Search for Ancestral Features in Genomes of Rhizobium leguminosarum bv. viciae Strains Isolated from the Relict Legume Vavilovia formosa

Vavilovia formosa is a relict leguminous plant growing in hard-to-reach habitats in the rocky highlands of the Caucasus and Middle East, and it is considered as the putative closest living relative of the last common ancestor (LCA) of the Fabeae tribe. Symbionts of Vavilovia belonging to Rhizobium leguminosarum bv. viciae compose a discrete group that differs from the other strains, especially in the nucleotide sequences of the symbiotically specialised (sym) genes. Comparison of the genomes of Vavilovia strains with the reference group composed of R. leguminosarum bv. viciae strains isolated from Pisum and Vicia demonstrated that the vavilovia strains have a set of genomic features, probably indicating the important stages of microevolution of the symbiotic system. Specifically, symbionts of Vavilovia (considered as an ancestral group) demonstrated a scattered arrangement of sym genes (>90 kb cluster on pSym), with the location of nodT gene outside of the other nod operons, the presence of nodX and fixW, and the absence of chromosomal fixNOPQ copies. In contrast, the reference (derived) group harboured sym genes as a compact cluster (<60 kb) on a single pSym, lacking nodX and fixW, with nodT between nodN and nodO, and possessing chromosomal fixNOPQ copies. The TOM strain, obtained from nodules of the primitive “Afghan” peas, occupied an intermediate position because it has the chromosomal fixNOPQ copy, while the other features, the most important of which is presence of nodX and fixW, were similar to the Vavilovia strains. We suggest that genome evolution from the ancestral to the derived R. leguminosarum bv. viciae groups follows the “gain-and-loss of sym genes” and the “compaction of sym cluster” strategies, which are common for the macro-evolutionary and micro-evolutionary processes. The revealed genomic features are in concordance with a relict status of the vavilovia strains, indicating that V. formosa coexists with ancestral microsymbionts, which are presumably close to the LCA of R. leguminosarum bv. viciae

Phyllobacterium zundukense sp. nov., a novel species of rhizobia isolated from root nodules of the legume species Oxytropis triphylla (Pall.) Pers.

Gram-negative strains Tri-36, Tri-38, Tri-48T and Tri-53 were isolated from root nodules of the relict legume Oxytropis triphylla (Pall.) Pers. originating from Zunduk Cape (Baikal Lake region, Russia). 16S rRNA gene sequencing showed that the novel isolates were phylogenetically closest to the type strains Phyllobacterium sophorae LMG 27899(T), Phyllobacterium brassicacearum LMG 22836(T), Phyllobacterium endophyticum LMG 26470(T) and Phyllobacterium bourgognense LMG 22837(T) while similarity levels between the isolates and the most closely related strain P. endophyticum LMG 26470(T) were 98.899.5 %. The recA and glnII genes of the isolates showed highest sequence similarities with P. sophorae LMG 27899(T) (95.4 and 89.5 %, respectively) and P. brassicacearum LMG 22836(T) (91.4 and 85.1 %, respectively). Comparative analysis of phenotypic properties between the novel isolates and the closest reference strains P. sophorae LMG 27899(T), P. brassicacearum LMG 22836(T) and P. endophyticum LMG 26470(T) was performed using a microassay system. Average nucleotide identities between the whole genome sequences of the isolates Tri-38 and Tri-48(T) and P. sophorae LMG 27899(T), P. brassicacearum LMG 22836(T) and P. endophyticum LMG 26470(T) ranged from 79.23% for P. endophyticum LMG 26470(T) to 85.74% for P. sophorae LMG 27899(T). The common nodABC genes required for legume nodulation were absent from strains Tri-38 and Tri-48(T), although some other symbiotic nod and fix genes were detected. On the basis of genotypic and phenotypic analysis, a novel species, Phyllobacterium zundukense sp. nov. (type strain Tri-48(T) = LMG 30371(T) = RCAM 03910(T)), is proposed

Microvirga ossetica sp. nov., a species of rhizobia isolated from root nodules of the legume species Vicia alpestris Steven

