. 1) and the TYGS tree based on whole genome sequences (Fig. S3) support the finding that strain A19T is placed in a highly supported novel lineage within the “photosynthetic clade” represented by B. oligotrophicum; B. oligotrophicum is also the closest relative.
Table 2 shows dDDH and ANI values for pair-wise comparison of the genome sequence of strain A19T with the genome sequences of five reference strains that are placed in the Bradyrhizobium “photosynthetic clade”. The largest values in these comparisons (33.4% (dDDH) and 88.8% (ANI)) are far lower than the threshold values (70% and ~ 96%, respectively) used for the definition of species boundaries. Based on these results, strain A19T represents a new species of Bradyrhizobium, with B. oligotrophicum as the most closely related species.
Genome sequence analyses revealed that strain A19T possesses a photosynthesis gene cluster (PGC) of size about 49 kb (co-ordinates 3,498,729–3,547,553 bp). The PGC contains key photosynthesis genes encoding bacteriochlorophyll (bchIDOCXYZGPFNBHLM and acsF), reaction centre L, M and H subunits (pufLM and puhA), light-harvesting protein alpha and beta subunits (pufBA), carotenoid (crtIBCDEF), bacteriophytochrome (bphP) and photosynthesis repressor (ppsR1 and ppsR2) proteins.
A Bayesian phylogenetic tree of concatenated photosynthetic reaction centre, pufLM, genes (Fig. 2A) shows that strain A19T is placed in a cluster together with other symbionts of A. indica; the closest relative of A19T is B. oligotrophicum S58T. The pufLM gene tree also shows that non-symbiotic species (B. cosmicum, B. amphicarpeae and B. betae) are placed in a separate cluster (clade) from the A. indica symbionts (B. denitrificans, B. oligotrophicum, ‘B. aeschynomenes’ and Bradyrhizobium sp. BTAi1 and novel strain A19T). It is notable that the phylogenetic division of symbiotic and nonsymbiotic bradyrhizobia on the basis of puf gene analysis corresponds to the two types of photosynthetic gene clusters (PGC1 and PGC2, respectively) defined by Avontuur et al. (2023). The organization of genes in the PGC of strain A19T is similar to close relative B. oligotrophicum possessing a type 1 PGC but differs from non-symbiotic bacteria such as B. amphicarpaea carrying type 2 PGCs (Fig. 2B).
Further analyses show that novel strain A19T lacks key nodulation (nodABC) and Type III Secretion System (T3SS) genes (Table 1) indicating that its symbiotic association with A. indica plants is initiated in a nod-factor and T3SS independent manner similar to the well characterised photosynthetic strains, Bradyrhizobium spp. BTAi1 and ORS278 (Giraud et al. 2007; Camuel et al. 2023).
In contrast to the absence of nod genes, the following key nitrogen fixation genes were found in the genome of strain A19T: nifDKEN, nifH, nifA and fixABCX (co-ordinates 7,793,281–7,842,331 bp). The phylogenetic tree of concatenated full length nifHDK gene sequences (Fig. S4) shows that strain A19T occupies a lineage that is well separated from other Bradyrhizobium species; the type strains of ‘B. aeschynomenes’ and B. oligotrophicum are closest relatives.
It should be noted that to date novel strain A19T represents only the fourth species to be placed in the “photosynthetic” clade (based on core gene sequence analysis—see Fig. 1) and as such represents a useful resource to further investigate the evolution of photosynthesis and symbiosis traits in the genus Bradyrhizobium.
