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

Abstract

A bacterial strain, designated T173T, was previously isolated from a root-nodule of a Melilotus albus plant growing in Canada and identified as a novel Ensifer lineage that shared a clade with the non-symbiotic species, Ensifer adhaerens. Strain T173T was also previously found to harbour a symbiosis plasmid and to elicit root-nodules on Medicago and Melilotus species but not fix nitrogen. Here we present data for the genomic and taxonomic description of strain T173T. Phylogenetic analyses including the analysis of whole genome sequences and multiple locus sequence analysis (MLSA) of 53 concatenated ribosome protein subunit (rps) gene sequences confirmed placement of strain T173T in a highly supported lineage distinct from named Ensifer species with E. morelensis Lc04T as the closest relative. The highest digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) values of genome sequences of strain T173T compared with closest relatives (35.7 and 87.9%, respectively) are well below the respective threshold values of 70% and 95–96% for bacterial species circumscription. The genome of strain T173T has a size of 8,094,229 bp with a DNA G + C content of 61.0 mol%. Six replicons were detected: a chromosome (4,051,102 bp) and five plasmids harbouring plasmid replication and segregation (repABC) genes. These plasmids were also found to possess five apparent conjugation systems based on analysis of TraA (relaxase), TrbE/VirB4 (part of the Type IV secretion system (T4SS)) and TraG/VirD4 (coupling protein). Ribosomal RNA operons encoding 16S, 23S, and 5S rRNAs that are usually restricted to bacterial chromosomes were detected on plasmids pT173d and pT173e (946,878 and 1,913,930 bp, respectively) as well as on the chromosome of strain T173T. Moreover, plasmid pT173b (204,278 bp) was found to harbour T4SS and symbiosis genes, including nodulation (nod, noe, nol) and nitrogen fixation (nif, fix) genes that were apparently acquired from E. medicae by horizontal transfer. Data for morphological, physiological and symbiotic characteristics complement the sequence-based characterization of strain T173T. The data presented support the description of a new species for which the name Ensifer canadensis sp. nov. is proposed with strain T173T (= LMG 32374T = HAMBI 3766T) as the species type strain.

Genome Analysis and Potential Ecological Functions of Members of the Genus Ensifer from Subsurface Environments and Description of Ensifer oleiphilus sp. nov.

“…On the phylogenetic tree ( Figure 2 ), the 16S rRNA gene sequences of the strains HO-A22 T and SHC 2-14 formed a branch with that of Ensifer morelensis Lc04 T [ 4 ]. At the time of writing this article, a new species ‘ Ensifer canadensis ’ has been described, also belonging to the E. morelensis Lc04 T branch [ 6 ]. The tree had low bootstrap values, which indicated the need for phylogenetic analysis.…”

“…It is important to highlight that the genome of strain SHC 2-14 is characterized by the absence of nifH , nifX , and nifE genes, which are associated with nitrogen fixation. These specific genes were previously identified in the genome of ‘ E. canadensis ’ T173 T and appear to have been acquired through horizontal transfer from Sinorhizobium medicae [ 6 ]. The strain HO-A22 T lacks these genes, so the terminal product of its denitrification was nitrous oxide.…”

“…Recent data revealed that despite the high phylogenetic similarity of all detected strains (cpAAI > 86%), some species from the clade differ significantly from the rest in their biology and ecology [ 5 ]. This fact has made it possible to separate these genera among themselves, to assign 17 validly described bacterial species to the genus Sinorhizobium and the species E. adhaerens , E. morelensis , E. sesbaniae , and ‘ E. canadensis ’ to the genus Ensifer [ 5 , 6 ]. Members of the genus Ensifer , originally described by Casida [ 7 ], are budding bacteria, able to grow in LB medium and to hydrolyze starch, with an optimal temperature at 27–28 °C; they are generally nonsymbiotic (except for symbiosis of E. sesbaniae with legumes and that of ‘ E. canadensis ’ with roots of Melilotus albus ), slightly acidophilic, and resistant to ampicillin and erythromycin [ 5 , 6 ].…”

Bradyrhizobium ontarionense sp. nov., a novel bacterial symbiont isolated from Aeschynomene indica (Indian jointvetch), harbours photosynthesis, nitrogen fixation and nitrous oxide (N2O) reductase genes

“…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 ).…”

“…Cells of strain A19 T 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 A19 T was grown for 96 h in YEM broth at 28 °C as outlined previously (Bromfield et al 2023 ).…”