Cell Plasticity and Genomic Structure of a Novel Filterable Rhizobiales Bacterium that Belongs to a Widely Distributed Lineage

Abstract

Rhizobiales bacterium strain IZ6 is a novel filterable bacterium that was isolated from a suspension filtrate (<0.22 µm) of soil collected in Shimane Prefecture, western Japan. Additional closely related isolates were recovered from filterable fractions of terrestrial environmental samples collected from other places in Japan; the Gobi Desert, north-central China; and Svalbard, Arctic Norway. These findings indicate a wide distribution of this lineage. This study reports the cell variation and genomic structure of IZ6. When cultured at lower temperatures (4 °C and 15 °C), this strain contained ultra-small cells and cell-like particles in the filtrate. PacBio sequencing revealed that this chromosome (3,114,641 bp) contained 3150 protein-coding, 51 tRNA, and three rRNA genes. IZ6 showed low 16S rRNA gene sequence identity (<97%) and low average nucleotide identity (<76%) with its closest known relative, Flaviflagellibacter deserti. Unlike the methylotrophic bacteria and nitrogen-fixing bacteria in related genera, there were no genes that encoded enzymes for one-carbon-compound utilization and nitrogen fixation in the IZ6 genome; the genes related to nitrate and nitrite reductase are retained and those related to the cell membrane function tend to be slightly enriched in the genome. This genomic information helps elucidate the eco-physiological function of a phenotypically heterogeneous and diverse Rhizobiales group.

Keywords: bacteria; filterable bacteria; genome; nitrogen cycling; novel lineage; phylogeny.

Figure 1

Genomic map of the Rhizobiales bacterium IZ6 chromosome. From outside to center: genes, specifically protein-coding sequences, on the forward strand are colored by clusters of orthologous group (COG) functional categories; genes on the reverse strand; rRNA and tRNA; guanine−cytosine (GC) skew metric (mean GC-skew value was used as a baseline, with higher-than-average values in green and lower-than-average values in red); and GC ratio metric (mean GC ratio was used as a baseline, with higher-than-average values in blue and lower-than-average values in yellow).

Figure 2

Phylogenetic tree based on near full-length 16S RNA gene sequences of IZ6 and its close relatives in the order Rhizobiales. The evolutionary relationship was inferred using the neighbor-joining method with the Kimura 2-parameter model. There was a total of 1128 positions in the final dataset. Members of the Terasakiella genus (Methylocystaceae Clade IV) were used as an outgroup. Five genera (Bartonella, Brucella, Phyllobacterium, Mabikibacter, and Notoacmeibacter) that are relatively distantly related to IZ6 are not described here. Accession numbers of nucleotide sequences registered in the DDBJ/ENA/NCBI databases are shown in parentheses. For the novel cluster presented in this study, the isolation source and country are shown in square brackets. Bootstrap values >50% based on 1000 replicates are shown at the nodes. Scale bar, 0.020 nucleotide substitutions per site.

Figure 3

(A) Heat map and unweighted pair group method with arithmetic mean (UPGMA) dendrogram based on gene content (presence/absence) of all protein-coding sequences (CDSs) of strain IZ6 and four related strains (Flaviflagellibacter deserti CPCC 101076, Hansschlegelia zhihuaiae S113^T, Methylosinus trichosporium OB3b^T, and Pleomorphomonas diazotrophica R5-392^T) in order Rhizobiales (class Alphaproteobacteria). The presence and absence of genes (pan-genome orthologous groups) on the heatmap are indicated in blue and red, respectively. (B) Venn diagram of shared and specific CDSs among the five strains.