Agricultural soils are a major source of N2O, a highly potent greenhouse gas and accelerant of ozone layer depletion (Montzka et al. 2011; Tian et al. 2020). The majority of N2O released from soils originates as a byproduct from the respiratory activity of nitrifying and denitrifying microorganisms (Thomson et al. 2012). While N2O can be generated by multiple mechanisms, the only known biological sink for N2O is the reduction of N2O to dinitrogen by the enzyme N2O reductase (encoded by the nosZ gene) found in some denitrifying bacteria (Torres et al. 2016; Minamisawa 2023). The nosZ gene has been detected infrequently in the rhizobia and has been found only in strains of the symbiotic species B. diazoefficiens (Sameshima-Saito et al. 2006; Itakura et al. 2013; Akiyama et al. 2016), B. ottawaense (Wasai-Hara et al. 2020a, b), Ensifer meliloti (Bueno, et al. 2015) and Rhizobium leguminosarum (Hénault et al. 2022). Recently nosZ gene containing strains of the nitrogen-fixing soybean symbiont, B. ottawaense, were found to be highly efficient with regard to the reduction of N2O to inert dinitrogen gas and the use of these strains as inoculants was suggested as a strategy for mitigating N2O emissions from agricultural soils (Wasai-Hara et al. 2023). In the current study we detected key genes encoding enzymes required for the complete denitrification of nitrate or nitrite to nitrogen gas in the genome sequence of strain A19T as follows: napEDABC (nitrate reductase); nirK (nitrite reductase); norCBQDE (nitric oxide reductase) and nosRZDFYLX (nitrous oxide reductase). Based on these findings, novel strain A19T represents a new resource for furthering studies on the reduction of N2O to inert gaseous nitrogen by members of the genus Bradyrhizobium.
We carried out further analyses to assess the frequency of occurrence of the nosZ gene (encoding N2O reductase) in the genus Bradyrhizobium by screening the genome sequences of type strains of named species. The results (Table S2) show that contrary to earlier reports, a substantial minority (i.e., 21 of 73 Bradyrhizobium species type strains) possess the nosZ gene. It is noteworthy that, with the exception of Bradyrhizobium sp. ORS 278, symbionts of the aquatic legume A. indica (B. oligotrophicum S58T,‘B. aeschynomenes’ 83002T, novel strain A19T (Table 1 and Table S2) and Bradyrhizobium spp. strains BTAi1 and ORS 285 (Table 1)) possess the nosZ gene, suggesting that reduction of N2O to nitrogen (where nitrate rather than oxygen is used as a terminal electron acceptor during respiration) might be an adaptation to oxygen limitation in environments subject to periodic waterlogging.
A Bayesian phylogenetic tree of the nosZ gene of strain A19T and reference strains of the genus Bradyrhizobium is presented in Fig. S5. The placement of strain A19T in a distinct lineage (closest neighbours, B. xenonodulans, B. lablabi and B. zhenyangense) is incongruent with its placement in trees based on core genes (Fig. 1) and whole genome sequences (Fig. S3) (closest neighbours B. oligotrophicum and ‘B. aeschynomenes’), suggesting that the nosZ gene was acquired by horizontal gene transfer from external sources.
B. denitrificans IFAM 1005T, a member of the “photosynthetic clade” and symbiont of the tropical legume, A. indica, was originally isolated from surface lake water in Germany (Hirsch and Müller 1985). Although novel strain A19T was also isolated in a temperate region (from a root-nodule of a tropical A. indica plant), we can only speculate as to its origin. The A. indica plants used in the present work had been raised from surface sterilized seeds and planted in rooting medium that was sterilized making it unlikely that A19T had been initially introduced on seed or in the rooting medium. However, as plants were maintained in a greenhouse without aseptic precautions it is possible that A19T was later accidentally introduced into the rooting medium by watering, fertilization or by aerial contamination from other sources such as soybeans (G. max) or corn (Zea mays) that had been grown in nearby facilities.
Phenotypic analyses
Colonies of strain A19T are circular, cream coloured, raised and with diameters ~ 0.5 mm after growth on YEM agar medium for 7 days at 28 °C. Cells of strain A19T are Gram-stain-negative, rod shaped, and based on examination by electron microscopy, possess at least one flagellum (Fig. 3 and Fig. S6). Growth on YEM agar medium at 28 °C is accompanied by an alkaline reaction (Table S3), which is characteristic of the genus Bradyrhizobium. Strain A19T, like close relative B. oligotrophicum S58T, shows growth at pH 5, but does not grow at pH 10, at 10 °C, or, in the presence of 0.5% NaCl, after 7 days incubation on YEM agar medium. However, strain A19T differed from B. oligotrophicum S58T, in that it did not grow at 37 °C (Table S3).
Strain A19T produced pink-pigmented colonies on modified HM agar medium (Okubo et al. 2013) after 7 days at 28 °C under natural light (14 h light, 10 h dark), typical of photosynthetic reference strains, B. oligotrophicum S58T and Bradyrhizobium sp. BTAi1.
