Draft genome sequence of type strain HBR26T and description of Rhizobium aethiopicum sp. nov

Abstract

Rhizobium aethiopicum sp. nov. is a newly proposed species within the genus Rhizobium. This species includes six rhizobial strains; which were isolated from root nodules of the legume plant Phaseolus vulgaris growing in soils of Ethiopia. The species fixes nitrogen effectively in symbiosis with the host plant P. vulgaris, and is composed of aerobic, Gram-negative staining, rod-shaped bacteria. The genome of type strain HBR26^T of R. aethiopicum sp. nov. was one of the rhizobial genomes sequenced as a part of the DOE JGI 2014 Genomic Encyclopedia project designed for soil and plant-associated and newly described type strains. The genome sequence is arranged in 62 scaffolds and consists of 6,557,588 bp length, with a 61% G + C content and 6221 protein-coding and 86 RNAs genes. The genome of HBR26^T contains repABC genes (plasmid replication genes) homologous to the genes found in five different Rhizobium etli CFN42^T plasmids, suggesting that HBR26^T may have five additional replicons other than the chromosome. In the genome of HBR26^T, the nodulation genes nodB, nodC, nodS, nodI, nodJ and nodD are located in the same module, and organized in a similar way as nod genes found in the genome of other known common bean-nodulating rhizobial species. nodA gene is found in a different scaffold, but it is also very similar to nodA genes of other bean-nodulating rhizobial strains. Though HBR26^T is distinct on the phylogenetic tree and based on ANI analysis (the highest value 90.2% ANI with CFN42^T) from other bean-nodulating species, these nod genes and most nitrogen-fixing genes found in the genome of HBR26^T share high identity with the corresponding genes of known bean-nodulating rhizobial species (96-100% identity). This suggests that symbiotic genes might be shared between bean-nodulating rhizobia through horizontal gene transfer. R. aethiopicum sp. nov. was grouped into the genus Rhizobium but was distinct from all recognized species of that genus by phylogenetic analyses of combined sequences of the housekeeping genes recA and glnII. The closest reference type strains for HBR26^T were R. etli CFN42^T (94% similarity of the combined recA and glnII sequences) and Rhizobium bangladeshense BLR175^T (93%). Genomic ANI calculation based on protein-coding genes also revealed that the closest reference strains were R. bangladeshense BLR175^T and R. etli CFN42^T with ANI values 91.8 and 90.2%, respectively. Nevertheless, the ANI values between HBR26^T and BLR175^T or CFN42^T are far lower than the cutoff value of ANI (> = 96%) between strains in the same species, confirming that HBR26^T belongs to a novel species. Thus, on the basis of phylogenetic, comparative genomic analyses and ANI results, we formally propose the creation of R. aethiopicum sp. nov. with strain HBR26^T (=HAMBI 3550^T=LMG 29711^T) as the type strain. The genome assembly and annotation data is deposited in the DOE JGI portal and also available at European Nucleotide Archive under accession numbers FMAJ01000001-FMAJ01000062.

Keywords: Average Nucleotide Identity; Common bean; Ethiopia; Genome; Rhizobium aethiopicum; Symbiotic.

Fig. 1

Neighbor-Joining phylogenetic tree reconstructed based partial 16S rRNA gene sequences (801 bp), showing the relationships between Rhizobium aethiopicum sp. nov (bold and highlighted) and recognized species of the genus Rhizobium. The tree was computed using the Kimura 2-parameter model using MEGA version 7. The rate variation among sites was modeled with a gamma distribution (shape parameter = 4). Bootstrap values (1000 replicates) are shown at the branching points. Reference type strains are indicated with superscript ‘T’. Bar, % estimated substitutions. GenBank accession numbers of the sequences are indicated in parentheses next to strains codes. The accession numbers of whole genome sequenced strains are indicated with bold*. Abbreviations: B, Bradyrhizobium; R, Rhizobium; N, Neorhizobium; sp., species

Fig. 2

Maximum Likelihood phylogenetic tree reconstructed based on recA-glnII concatenated nucleotide sequences, showing the relationships between Rhizobium aethiopicum sp. nov. (in bold) and recognized species of the genus Rhizobium. The tree was constructed by using Tamura-Nei model using MEGA version 7. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.3397). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 32.0253% sites). Bootstrap values (100 replicates) are indicated at the branching points. Reference type strains are indicated with superscript ‘T’. Bar, % estimated substitutions. GenBank accession numbers of the sequences (recA, glnII in order) are listed in parentheses next to strains codes. The accession numbers of whole genome sequenced strains are indicated with bold*. Abbreviations: B, Bradyrhizobium; R, Rhizobium; sp., species

Fig. 3

Gram stain of Rhizobium aethiopicum sp. nov. strain HBR26^T

Fig. 4

Venn diagram plotted by OrthoVenn program shows shared orthologous protein clusters among the genomes of bean-nodulating rhizobial strains (in the center): Rhizobium etli CFN42^T, Rhizobium phaseoli Ch24-10, Rhizobium phaseoli CIAT652, Rhizobium leguminosarum CCGM1 and Rhizobium aethiopicum type strain HBR26^T. The number of protein clusters comprising multiple protein families is indicated for each genome and also the number of singletons i.e., protein with no orthologous of each strain are shown in parenthesis. The total number of protein sequences of each genome are indicated in parentheses next to strains codes

Fig. 5

Mauve alignment comparing the genome of Rhizobium aethiopicum type strain HBR26^T with the genome of Rhizobium etli CFN42^T, Rhizobium phaseoli CIAT652 and Rhizobium phaseoli Ch24-10. The module of nodDIJSCB genes are indicated by the arrows