Vol.: (0123456789) 1 3 Antonie van Leeuwenhoek (2024) 117:69 https://doi.org/10.1007/s10482-024-01940-6 ORIGINAL PAPER Bradyrhizobium ontarionense sp. nov., a novel bacterial symbiont isolated from Aeschynomene indica (Indian jointvetch), harbours photosynthesis, nitrogen fixation and nitrous oxide (N2O) reductase genes Eden S. P. Bromfield  · Sylvie Cloutier Received: 22 November 2023 / Accepted: 2 February 2024 © His Majesty the King in Right of Canada as represented by the Minister of Agriculture and Agri-Food 2024 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 Bradyrhizo- bium. 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. oligo- trophicum 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 thresh- old 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 rhizo- bia, 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 pre- sented, a new species named Bradyrhizobium ontari- onense sp. nov. is proposed with strain A19T (= LMG 32638T = HAMBI 3761T) as the type strain. Keywords Aeschynomene indica (Indian jointvetch) · Bradyrhizobium ontarionense sp. nov. · Nitrous oxide reductase gene · Photosynthetic gene cluster · Symbiotic nitrogen fixation 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 Introduction Bacteria belonging to the genus Bradyrhizobium rep- resent one of the most abundant taxa in soils glob- ally and are considered a priority group for research Supplementary Information The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s10482- 024- 01940-6. E. S. P. Bromfield (*) · S. Cloutier  Agriculture and Agri-Food Canada, 960 Carling Ave., Ottawa K1A 0C6, Canada e-mail: eden.bromfield@agr.gc.ca http://crossmark.crossref.org/dialog/?doi=10.1007/s10482-024-01940-6&domain=pdf http://orcid.org/0000-0001-5385-7815 http://orcid.org/0000-0002-3349-4604 https://doi.org/10.1007/s10482-024-01940-6 https://doi.org/10.1007/s10482-024-01940-6 Antonie van Leeuwenhoek (2024) 117:69 1 3 69 Page 2 of 14 Vol:. (1234567890) on furthering the understanding of the contribution of soil microbes to ecosystem functioning (Del- gado-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 photosyn- thesis (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. amphi- carpaeae (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 spe- cies 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 nod- ules 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 modi- fied yeast extract-mannitol (YEM) agar medium hav- ing 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; Bacte- riological agar (Thermo Scientific™ Oxoid™), 18.0. Bacterial cultures were maintained in 20% w/v glyc- erol 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 Sin- gle-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 Antonie van Leeuwenhoek (2024) 117:69 1 3 Page 3 of 14 69 Vol.: (0123456789) genome sequences (where available). Analysis of photosynthesis reaction center (pufLM) genes, nitro- gen 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 sin- gle-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 par- alogous 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 substi- tution models. Phylogenetic analyses using MrBayes (software version 3.2.1) were done employing default priors as described previously (Yu et al. 2014). Maxi- mum-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 digi- tal DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) are routinely employed in bacterial taxonomic studies to facilitate species cir- cumscription (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. genei ous. 