Results for fatty acid profiles of strain A19T and four reference strains are presented in Table S4. Fatty acids C16:0, 18:1ω7c 11-methyl and C18:1 ω6c/C18:1 ω7c (summed feature 8), were detected in A19T and all four reference strains. The dominance of fatty acids C16:0 and C18:1 ω6c/C18:1 ω7c (summed feature 8) in strain A19T is typical of the genus Bradyrhizobium (Tighe et al. 2000).
Table S5 shows the results for assays of carbon source utilization and chemical sensitivity utilizing Biolog™ phenotype microarrays. The data show that strain A19T can be readily differentiated from photosynthetic symbionts of A. indica (B. oligotrophicum S58T, B. denitrificans IFAM 1005T and ‘B. aeschynomenes’ 83002T), as well as from the (non-photosynthetic) genus type strain (B. japonicum USDA6T) based on multiple tests.
Plant tests showed that strain A19T was able to elicit efficient nitrogen fixing nodules on the stems and roots of A. indica plants (Fig. 4) but did not form nodules on ‘Glengarry’ soybeans or Macroptilium atropurpureum ‘siratro’.
Description of Bradyrhizobium ontarionense sp. nov.
Bradyrhizobium ontarionense (on.ta.ri.o.nen′se. N.L. neut. adj. ontarionense, of or belonging to the province of Ontario). Bacterial cells are aerobic, non-spore-forming rods, Gram-stain-negative and possess one or more flagella. Colonies on YEM agar medium are cream colored, raised and circular with diameters ~ 0.5 mm after growth for 7 days at 28 °C. Produces an alkaline reaction on YEM agar medium. Grows at pH 5 but not at pH 10 (optimum ~ pH 7.0). Does not grow at 10 °C or 37 °C (optimal at ~ 28 °C) or in the presence of 0.5% (w/v) NaCl. Produces pink-pigmented colonies on modified HM agar medium after 7 days of light–dark cycles at 28 °C. Dominant fatty acids are C16:0 and C18:1 ω6c/C18:1 ω7c (summed feature 8). The type strain is able to utilize 17 carbon sources including α-d-Glucose, d-Galactose, d-Sorbitol, d-Mannitol, d-Arabitol, d-Gluconic Acid, d-Malic Acid, l-Malic Acid, Tween 40, Propionic Acid, Acetic Acid and Formic Acid. The type strain does not utilize 53 carbon sources including d-Fructose, l-Fucose, myo-Inositol, d-Glucose- 6-PO4, d-Fructose- 6-PO4, d-Aspartic Acid, Gelatin, Pectin, d-Galacturonic Acid, l-Galactonic Acid Lactone, Mucic Acid, l-Lactic Acid, Citric Acid, and γ-Amino-Butryric Acid. The type strain is resistant to 1% Sodium Lactate, Troleandomycin, Rifamycin SV, Minocycline, Lincomycin, Tetrazolium Violet, Tetrazolium Blue, Nalidixic Acid and Aztreonam. Susceptible to Fusidic Acid, Niaproof 4, d-Serine, Guanidine HCl, Vancomycin, Potassium Tellurite, Lithium Chloride, Sodium Butyrate and Sodium Bromate.
Elicits efficient nitrogen fixing root- and stem-nodules on plants of the aquatic legume Aeschynomene indica. Does not elicit nodules G. max (soybeans) or Macroptilium atropurpureum.
The type strain, A19T (= LMG 32638T = HAMBI 3761T) was isolated from a root-nodule of an Aeschynomene indica plant grown in a greenhouse. The size of the genome is 8.44 Mbp and the DNA G+C content is 64.9 mol%. The type strain does not possess nodulation or type III secretion system genes but contains photosynthesis genes, nitrogen-fixation genes and genes encoding a complete denitrifying enzyme system including nitrous oxide reductase.
Data availability
All data required for this study are included in this paper and Supplementary Information. The whole genome shotgun project for Bradyrhizobium ontarionense sp. nov. strain A19T was deposited at DDBJ/ENA/ GenBank as accession number CP088156. Raw PacBio data was deposited in the NCBI Sequence Read Archive as BioProject accession number PRJNA783021. A culture of Bradyrhizobium ontarionense sp. nov. strain A19T was deposited in the Culture Collection of Bacteria (BCCM/LMG), University of Ghent, Belgium as LMG 32638T and in the Microbial Culture Collection (HAMBI), University of Helsinki, Finland as HAMBI 3761T.
