Roseomonas rosulenta sp. nov., isolated from rice paddy soil

Three bacterial isolates, Gram-stain-negative, non-motile, coccobacilli-shaped bacteria, strains OP-27T, OP-5 and OP-30, were isolated from rice paddy soil. Phylogenetic analyses based on 16S rRNA gene sequences revealed that three isolates belonged to the genus Roseomonas, showing the highest sequence similarities to Roseomonas sediminicola FW-3T (98.1%) and Roseomonas lacus TH-G33T (98.0%). The genome size of strain OP-27T was 5.2 Mb in a single contig with DNA G+C content of 71.2%. The genome included 5164 predicted protein-coding genes, as well as 48 tRNA, 4 rRNA and 4 mRNA genes. The average nucleotide identity value between strain OP-27T and type strains of related species of the genus Roseomonas were 81.1–83.1%, and the digital DNA–DNA hybridization values of strain OP-27T and the related strains were 24.6–26.8%, respectively. The DNA–DNA hybridization values between strains OP-27T, OP-5 and OP-30 were 84–100% and its closest relative, Roseomonas sediminicola KACC 16616T was 21.1%. The major fatty acids were C18:1ω7c, C18:1 2-OH and C16:0 and predominant quinone was Q-10. Based on its distinctive phenotypic, phylogenetic, and chemotaxonomic characteristics, the three strains are considered to represent novel species of the genus Roseomonas, for which the name Roseomonas rosulenta sp. nov. is proposed. The type strain is OP-27T (=KACC 21501T= NBRC 114497T).


Introduction
The genus Roseomonas was first classified by Rihs et al. (1993). Currently, 50 species of the genus Roseomonas have been reported (https:// lpsn. dsmz. de/ genus/ Roseo monas) and isolated from various environmental habitats (air, freshwater sediment, plants, soil, surfaces and water etc.). Most members of the genus Roseomonas are Gram stain negative, red, orange, pink, yellow, white, brown pigmented, coccobacilli shaped and occasionally rod shaped (Yoon et al. 2007;Ramana et al. 2010;Subhash et al. 2016Subhash et al. , 2017Wang et al. 2016). Among them, 42 strains are pink pigmented bacteria. In the previous study, we isolated purple phototrophic bacteria (PPB) from paddy soil samples managed long-term for over 20 years with different chemical fertilizer. Researchers have tried to improve cultivation methods to isolate diverse and novel PPB in rice paddy soil. We succeeded in isolating purple phototrophic bacteria belonging to diversity of genera. Phylogenetic analysis indicated that some isolates belonged to be a member of novel genera and species. Currently, there are published data on four novel species Whang 2020, 2022;Cho and Whang 2021). In this study, we propose that strains OP-27 T , OP-5 and OP-30 represent a novel species of the genus Roseomonas based on polyphasic taxonomic approach.

Phylogenetic analysis and genome information
The genomic DNA of strains OP-27 T , OP-5 and OP-30 were extracted using the Genomic DNA Prep Kit for Bacterium, Cultured Cell (BIOFACT). The 16S rRNA gene sequence was amplified by PCR using the universal primers 27F, and 1492R (Lee et al. 2012). The identification of phylogenetic neighbors and calculations of pairwise 16S rRNA gene sequence similarity was determined using the EzBioCloud server (www. ezbio cloud. net/ ident ify) (Yoon et al. 2017). The 16S rRNA gene sequence was aligned with the published sequences of closely related bacteria with ClustalW 2.1 software (Larkin et al. 2007). Phylogenetic trees were reconstructed by the neighbor-joining (Saitou and Nei 1987), maximum-likelihood (Felsenstein 1981) and maximum-parsimony (Fitch 1971), algorithms within the MEGA 7.0 program (Kumar et al. 2016). Evolutionary distance matrices for the neighbor-joining method were calculated using the algorithm of Kimura's two-parameter model (Kimura 1980). To evaluate the stability of the phylogenetic tree, a bootstrap analysis was performed based on 1000 replications (Felsenstein 1985).
To determine genomic relatedness, DNA-DNA hybridization (DDH) was performed using the modified method of Ezaki et al. (1989). Probe labeling for DDH was conducted using the non-radioactive DIG-High prime system (Roche); hybridized DNA was visualized using the DIG luminescent detection kit (Roche) and the level of DNA-DNA relatedness was quantified using a densitometer (Bio-Rad). The genome sequence of strain OP-27 T was determined using the Illumina Miseq 300 platform at ChunLab. Illumina reads was assembled by SPAdes 3.13.0 (Bankevich 2012) to obtain the draft genomes. The Rapid Annotation using Subsystem Technology (RAST) (Aziz et al. 2008) server was used for the genome annotation. The average nucleotide identity (ANI) score was by calculated using the ANI calculator (www. ezbiocloud.net/tools/ ani) . In silico digital DNA-DNA hybridization (dDDH) were calculated using Genome-to-Genome Distance Calculator 3.0 method, with the recommended formula 2, available at the TYGS web service, respectively (Meier-Kolthoff and Göker 2019).