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 Micro- Plates (Biolog™, United States) as detailed in the manufacturer’s instructions. Cells of strain A19T were examined using a scan- ning electron microscope (model, Hitachi SU7000 FESEM) and a transmission electron microscope (model, H-7000; Hitachi). For microscopic examina- tion 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 Bro- mfield et al. (2010). Plant tests were carried out with modified Leon- ard jars (Vincent 1970) (two plants per jar with three replicate jars for each inoculation treatment) using nitrogen-free nutrient solution as detailed by Bro- mfield et al. (2010). Seeds of A. indica were vernal- ized 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 https://www.geneious.com Antonie van Leeuwenhoek (2024) 117:69 1 3 69 Page 4 of 14 Vol:. (1234567890) water agar plates (Bacteriological agar (Thermo Sci- entific™ 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 cov- ered 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 posi- tive 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 ‘sira- tro’). 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 nec- essary 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 cover- age 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 tra- ditionally 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 analy- sis of 16S rRNA gene sequences is considered to be useful for verifying the genus level identity of bacte- ria (Young et al. 2023). The phylogenetic tree of 16S rRNA gene sequences (Fig. S1) of strain A19T and 86 Bradyrhizobium spe- cies (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. oligotrophi- cum as the most closely related species. Multiple Locus Sequence Analysis (MLSA) of sin- gle 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. oligotrophi- cum S58T as the closest relative. Fig. S2 also shows that strain A19T is placed in the “photosynthetic clade” (represented by B. oligotrophicum) contain- ing 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. den- itrificans and therefore represents a potential mem- ber 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 bac- terial ribosome protein subunit (rps) gene sequences (Jolley et al. 2012) as well as a phylogenomic analy- sis (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 poten- tial member of the species, B. denitrificans, we used BTAi1 as a proxy for IFAM 1005T in subsequent phy- logenetic 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 line- age within the “photosynthetic clade” represented by B. oligotrophicum; B. oligotrophicum is also the clos- est 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 “photosyn- thetic clade”. The largest values in these comparisons (33.4% (dDDH) and 88.8% (ANI)) are far lower than Antonie van Leeuwenhoek (2024) 117:69 1 3 Page 5 of 14 69 Vol.: (0123456789) Ta bl e 1 G en om e ch ar ac te ris tic s o f B ra dy rh iz ob iu m o nt ar io ne ns e sp . n ov . A 19 T a nd re fe re nc e str ai ns C ha ra ct er - ist ic St ra in B. o nt ar io n- en se sp . n ov A 19 T B. o lig o- tro ph ic um S5 8T ‘B . ae sc hy no m en es ’ 83 00 2T Br ad yr hi zo - bi um sp . B TA i1 Br ad yr hi zo - bi um sp . O R S 27 8 Br ad yr hi zo - bi um sp . O R S 28 5 ‘B . g ua ng - zh ou en se ’ C C BA U 51 67 0T B. a m ph i- ca rp ae ae 39 S1 M B T B. b et ae PL 7H G 1T B. c os m i- cu m 58 S1 T B. ja po ni cu m U SD A 6 T G en om e as se m bl y (n o. c on tig s) C om pl et e (1 ) C om pl et e (1 ) D ra ft (5 8) C om pl et e (2 ) C om pl et e (1 ) C om pl et e (1 ) C om pl et e (2 ) C om pl et e (1 ) C om pl et e (2 ) C om pl et e (1 ) C om pl et e (1 ) G en om e si ze (b p) 84 35 84 5 82 64 16 5 75 22 25 4 84 93 51 3 74 56 58 7 77 97 09 8 81 38 17 7 70 44 51 7 74 19 40 2 73 04 13 6 92 07 38 4 Pl as m id n o. (s iz e, b p) 0 0 na 1 (2 28 82 6) 0 0 1 (9 79 29 1) * 0 1 (2 69 30 7) 0 0 G en es (t ot al ) 74 56 72 76 68 08 77 00 66 24 69 37 76 83 66 04 71 67 69 85 86 52 C D Ss (t ot al ) 73 90 72 12 67 39 76 38 66 72 68 77 76 29 65 47 67 80 67 57 84 38 G + C c on te nt % 64 .9 65 .1 65 64 .8 65 .5 65 .2 63 .4 64 .7 64 .4 64 .0 63 .7 N o. o f r R N A op er on s (5 S, 2 3S , 16 S) 2 2 na 2 2 2 1 2 1 1 2 Ph ot os yn - th et ic g en e cl us te r Ye s Ye s Ye s Ye s Ye s Ye s Ye s Ye s Ye s Ye s N o N od ul at io n (n od AB C ) ge ne s N o N o N o N o N o Ye s Ye s N o N o N o Ye s N itr og en fix at io n (n ifH D K ) ge ne s Ye s Ye