Abbreviations
- ANI:
-
Average nucleotide identity
- dDDH:
-
Digital DNA DNA hybridization
- GBDP:
-
Genome blast distance phylogeny
- MLSA:
-
Multiple locus sequence analysis
- TYGS:
-
Type strain genome server
- YEM:
-
Yeast-extract mannitol medium
References
Akiyama H, Hoshino Y, Itakura M, Shimomura Y, Wang Y, Yamamoto A et al (2016) Mitigation of soil N2O emission by inoculation with a mixed culture of indigenous Bradyrhizobium diazoefficiens. Sci Rep 6:32869. https://doi.org/10.1038/srep32869
Ardui S, Ameur A, Vermeesch JR, Hestand MS (2018) Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics. Nucleic Acids Res 46:2159–2168. https://doi.org/10.1093/nar/gky066
Arkin AP, Cottingham RW, Henry CS, Harris NL, Stevens RL, Maslov S et al (2018) KBase: the United States Department of Energy Systems Biology Knowledgebase. Nat Biotechnol 36:566–569. https://doi.org/10.1038/nbt.4163
Avontuur JR, Wilken PM, Palmer M, Coetzee MPA, Stępkowski T, Venter SN, Steenkamp ET (2023) Complex evolutionary history of photosynthesis in Bradyrhizobium. Microb Genomics 9:001105. https://doi.org/10.1099/mgen.0.001105
Bromfield ESP, Tambong JT, Cloutier S, Prévost D, Laguerre G, van Berkum P et al (2010) Ensifer, Phyllobacterium and Rhizobium species occupy nodules of Medicago sativa (alfalfa) and Melilotus alba (sweet clover) grown at a Canadian site without a history of cultivation. Microbiology 156:505–520. https://doi.org/10.1099/mic.0.034058-0
Bromfield ESP, Cloutier S, Nguyen HDT (2019) Description and complete genome sequence of Bradyrhizobium amphicarpaeae sp. nov., harbouring photosystem and nitrogen-fixation genes. Int J Syst Evol Microbiol 69:2841–2848. https://doi.org/10.1099/ijsem.0.003569
Bromfield ESP, Cloutier S, Hynes MF (2023) Ensifer canadensis sp. Nov. strain T173T isolated from Melilotus albus (sweet clover) in Canada possesses recombinant plasmid pT173b harbouring symbiosis and type IV secretion system genes apparently acquired from Ensifer medicae. Front Microbiol 14:1195755. https://doi.org/10.3389/fmicb.2023.1195755
Buck JD (1982) Nonstaining KOH method for determination of gram reactions of marine bacteria. Appl Environ Microbiol 44:992–993. https://doi.org/10.1128/aem.44.4.992-993.1982
Bueno E, Mania D, Frostegard A, Bedmar EJ, Bakken LR, Delgado MJ (2015) Anoxic growth of Ensifer meliloti 1021 by N2O-reduction, a potential mitigation strategy. Front Microbiol 6:537. https://doi.org/10.3389/fmicb.2015.00537
Camuel A, Teulet A, Carcagno M, Haq F, Pacquit V, Gully D et al (2023) Widespread Bradyrhizobium distribution of diverse Type III effectors that trigger legume nodulation in the absence of Nod factor. ISME J 17:1416–1429. https://doi.org/10.1038/s41396-023-01458-1
Chun J, Oren A, Ventosa A, Christensen H, Arahal DR, da Costa MS et al (2018) Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes. Int J Syst Evol Microbiol 68:461–466. https://doi.org/10.1099/ijsem.0.002516
Ciufo S, Kannan S, Sharma S, Badretdin A, Clark K, Turner S et al (2018) Using average nucleotide identity to improve taxonomic assignments in prokaryotic genomes at the NCBI. Int J Syst Evol Microbiol 68:2386–2392. https://doi.org/10.1099/ijsem.0.002809
Cloutier S, Bromfield ESP (2019) Analysis of the complete genome sequence of the widely studied strain Bradyrhizobium betae PL7HG1Treveals the presence of photosynthesis genes and a putative plasmid. Microbiol Resour Announc 8:e01282-e1319. https://doi.org/10.1128/MRA.01282-19