Morphological, physiological and biochemical characteristics
Morphological, physiological and biochemical characteristics of strains OP-27 T , OP-5 and OP-27 were determined after 5 days at 30 °C using R2A agar. Cell morphology was observed with stereoscopy (Leica EZ4) and transmission electron microscopy (FEI Tecnai G2 spirit TWIN) of cultures on R2A agar incubated at 30 °C for 5 days. Growth at different temperatures (4, 10, 15, 20, 25, 30, 37, 40 and 45 °C) was examined using R2A agar for 10 days. The pH ranges for growth at pH 4.0-11.0 (in increments of 0.5 pH units, adjusted with HCl or NaOH, 1 M, respectively) was assessed in R2A broth after 10 days. The tolerance range for salts was tested at 0, 0.5, 1.0, 2.0, 3.0, 5.0 and 7.0% NaCl (w/v) in R2A broth at pH 7.0. Gram staining was performed by the Hucker method (Gerhardt et al. 1994). Motility was determined by the hanging-drop method. Catalase activity was determined by assessing bubble production in 3.0% (w/v) H 2 O 2 . Oxidase activity was tested using oxidase reagent (bioMérieux) according to the instructions of the manufacturer. Enzyme activity and other biochemical characteristics were tested using API 20NE and API ZYM test kits (bioMérieux) at 30 °C for 5 days.

Chemotaxonomic analysis
Cellular fatty acids were extracted from cells grown on R2A agar for 5 days at 30 °C. The cellular fatty acid pattern analysis was performed as described by Sasser (1990), using the Microbial Identification System (Sherlock Version 4.5; MIDI database: TSBA 40). For chemotaxonomic analysis, freeze-dried cells were obtained from a culture grown in R2A agar for 10 days at 30 °C. The isoprenoid quinones were extracted as described by Collins (1985) and analyzed by high-performance liquid chromatography (SPD-10AV; Shimadzu) (Kroppenstedt 1982). Polar lipids were examined by two-dimensional thin-layer chromatography and identified according to the method of Minnikin et al. (1984).

Phylogenetic analysis and genome information
Phylogenetic analyses based on 16S rRNA gene sequences showed that the strains OP-27 T , OP-5, OP-30 belongs to the genus Roseomonas and it was closely related to Roseomonas sediminicola FW-3 T (98.1%) and Roseomonas lacus TH-G33 T (98.0%). The 16S rRNA gene sequences of strain OP-27 were the same as those of strains OP-5 and OP-30. In the maximum-likelihood tree, strains OP-27 T , OP-5 and OP-30 clustered with members of the genus Roseomonas and were most closely related to R. lacus TH-G33 T and R. terrae DS-48 T (Fig. 1). The phylogenetic position in the neighbor-joining (Fig. S1) and maximum-parsimony (Fig. S2) trees showed that strains OP-27, OP-5 and OP-30 monophyletic cluster with recognized members of the genus Roseomonas. The complete genome of strain OP-27 T obtained 671 contigs, with a total length of 5,277,075 bp and an N50 length of 15,298 bp (GenBank accession no. JACADR000000000). A total of 5458 genes were predicted, which included 5164 proteincoding genes and 56 RNA genes (Table S1). As the result of categorizing functional genes using the RAST subsystem, 5735 genes were predicted as coding sequence by the RAST server; 1198 CDSs (21%) were in the subsystem and 4537 CDSs (79%) were not in the subsystem. According to the functional subsystem distribution, a total of 1147 genes were counted as subsystem features. Strain OP-27 T had the photosynthetic genes for chlorophyllide synthase subunit encoding genes (bchF, bchJ, bchX, bchY and bchZ) in the chromosome. The DNA G+C content of strain OP-27 T was 71.2 mol% obtained from the genomic sequence.   (Richter and Rosselló-Móra 2009). In addition, digital DNA-DNA relatedness values between strain OP-27 T and the type strains of Roseomonas species were 24.6-26.8%, respectively (Table S2). The DNA-DNA relatedness values for strains OP-27 T , OP-5 and OP-30 were within the range 84-100% and were identified as the same strain. The DNA-DNA hybridization values between strain OP-27 T and its closest relative, Roseomonas sediminicola KACC 16616 T was 21.1% and related species of the genus Roseomonas were 19.2-31.2%, which are lower than the 70% proposed threshold for species designation (Wayne et al. 1987). These results further suggest also that strain OP-27 T represents a novel species of the genus Roseomonas.

Morphological, physiological and biochemical characteristics
The morphological characteristics of strains OP-27 T , OP-5 and OP-30 by stereoscopy and transmission electron microscopy of 5-day cultures on R2A agar. Colonies are circular with smooth, convex, with entire margins and deep pink or rose on R2A agar. Cells are Gram stain negative, aerobic, non-motile without flagella and coccobacilli shaped (1.4-1.6 × 2.5-2.9 μm in size) (Fig. S4). The results of the physiological and biochemical analyses are presented in Table 1.
Negative results in API 20NE and API ZYM tests are listed in Table S3.

Taxonomic conclusion
In this study, phylogenetic analysis, morphological, physiological and biochemical characteristics, chemotaxonomic analysis, strains OP-27 T , OP-5 and OP-30 should be affiliated to the genus Roseomonas (Table 1). Moreover, the low ANI values (81.1-83.1%) and DDH values (24.6-26.8%) and the phenotypic and chemotaxonomic properties presented above, we suggest that strains OP-27 T , OP-5 and OP-30 is a novel species of the genus Roseomonas, and the name Roseomonas rosulenta sp. nov. is proposed.
The type strain, OP-27 T (= KACC 21501 T = NBRC 114497 T ) which was isolated from rice paddy soil. The Gen-Bank accession numbers of 16S rRNA gene sequence and the whole-genome sequence of strain OP-27 T are LC505025 and JACADR000000000, respectively.