s Ye s Ye s Ye s Ye s Ye s Ye s N o Ye s Ye s Ty pe II I Se cr et io n Sy ste m (T 3S S) ge ne s N o N o N o N o N o Ye s Ye s N o N o N o Ye s Antonie van Leeuwenhoek (2024) 117:69 1 3 69 Page 6 of 14 Vol:. (1234567890) 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 clus- ter (PGC) of size about 49  kb (co-ordinates 3,498,729–3,547,553 bp). The PGC contains key pho- tosynthesis genes encoding bacteriochlorophyll (bchI- DOCXYZGPFNBHLM and acsF), reaction centre L, M and H subunits (pufLM and puhA), light-harvest- ing protein alpha and beta subunits (pufBA), carote- noid (crtIBCDEF), bacteriophytochrome (bphP) and photosynthesis repressor (ppsR1 and ppsR2) proteins. A Bayesian phylogenetic tree of concatenated pho- tosynthetic reaction centre, pufLM, genes (Fig.  2A) shows that strain A19T is placed in a cluster together with other symbionts of A. indica; the closest rela- tive 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 sym- bionts (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 bradyrhizo- bia 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. oligotrophi- cum 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 Sys- tem (T3SS) genes (Table  1) indicating that its sym- biotic association with A. indica plants is initiated in a nod-factor and T3SS independent manner simi- lar 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 fol- lowing key nitrogen fixation genes were found in the genome of strain A19T: nifDKEN, nifH, nifA and fix- ABCX (co-ordinates 7,793,281–7,842,331  bp). The phylogenetic tree of concatenated full length nif- HDK gene sequences (Fig. S4) shows that strain Ta bl e 1 (c on tin ue d) C ha ra ct er - ist ic St ra in B. o nt ar io n- en se sp . n ov A 19 T B. o lig o- tro ph ic um S5 8T ‘B . ae sc hy no m en es ’ 83 00 2T Br ad yr hi zo - bi um sp . B TA i1 Br ad yr hi zo - bi um sp . O R S 27 8 Br ad yr hi zo - bi um sp . O R S 28 5 ‘B . g ua ng - zh ou en se ’ C C BA U 51 67 0T B. a m ph i- ca rp ae ae 39 S1 M B T B. b et ae PL 7H G 1T B. c os m i- cu m 58 S1 T B. ja po ni cu m U SD A 6 T N itr ou s ox id e re du ct as e (n os RZ D - FY L) g en es Ye s Ye s Ye s Ye s N o Ye s N o N o Ye s Ye s N o tR N A s 56 54 51 52 52 50 47 50 47 48 56 * Sy m bi os is p la sm id na , d at a no t a va ila bl e Antonie van Leeuwenhoek (2024) 117:69 1 3 Page 7 of 14 69 Vol.: (0123456789) Fig. 1 Bayesian tree (GTR+G+I substitution model) inferred from 50 full-length concatenated ribosome protein subunit (rps) gene sequences of Bradyrhizobium ontarion- ense sp. nov. strain A19T and reference taxa. Align- ment length, 22,500 bp. Black dots designate species possessing photo- synthesis genes. Posterior probabilities ≥ 0.90 are indicated. Bar represents expected substitutions per site Antonie van Leeuwenhoek (2024) 117:69 1 3 69 Page 8 of 14 Vol:. (1234567890) 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 use- ful 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 respira- tory activity of nitrifying and denitrifying microor- ganisms (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 bac- teria (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 Rhizo- bium 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 mitigat- ing N2O emissions from agricultural soils (Wasai- Hara et al. 2023). In the current study we detected key genes encoding enzymes required for the com- plete denitrification of nitrate or nitrite to nitrogen gas in the genome sequence of strain A19T as fol- lows: 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 fre- quency 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 con- trary to earlier reports, a substantial minority (i.e., 21 of 73 Bradyrhizobium species type strains) pos- sess the nosZ gene. It is noteworthy that, with the exception of Bradyrhizobium sp. ORS 278, symbi- onts of the aquatic legume A. indica (B. oligotrophi- cum 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 place- ment of strain A19T in a distinct lineage (closest