Darriba D, Posada D, Kozlov AM, Stamatakis A, Morel B, Flouri T (2020) Model test-NG: a new and scalable tool for the selection of DNA and protein evolutionary models. Mol Biol Evol 37:291–294. https://doi.org/10.1093/molbev/msz189
de Lajudie PM, Andrews M, Ardley J, Eardly B, Jumas-Bilak E, Kuzmanović N et al (2019) Minimal standards for the description of new genera and species of rhizobia and agrobacteria. Int J Syst Evol Microbiol 69:1852–1863. https://doi.org/10.1099/ijsem.0.003426
Delgado-Baquerizo M, Oliverio AM, Brewer TE, Benavent-González A, Eldridge DJ, Bardgett RD et al (2018) A global atlas of the dominant bacteria found in soil. Science 359:320–325. https://doi.org/10.1126/science.aap9516
Edgar RC (2004) MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformat 5:113. https://doi.org/10.1186/1471-2105-5-113
Giraud E, Moulin L, Vallenet D, Barbe V, Cytryn E, Avarre J-C et al (2007) Legumes symbioses: absence of Nod genes in photosynthetic bradyrhizobia. Science 316:1307–1312. https://doi.org/10.1126/science.1139548
Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol 59:307–321. https://doi.org/10.1093/sysbio/syq010
Hénault C, Barbier E, Hartmann A, Revellin C (2022) New insights into the use of rhizobia to mitigate soil N2O emissions. Agriculture 12:271. https://doi.org/10.3390/agriculture12020271
Hirsch P, Müller M (1985) Blastobacter aggregatus sp. nov., Blastobacter capsulatus sp. nov., and Blastobacter denitrificans sp. nov., new budding bacteria from freshwater habitats. Syst Appl Microbiol 6:281–286. https://doi.org/10.1016/S0723-2020(85)80032-1
Holland BR, Huber KT, Dress A, Moulton V (2002) Delta plots: a tool for analyzing phylogenetic distance data. Mol Biol Evol 19:2051–2059. https://doi.org/10.1093/oxfordjournals.molbev.a004030
Itakura M, Uchida Y, Akiyama H, Hoshino YT, Shimomura Y, Morimoto S et al (2013) Mitigation of nitrous oxide emissions from soils by Bradyrhizobium japonicum inoculation. Nat Clim Change 3:208–212. https://doi.org/10.1038/nclimate1734
Jain C, Rodriguez-R LM, Phillippy AM, Konstantinidis KT, Aluru S (2018) High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nat Commun 9:5114. https://doi.org/10.1038/s41467-018-07641-9
Jolley KA, Maiden MCJ (2010) BIGSdb: scalable analysis of bacterial genome variation at the population level. BMC Bioinform 11:595. https://doi.org/10.1186/1471-2105-11-595
Jolley KA, Bliss CM, Bennett JS, Bratcher HB, Brehony C, Colles FM et al (2012) Ribosomal multilocus sequence typing: universal characterisation of bacteria from domain to strain. Microbiology 158:1005–1015. https://doi.org/10.1099/mic.0.055459-0
Kolmogorov M, Yuan J, Lin Y, Pevzner PA (2019) Assembly of long, error-prone reads using repeat graphs. Nat Biotechnol 37:540–546. https://doi.org/10.1038/s41587-019-0072-8
Lee I, Ouk Kim Y, Park SC, Chun J (2016) OrthoANI: an improved algorithm and software for calculating average nucleotide identity. Int J Syst Evol Microbiol 66:1100–1103. https://doi.org/10.1099/ijsem.0.000760
Lefort V, Desper R, Gascuel O (2015) Fast ME 2.0: a comprehensive, accurate, and fast distance-based phylogeny inference program. Mol Biol Evol 32:2798–2800. https://doi.org/10.1093/molbev/msv150
Lindström K, Mousavi SA (2019) Effectiveness of nitrogen fixation in rhizobia. Microb Biotechnol 13:1751–7915. https://doi.org/10.1111/1751-7915.13517
Meier-Kolthoff JP, Göker M (2019) TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. Nat Commun 10:2182. https://doi.org/10.1038/s41467-019-10210-3
Meier-Kolthoff JP, Auch AF, Klenk HP, Göker M (2013) Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinform 14:60. https://doi.org/10.1186/1471-2105-14-60
Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES science gateway for inference of large phylogenetic trees. In: Proceedings of the gateway computing environments workshop. New Orleans, LA
Minamisawa K (2023) Mitigation of greenhouse gas emission by nitrogen-fixing bacteria. Biosci Biotechnol Biochem 87:7–12. https://doi.org/10.1093/bbb/zbac177
Montzka SA, Dlugokencky EJ, Butler JH (2011) Non-CO2 greenhouse gases and climate change. Nature 476:43–50. https://doi.org/10.1038/nature10322
Ohtsubo Y, Ikeda-Ohtsubo W, Nagata Y, Tsuda M (2008) Genome matcher: a graphical user interface for DNA sequence comparison. BMC Bioinform 9:376. https://doi.org/10.1186/1471-2105-9-376
Okubo T, Fukushima S, Itakura M, Oshima K, Longtonglang A, Teaumroong N et al (2013) Genome analysis suggests that the soil oligotrophic bacterium Agromonas oligotrophica (Bradyrhizobium oligotrophicum) is a nitrogen-fixing symbiont of Aeschynomene indica. Appl Environ Microbiol 79:2542–2551. https://doi.org/10.1128/AEM.00009-13
Ramirez-Bahena MH, Chahboune R, Peix A, Velazquez E (2013) Reclassification of Agromonas oligotrophica into the genus Bradyrhizobium as Bradyrhizobium oligotrophicum comb. nov. Int J Syst Evol Microbiol 63:1013–1016. https://doi.org/10.1099/ijs.0.041897-0
Renier A, Maillet F, Fardoux J, Poinsot V, Giraud E, Nouwen N (2011) Photosynthetic Bradyrhizobium sp. strain ORS285 synthesizes 2-O-methylfucosylated lipochitooligosaccharides for nod gene-dependent interaction with Aeschynomene plants. Mol Plant Microbe Interact 12:1440–1447. https://doi.org/10.1094/MPMI-05-11-0104
Richter M, Rosselló-Móra R (2009) Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci U S A 106:19126–19131. https://doi.org/10.1073/pnas.0906412106
Rivas R, Willems A, Palomo JL, García-Benavides P, Mateos PF, Martínez-Molina E et al (2004) Bradyrhizobium betae sp. nov., isolated from roots of Beta vulgaris affected by tumour-like deformations. Int J Syst Evol Microbiol 54:1271–1275. https://doi.org/10.1099/ijs.0.02971-0
Sameshima-Saito R, Chiba K, Hirayama J, Itakura M, Mitsui H, Eda S, Minamisawa K (2006) Symbiotic Bradyrhizobium japonicum reduces N2O surrounding the soybean root system via nitrous oxide reductase. Appl Environ Microbiol 72:2526–2625. https://doi.org/10.1128/AEM.72.4.2526-2532.2006
Sasser M (1990) Identification of bacteria by gas chromatography of cellular fatty acids. MIDI Technical Note 101. MIDI Inc., Newark, DE
Sun L, Zhang Z, Dong X, Tang Z, Ju B, Zongjun D et al (2022) Bradyrhizobium aeschynomenes sp. nov., a root and stem nodule microsymbiont of Aeschynomene indica. Syst Appl Microbiol 45:126337. https://doi.org/10.1016/j.syapm.2022.126337
Thomson AJ, Giannopoulos G, Pretty J, Baggs EM, Richardson DJ (2012) Biological sources and sinks of nitrous oxide and strategies to mitigate emissions. Philos Trans R Soc 12:B3671157–B3671168. https://doi.org/10.1098/rstb.2011.0415
Tian H, Xu R, Canadell JG, Thompson RL, Winiwarter W, Suntharalingam P et al (2020) A comprehensive quantification of global nitrous oxide sources and sinks. Nature 586:248–256. https://doi.org/10.1038/s41586-020-2780-0
Tighe SW, de Lajudie P, Dipietro K, Lindström K, Nick G, Jarvis BD (2000) Analysis of cellular fatty acids and phenotypic relationships of Agrobacterium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium species using the Sherlock microbial identification system. Int J Syst Evol Microbiol 50:787–801. https://doi.org/10.1099/00207713-50-2-787
