Genome Sequences of Rhodoplanes serenus and Two Thermotolerant Strains, Rhodoplanes tepidamans and “Rhodoplanes cryptolactis,” Further Refine the Genus

ABSTRACT

Rhodoplanes is a small genus of anoxygenic purple nonsulfur bacteria belonging to the family Nitrobacteraceae in the class Alphaproteobacteria. We sequenced the genomes of one mesophilic and two thermotolerant strains, and whole-genome-based comparisons confirmed suspected close similarities and synonyms between different species in the genus.

ANNOUNCEMENT

The phototrophic genus Rhodoplanes contains 10 named species (1); however, until now, only 3 had been sequenced: Rhodoplanes piscinae, R. roseus, and R. elegans (2). We have now sequenced the genomes of Rhodoplanes serenus, R. tepidamans, and “Rhodoplanes cryptolactis.” Earlier studies proposed that the species R. cryptolactis be included in the new species R. tepidamans, based on the loss of R. cryptolactis from the ATCC collection (3). Whole-genome sequencing was performed to determine the genomic differences between the known strains of Rhodoplanes and to further refine the genus.

Rhodoplanes serenus DSM 18633 was originally isolated from pond water at the University of Tokyo, Japan (4), while R. tepidamans DSM 9987 and R. cryptolactis are thermotolerant species isolated from a soil sample near a hot spring in Wyoming (5). Genomic DNA (gDNA) of R. serenus and R. tepidamans was obtained from the DSMZ culture collection, while frozen cultures of R. cryptolactis TEM were obtained from T. E. Meyer, who received a direct transfer from the original isolation by R. Stadtwald-Demchick, F. R. Turner, and H. Gest (5). The cultures were grown in DSMZ medium 27, supplemented with 20 μg/L vitamin B21, and DNA was extracted using the GeneJET genomic DNA isolation kit (Thermo Scientific). DNA analysis showed ratios of absorption at 260/280 nm of 1.96 for R. serenus, 1.85 for R. tepidamans, and 2.0 for R. cryptolactis.

Sequencing libraries were prepared using the Illumina Nextera DNA Flex library prep kit and sequenced using an Illumina MiniSeq instrument, using 500 μL of a 1.8 pM library. Paired-end (2 × 150-bp) sequencing generated 3,015,458 reads and 227 Mbp for R. serenus, 1,239,460 reads and 187.2 Mbp for R. tepidamans, and 3,081,022 reads and 465.2 Mbp for R. cryptolactis. Quality control of the reads was performed using FastQC version 1.0.0 within BaseSpace (Illumina), using a k-mer size of 5 and contamination filtering. We assembled the genome de novo through BV-BRC (6) using Unicycler (7). A comparison of the features of these new genomes to the other Rhodoplanes genomes is provided in Table 1. The genomes were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (8). The resulting coding sequences and tRNAs are also available in Table 1. Default parameters were used for all software applications unless otherwise noted.

TABLE 1.

Overview of the features of all the Rhodoplanes genome sequences

Species	Genome size (bp)	GC content (%)	No. of contigsa	N50 (bp)	Coverage (×)	No. of CDSsb	No. of tRNAs	Reference	GenBank accession no.
R. tepidamans DSM 9987	6,800,548	71.1	132	94,445	28	6,095	48	This study	JAQQLI000000000
R. cryptolactis TEM	6,781,861	71	238	50,670	69	6,150	48	This study	JAQQSA000000000
R. roseus DSM 5909	6,879,536	69.4	1,040	19,390	308	6,585	48	9	NPEX00000000
R. elegans DSM 11907	6,538,588	69.5	320	32,496	33	6,093	48	2	NHSK00000000
R. serenus DSM 18633	5,501,108	70.3	51	262,047	41	4,965	49	This study	WNKV00000000
R. piscinae DSM 19946	5,370,996	70.2	880	15,387	385	5,201	46	10	NPEW00000000

^aNumber of contigs >300 bp.

^bCDSs, coding DNA sequences.

A JSpecies comparison (11) of the average nucleotide identity (ANIb) showed that the 3 new genomes all have ≤86% ANI with the earlier sequenced Rhodoplanes genomes, except for R. serenus and R. piscinae, which share 97.4% ANI. The latter is above the proposed 95% cutoff for genome definition of a species (11). R. tepidamans and R. cryptolactis are about equidistant to all the Rhodoplanes species; however, they share 99.9% ANI, which indicates that they belong to the same species. Whole-genome-based phylogenetic analysis was performed using RAxML (12, 13) version 8.2.11 with all the Rhodoplanes genomes (Fig. 1). Protein sequences were aligned using MUSCLE (14), and the nucleotide coding gene sequences were aligned using the Codon_align function of Biopython (15). A concatenated alignment of all proteins and nucleotides was written to a partition file for RAxML. Consistent with the ANI analysis, this analysis also closely grouped R. serenus with R. piscinae and R. tepidamans with R. cryptolactis. This finding agrees with the suggestion that R. piscinae should be a later synonym of R. serenus (16) and confirms that R. tepidamans and R. cryptolactis belong to the same species. The addition of these new Rhodoplanes genomes has substantially strengthened the phylogenetic tree of this genus.

FIG 1.

Whole-genome-based phylogenetic tree of all sequenced Rhodoplanes species. The phylogenetic tree was generated using the codon tree method within BV-BRC (6), which used PGfams as homology groups; 875 PGfams were found among these selected genomes using the CodonTree analysis, and the aligned proteins and coding DNA from single-copy genes were used for RAxML analysis (12, 13). One hundred rounds of the rapid bootstrapping option in RAxML were used to generate the support values for the phylogenetic tree. The branch length tree scale is defined as the mean number of substitutions per site, which is an average across both nucleotide and amino acid changes. New genomes are in red. The Rhodopseudomonas sp. strain ATH 2.1.18 genome was used as an outgroup (17). iTOL was used for the tree visualization (18).

Data availability.

These whole-genome shotgun projects have been deposited at DDBJ/ENA/GenBank under the accession numbers WNKV00000000 for R. serenus, JAQQLI000000000 for R. tepidamans, and JAQQSA000000000 for R. cryptolactis. The versions described in this paper are versions WNKV01000000, JAQQLI010000000, and JAQQSA010000000, respectively. The raw sequencing reads have been submitted to the SRA under the accession numbers SRR23308057 for R. serenus, SRR23308056 for R. tepidamans, and SRR23308055 for R. cryptolactis.