neighbours, B. xenonodulans, B. lablabi and B. zhen- yangense) is incongruent with its placement in trees based on core genes (Fig.  1) and whole genome sequences (Fig. S3) (closest neighbours B. oligo- trophicum and ‘B. aeschynomenes’), suggesting that Table 2 ANI and dDDH values for pair-wise comparisons of the genome sequence of Bradyrhizobium ontarionense sp. nov A19T (Accession no. CP088156) with members of the “photo- synthetic clade” * Values represent averages of reciprocal comparisons † dDDH values and confidence intervals [C.I.] based on Genome BLAST Distance Phylogeny (GBDP) formula 4 implemented in the Type Strain Genome Server (TYGS) (Meier-Kolthoff and Göker 2019); formula 4 is independent of genome length and is robust against use of incomplete draft genomes Reference strain (sequence accession no.) Fast ANI* dDDH % [C.I.]† A19T A19T B. oligotrophicum S58T (AP012603) 88.8 33.4 [31.0–35.9] ‘B. aeschynomenes’ 83002T (JABFDM01) 87.5 30.7 [28.3–33.2] Bradyrhizobium sp. BTAi1 (CP00494-CP000495) 86.8 30.2 [27.8–32.7] Bradyrhizobium sp. ORS 285 (LT859959) 87.1 29.8 [27.4–32.3] Bradyrhizobium sp. ORS 278 (CU234118) 86.9 29.4 [27.0–31.9] Antonie van Leeuwenhoek (2024) 117:69 1 3 Page 9 of 14 69 Vol.: (0123456789) the nosZ gene was acquired by horizontal gene trans- fer from external sources. B. denitrificans IFAM 1005T, a member of the “photosynthetic clade” and symbiont of the tropi- cal 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 tropi- cal 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 mak- ing 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 pre- cautions it is possible that A19T was later accidentally introduced into the rooting medium by watering, ferti- lization 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 utiliz- ing Biolog™ phenotype microarrays. The data show that strain A19T can be readily differentiated from photosynthetic symbionts of A. indica (B. oligo- trophicum 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 prov- ince 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-pig- mented 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 car- bon 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-Fruc- tose, l-Fucose, myo-Inositol, d-Glucose- 6-PO4, d-Fructose- 6-PO4, d-Aspartic Acid, Gelatin, Pec- tin, d-Galacturonic Acid, l-Galactonic Acid Lac- tone, 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, Tetra- zolium Blue, Nalidixic Acid and Aztreonam. Suscep- tible to Fusidic Acid, Niaproof 4, d-Serine, Guanidine Antonie van Leeuwenhoek (2024) 117:69 1 3 69 Page 10 of 14 Vol:. (1234567890) ‘B. aeschynomenes’ 83012 ‘B. aeschynomenes’ 83013 ‘B. aeschynomenes’ 83002T Bradyrhzobium sp. ORS278 Bradyrhizobium sp. ORS285 Bradyrhizobium sp. ORS375 Bradyrhizobium sp. BTAi1 B. denitrificans K2 B. denitrificans SZCCT0094 B. ontarionense sp. nov. A19T B. oligotrophicum S58T Bradyrhizobium sp. SSBR45G ‘B. guangzhouense’ CCBAU 51670T ‘B. guangzhouense’ CCBAU53424 ‘B. guangzhouense’ CCBAU53426 B. cosmicum 58S1T B. cosmicum S23321 B. betae PL7HG1T Bradyrhizobium sp. PSBB068 B. amphicarpaeae 39S1MBT Rhodopseudomonas palustris HAa2 1.00 1.00 0.991.00 1.00 1.00 1.00 0.96 1.00 0.99 0.99 0.05 I D O I B C D E F C X Y Z L M BA I D O I B C D E F C X Y Z L M BA I D O I B C D E F C X Y Z L M BA P R2 P G R1 F N B H L A BC EM P R2 P G R1 F N B H L A B C EM PR2PGR1 F N B H L A B C EM A B B. ontarionense sp. nov. A19T 3,499,937-3,547,553 (complement) B. oligotrophicum S58T 6,666,590-6,714,470 (complement) B. amphicarpaeae 39S1MBT 5,082,884-5,132,814 (complement) Bacteriochlorophyll synthesis (bch) Aerobic cyclase gene (acsF) Carotenoid synthesis (crt) Hypothetical proteins Light-harvesting protein (puf) Bacteriophytochrome (bph) Photosynthesis repressor protein (pps) Core light-harvesting subunits (puh) 30 40 50 60 70 80 90 100 Identity % (amino acid sequences) PGC1 PGC2 B. ontarionense sp. nov. A19T 3,499,937-3,547,553 (complement) B. oligotrophicum S58T 6,666,590-6,714,470 (complement) B. amphicarpaeae 39S1MBT 5,082,884-5,132,814 (complement) (PGC1) Other (PGC1) (PGC2) (PGC1) (PGC1) (PGC2) Antonie van Leeuwenhoek (2024) 117:69 1 3 Page 11 of 14 69 Vol.: (0123456789) HCl, Vancomycin, Potassium Tellurite, Lithium Chloride, Sodium Butyrate and Sodium Bromate. Elicits efficient nitrogen fixing root- and stem-nod- ules 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. 