Gram-stain-negative strains V5/3M(T), V5/5K, V5/5M and V5/13 were isolated from root nodules of Vicia alpestris plants growing in the North Ossetia region (Caucasus). Sequencing of the partial 16S rRNA gene (rrs) and four housekeeping genes (dnaK, gyrB, recA and rpoB) showed that the isolates from V. alpestris were most closely related to the species Microvirga zambiensis (order Rhizobiales, family Methylobacteriaceae) which was described for the single isolate from root nodule of Listia angolensis growing in Zambia. Sequence similarities between the Microvirga-related isolates and M. zambiensis WSM3693(T) ranged from 98.5 to 98.7% for rrs and from 79.7 to 95.8% for housekeeping genes. Cellular fatty acids of the isolates V5/3M(T), V5/5K, V5/5M and V5/13 included important amounts of C-18 : 1 omega 7c (54.0-67.2 %), C-16 : 0 (6.0-7.8 %), C-19 : 0 cyclo omega 8c (3.1-10.2 %), summed feature 2 (comprising one or more of iso-C-16 : 1 I, C-14 : 0 3-OH and unknown ECL 10.938, 5.8-22.5 %) and summed feature 3 (comprising C-16 : 1 omega 7c and/or iso-C(15 : 0)2-OH, 2.9-4.0 %). DNA-DNA hybridization between the isolate V5/3M(T) and M. zambiensis WSM3693(T) revealed DNA-DNA relatedness of 35.3 %. Analysis of morphological and physiological features of the novel isolates demonstrated their unique phenotypic profile in comparison with reference strains from closely related species of the genus Microvirga. On the basis of genotypic and phenotypic analysis, a novel species named Microvirga ossetica sp. nov. is proposed. The type strain is V5/3M(T) (= LMG 29787(T) = RCAM 02728(T)). Three additional strains of the species are V5/5K, V5/5M and V5/13

Bosea caraganae sp. nov. a new species of slow-growing bacteria isolated from root nodules of the relict species Caragana jubata (Pall.) Poir. originating from Mongolia

Two Gram-stain-negative strains, RCAM04680(T) and RCAM04685, were isolated from root nodules of the relict legume Caragana jubata (Pall.) Poir. originating from the south-western shore of Lake Khuvsgul (Mongolia). The 16S rRNA gene (rrs) sequencing data showed that these novel isolates belong to the genus Bosea and are phylogenetically closest to the type strains Bosea lathyri LMG 26379(T), Bosea vaviloviae LMG 28367(T), Bosea massiliensis LMG 26221(T) and Bosea lupini LMG 26383(T) (the rrs-similarity levels were 98.7-98.8 %). The recA gene of strain RCAM04680(T) showed the highest sequence similarity to the type strain B. lupini LMG 26383(T) (95.4 %), while its atpD gene was closest to that of B. lathyri LMG 26379(T) (94.4 %). The ITS, dnaK and gyrB sequences of this isolate were most similar to the B. vaviloviae LMG 28367(T) (86.8% for ITS, 90.4% for the other genes). The most abundant fatty acid was C-18:1 omega 7c (40.8 %). The whole genomes of strains RCAM04680(T) and RCAM04685 were identical (100% average nucleotide identity). The highest average nucleotide identity value (82.8 %) was found between the genome of strain RCAM04680(T) and B. vaviloviae LMG 28367(T). The common nodABC genes required for legume nodulation were absent in both strains; however, some other symbiotic nol, nod, nif and fix genes were detected. Based on the genetic study, as well as analyses of the whole-cell fatty acid compositions and phenotypic properties, a new species, Bosea caraganae sp. nov. (type strain RCAM04680(T) (=LMG 31125(T)), is proposed

Rhizobia Isolated from the Relict Legume Vavilovia formosa Represent a Genetically Specific Group within Rhizobium leguminosarum biovar viciae

Twenty-two rhizobia strains isolated from three distinct populations (North Ossetia, Dagestan, and Armenia) of a relict legume Vavilovia formosa were analysed to determine their position within Rhizobium leguminosarum biovar viciae (Rlv). These bacteria are described as symbionts of four plant genera Pisum, Vicia, Lathyrus, and Lens from the Fabeae tribe, of which Vavilovia is considered to be closest to its last common ancestor (LCA). In contrast to biovar viciae, bacteria from Rhizobium leguminosarum biovar trifolii (Rlt) inoculate plants from the Trifolieae tribe. Comparison of house-keeping (hkg: 16S rRNA, glnII, gltA, and dnaK) and symbiotic (sym: nodA, nodC, nodD, and nifH) genes of the symbionts of V. formosa with those of other Rlv and Rlt strains reveals a significant group separation, which was most pronounced for sym genes. A remarkable feature of the strains isolated from V. formosa was the presence of the nodX gene, which was commonly found in Rlv strains isolated from Afghanistan pea genotypes. Tube testing of different strains on nine plant species, including all genera from the Fabeae tribe, demonstrated that the strains from V. formosa nodulated the same cross inoculation group as the other Rlv strains. Comparison of nucleotide similarity in sym genes suggested that their diversification within sym-biotypes of Rlv was elicited by host plants. Contrariwise, that of hkg genes could be caused by either local adaptation to soil niches or by genetic drift. Long-term ecological isolation, genetic separation, and the ancestral position of V. formosa suggested that symbionts of V. formosa could be responsible for preserving ancestral genotypes of the Rlv biovar