Torres MJ, Simon J, Rowley G, Bedmar EJ, Richardson DJ, Gates AJ, Delgado MJ (2016) Chapter Seven—Nitrous oxide metabolism in nitrate-reducing bacteria: physiology and regulatory mechanisms. In: Poole RK (ed) Advances in Microbial Physiology, vol 68. Elsevier, pp 353–432. https://doi.org/10.1016/bs.ampbs.2016.02.007
van Berkum P, Leibold JM, Eardly BD (2006) Proposal for combining Bradyrhizobium spp. (Aeschynomene indica) with Blastobacter denitrificans and to transfer Blastobacter denitrificans (Hirsch and Muller, 1985) to the genus Bradyrhizobium as Bradyrhizobium denitrificans (comb. nov.). Syst Appl Microbiol 29:207215. https://doi.org/10.1099/ijsem.0.004380
Vincent JM (1970) A manual for the practical study of root-nodule bacteria. Blackwell Scientific, Oxford
Wasai-Hara S, Hara S, Morikawa T, Sugawara M, Takami H, Yoneda J et al (2020a) Diversity of Bradyrhizobium in non-leguminous sorghum plants: B. ottawaense isolates unique in genes for N2O reductase and lack of the type VI secretion system. Microbes Environ 35:1–6. https://doi.org/10.1264/jsme2.ME19102
Wasai-Hara S, Minamisawa K, Cloutier S, Bromfield ESP (2020b) Strains of Bradyrhizobium cosmicum sp. nov., isolated from contrasting habitats in Japan and Canada possess photosynthesis gene clusters with the hallmark of genomic islands. Int J Syst Evol Microbiol 70:5063–5074. https://doi.org/10.1099/ijsem.0.004380
Wasai-Hara S, Itakura M, Siqueira AF, Takemoto D, Sugawara M, Mitsui H et al (2023) Bradyrhizobium ottawaense efficiently reduces nitrous oxide through high nosZ gene expression. Sci Rep 13:18862. https://doi.org/10.1038/s41598-023-46019-w
Young JPW, Jorrin B, Moeskjær S, James EK (2023) Rhizobium brockwellii sp. nov., Rhizobium johnstonii sp. nov. and Rhizobium beringeri sp. nov., three genospecies within the Rhizobium leguminosarum species complex. Int J Syst Evol Microbiol 73:005979. https://doi.org/10.1099/ijsem.0.005979
Yu X, Cloutier S, Tambong JT, Bromfield ESP (2014) Bradyrhizobium ottawaense sp. nov., a symbiotic nitrogen fixing bacterium from root nodules of soybeans in Canada. Int J Syst Evol Microbiol 64:3202–3207. https://doi.org/10.1099/ijs.0.065540-0
Acknowledgements
The authors are thankful to Rachelle Gendron, Greenhouse Supervisor, Agriculture and Agri-Food Canada, Ottawa for maintaining Aeschynomene indica plants for seed production and to Keith Hubbard, Microscopy Centre, Agriculture and Agri-Food Canada, Ottawa for preparing electron microscope images.
Funding
Open access funding provided by Agriculture & Agri-Food Canada library. This research was supported by grant J-002272 from Agriculture and Agri-Food Canada.
Author information
Authors and Affiliations
Contributions
ESPB, conceptualization, funding acquisition and draft manuscript writing. SC and ESPB, experimentation, data analyses, writing-review and editing. Both authors read and approved the final manuscript.
Corresponding author
Ethics declarations
Conflict of interest
The authors have no competing interests to declare that are relevant to the content of this article.
Ethical approval
Not applicable.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Bromfield, E.S.P., Cloutier, S. Bradyrhizobium ontarionense sp. nov., a novel bacterial symbiont isolated from Aeschynomene indica (Indian jointvetch), harbours photosynthesis, nitrogen fixation and nitrous oxide (N2O) reductase genes. Antonie van Leeuwenhoek 117, 69 (2024). https://doi.org/10.1007/s10482-024-01940-6
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s10482-024-01940-6