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 elec- tron microscope images. Author contributions ESPB,  conceptualization, funding acquisition and draft manuscript writing. SC and ESPB, exper- imentation, data analyses, writing-review and editing. Both authors read and approved the final manuscript. 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. 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 depos- ited in the NCBI Sequence Read Archive as BioProject acces- sion number PRJNA783021. A culture of Bradyrhizobium Fig. 2 A Bayesian phylogenetic tree (GTR+G+I substitu- tion model) of concatenated photosynthetic reaction centre, pufLM, genes (1739  bp) for Bradyrhizobium ontarionense sp. nov. strain A19T and reference taxa showing division of Bradyrhizobium strains into two clades corresponding to pho- tosynthetic gene cluster type 1 (PGC1) and type 2 (PGC2) as defined by Avontuur et al. (2023). B Comparative arrangement of the photosynthesis gene cluster of Bradyrhizobium ontari- onense sp. nov. strain A19T (PGC1) relative to Bradyrhizo- bium. oligotrophicum (PGC1) and Bradyrhizobium amphicar- paeae (PGC2). Color coding represents % identity based on amino acid sequences of PGC genes calculated by Genom- eMatcher software (Ohtsubo et al. 2008) ◂ Fig. 3 Scanning electron microscope image showing morpho- logical features of a cell of Bradyrhizobium ontarionense sp. nov A19T. Scale bar (μm) is indicated Fig. 4 Effective nitrogen-fixing root (A) and stem (B and C) nodules on Aeschynomene indica plants inoculated with Bradyrhizobium ontarionense sp. nov. A19T. Panel (C) shows the cut surface of an effective stem nodule exhibiting red colored pigment characteristic of leghaemoglobin that is required for nitrogen fixation Antonie van Leeuwenhoek (2024) 117:69 1 3 69 Page 12 of 14 Vol:. (1234567890) 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 Col- lection (HAMBI), University of Helsinki, Finland as HAMBI 3761T. 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. Open Access This article is licensed under a Creative Com- mons 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 Crea- tive 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:// creat iveco mmons. org/ licen ses/ by/4. 0/. 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Int J Syst Evol Microbiol 64:3202–3207. https:// doi. org/ 10. 1099/ ijs.0. 065540-0 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. https://doi.org/10.1098/rstb.2011.0415 https://doi.org/10.1098/rstb.2011.0415 https://doi.org/10.1038/s41586-020-2780-0 https://doi.org/10.1099/00207713-50-2-787 https://doi.org/10.1099/00207713-50-2-787 https://doi.org/10.1016/bs.ampbs.2016.02.007 https://doi.org/10.1099/ijsem.0.004380 https://doi.org/10.1099/ijsem.0.004380 https://doi.org/10.1264/jsme2.ME19102 https://doi.org/10.1264/jsme2.ME19102 https://doi.org/10.1099/ijsem.0.004380 https://doi.org/10.1038/s41598-023-46019-w https://doi.org/10.1038/s41598-023-46019-w https://doi.org/10.1099/ijsem.0.005979 https://doi.org/10.1099/ijsem.0.005979 https://doi.org/10.1099/ijs.0.065540-0 https://doi.org/10.1099/ijs.0.065540-0 Bradyrhizobium ontarionense sp. nov., a novel bacterial symbiont isolated from Aeschynomene indica (Indian jointvetch), harbours photosynthesis, nitrogen fixation and nitrous oxide (N2O) reductase genes Abstract Introduction Materials and methods Bacterial strains Genomic DNA sequencing and phylogenetic analysis Genomic analyses Phenotypic characterisation Results and discussion Genomic and phylogenetic characterisation Phenotypic analyses Description of Bradyrhizobium ontarionense sp. nov. Acknowledgements References