Genome Sequence of Rhizobium jaguaris CCGE525^T, a Strain Isolated from Calliandra grandiflora Nodules from a Rain Forest in Mexico

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Rhizobium jaguaris CCGE525^T was isolated from nodules of the medicinal legume Calliandra grandiflora growing in a rain forest in Chiapas, Mexico, and was described as related to Rhizobium tropici group A (1). R. tropici group A was defined by 16S rRNA gene sequences and distinctive phenotypic characteristics (2). We report the genome sequence of strain CCGE525, the type strain of Rhizobium jaguaris.

A single colony from a freeze-dried culture sample of R. jaguaris CCGE525^T was incubated on peptone yeast (PY) medium (5 g/liter peptone, 3 g/liter yeast extract, and 0.6 g/liter CaCl₂) for 3 days at 30°C. DNA was extracted from 3 ml of culture using a kit for cells and tissues (Roche Applied Science, Germany). A SMRTbell library of 15- to 20-kb insert size was constructed using standard protocols. The library was sequenced on a PacBio RS II sequencer (3) using P6-C4 chemistry, which yielded 3.4 Gb of data. Reads were filtered and assembled de novo using Canu v.1.5 (4). Annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline (https://www.ncbi.nlm.nih.gov/genome/annotation_prok/) (5). Amino acid sequences served as input to PhyloPhlAn (6) to predict evolutionary relationships. The progressive Mauve tool was used for genome alignments (7). DNA-DNA hybridization (DDH) values were computed using the Genome-to-Genome Distance Calculator v.2.1 (8). Average nucleotide identity (ANI) values were calculated as previously proposed (9) using the ANI calculator from the Konstantinidis Lab (http://enve-omics.ce.gatech.edu/ani/) (10). Default parameters were used for all programs.

The genome of R. jaguaris CCGE525^T (8,025,568 bp, 58.95% G+C content, and ∼278-fold coverage) consisted of a chromosome (4,575,315 bp), a chromid (2,584,926 bp), a symbiotic plasmid required for establishing interactions with legumes (550,563 bp), and an additional plasmid (314,764 bp). The genome coded for 8,400 predicted genes.

The R. tropici group A affiliation of R. jaguaris CCGE525^T was supported by its position in a genome tree (Fig. 1A). This phylogenomic approach increased resolution and confirmed the placement of R. jaguaris CCGE525^T as an isolated branch in the vicinity of Rhizobium leucaenae USDA 9039^T.

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FIG 1

(A) Phylogenomic analysis showing the evolutionary relationships between sequenced Rhizobium strains belonging to the R. tropici type A and type B groups. The phylogeny is based on 373 marker proteins. Branch labels correspond to Shimodaira-Hasegawa-like support values. (B) Genome alignment of symbiotic plasmid sequences of the type strains R. tropici CIAT 899, R. leucaenae USDA 9039, and R. jaguaris CCGE525. Conserved regions are shown in colored blocks.

R. jaguaris CCGE525^T is classified within the symbiovar calliandrae and has the capacity to form nitrogen-fixing nodules with common bean (1). A multiple sequence alignment revealed that the symbiotic plasmid of R. jaguaris CCGE525^T was less conserved and presented rearrangements compared to the similar symbiotic plasmids of Rhizobium tropici CIAT 899^T and R. leucaenae USDA 9039^T (Fig. 1B).

Sequence comparisons between the symbiotic plasmid of R. jaguaris CCGE525^T and the counterparts of R. tropici CIAT 899^T and R. leucaenae USDA 9039^T revealed ANI values of 85.40% and 85.48%, respectively. DDH estimates were 29.00% and 29.20% between the corresponding symbiotic plasmids. Thus, the symbiovar calliandrae is further validated.

Full-genome comparisons of R. jaguaris CCGE525^T revealed DDH estimates of 33.90% and 35.00% against R. leucaenae USDA 9039^T and Rhizobium sp. strain NXC24, respectively. ANI values of 87.07% and 87.50% were obtained when performing the same comparisons. These DDH and ANI values are below the thresholds for species boundaries of 70% and 95 to 96%, respectively (8, 9, 11–13). Thus, genome-based metrics allowed an accurate taxonomic circumscription of Rhizobium jaguaris.