Roseomonas ponticola sp. nov., a novel bacterium isolated from Pearl River estuary

A novel species of the genus Roseomonas , designated SYSU M41301T, was isolated from water sample of the Pearl River estuary in Guangdong, China. Polyphasic, taxonomic and phylogenomic analyses were used to determine the taxonomy position of the strain. Phylogenetic analysis using 16S rRNA gene sequence indicated that strain SYSU M41301T showed the highest sequence similarity to Roseomonas stagni KCTC 22213T (97.9 %) and Roseomonas riguiloci KCTC 23339T (96.4 %). The novel species could be differentiated from other species of the genus Roseomonas by its distinct phenotypic and genotypic characteristics. The isolate was Gram-staining-negative, aerobic, short rod-shape, oxidase-positive and non-motile. The predominant respiratory quinone was ubiquinone 8 (Q-8). The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, and one unidentified polar lipid. The major fatty acids (>10 % of total) were 11-methyl C18 : 1  ω7c, summed feature 3 (C16 : 1  ω7c and/ or C16 : 1  ω6c) and summed feature 8 (C18:  :1  ω7c and/or C18 : 1  ω6c). The G+C content of the novel isolate based on genomic DNA was 72.0 mol%. On the basis of phenotypic, genotypic and phylogenetic data, strain SYSU M41301T should be considered to represent a novel species in the genus Roseomonas, for which the name Roseomonas ponticola sp. nov. is proposed with the type strain SYSU M41301T (=KCTC 72726T=CGMCC 1.18613T).

The genus Roseomonas, which belongs to the family Acetobacteraceae of the order Rhodospirillales in the Alphaproteobacteria, was first proposed by Rihs et al. [1]. At the time of writing, the genus Roseomonas comprises 48 species and two subspecies with validly published names. Members of this genus are Gram-staining-negative, aerobic and pink pigmented, which have been isolated from various sources. Species of the genus Roseomonas were initially isolated from clinical samples that cause infection in children and adults [2]. Members of the genus Roseomonas have also been isolated frequently from environment samples, such as drinking water distribution systems [3], freshwater [4], water-cooling system [5], deep-water marine invertebrates [6].
In order to investigate the cultivable planktonic bacterial community in the Pearl River estuary, sea water samples were collected from the Pearl River estuary, Guangdong, China (22° 20′ 56′ N, 113° 44′ 33′ E). The temperature and pH at the sampling site were 28 °C and pH 7.5, respectively. The site also depicted the following chemical parameters during the sampling period (mg l −1 ): NH 4 + (0.1), NO 3 -(0.1), NO 2 -(0.1), TOC (4.7), TON (3.0). During this study, one isolate designated as SYSU M41301 T was isolated from estuary sea water sample. The analysis based on 16S rRNA gene sequence of strain SYSU M41301 T indicated the isolate belonged to the genus Roseomonas with a low sequence similarity identity (<98.0%). This study is an attempt to analyse the phenotypic, chemotaxonomic and molecular characteristics of the novel isolate.
The sample was plated on R2A agar medium and incubated at 28 °C. Distinct colonies were selected and purified on R2A agar medium. One pink-coloured strain SYSU M41301 T among the purified isolates showed low 16S rRNA sequence OPEN ACCESS similarity with known members of the genus Roseomonas, and was further selected for characterizing of its taxonomic position. Cells of strain SYSU M41301 T were maintained in 20 % glycerol (v/v) at -80 °C. The Gram-reaction was tested by the non-staining KOH method [7]. Micromorphology of the cells grown on R 2 A agar for 2 days was observed by using a transmission electron microscope (JEM1400FLASH, JEOL, JAPAN). Cell motility was checked by inoculating the strain in a tube containing semi-solid medium [8]. Growth was also tested on Luria-Bertani (LB) agar, Tryptic Soy Agar (TSA, Difco), nutrient agar and R 2 A agar, following 5 days of incubation at 28 °C. Oxidase and catalase activities were determined by assessing the oxidation of 1.0 % (w/v) tetramethyl-p-phenylenediamine [9] and the formation of bubbles upon addition of 3.0 % (v/v) H 2 O 2 , respectively. Growth temperature (4,14,23,28,37,45,50, 55 and 60 °C) and NaCl tolerance (0-15.0 %, at intervals of 0.5 %, w/v) were checked using R 2 A agar for 1 week. The pH range for growth was tested from pH 4.0 to 10.0 (at intervals of 1.0 pH unit, prepared using the buffer system indicated in Nie et al. [10]) in R 2 A broth for 1 week. Cellulose, gelatin and starch hydrolysis, H 2 S production, milk coagulation and peptonization, nitrate reduction, Tweens 20, 40, 60 and 80 degradation and urease activity were tested as previously described [11,12]. Utilization of sole carbon and nitrogen sources were determined as previously described [13]. Other phenotypic characteristics were determined by the API ZYM (bioMérieux), API 20NE (bioMérieux) and GEN III MicroPlate (Biolog) systems using the instructions provided by the manufacturers.
Cells of strain SYSU M41301 T were Gram-staining-negative, short rod-shape, and non-motile (Fig. S1, available in the online version of this article). The strain grew well on nutrient agar, R 2 A agar, but not on Luria-Bertani agar and TSA. Colonies on R 2 A agar after 2 days of growth at 28 °C were non-translucent, pink-coloured, circular, entire margin, low convex and smooth, measuring up to 4 mm in diameter. Strain SYSU M41301 T was catalase-positive and oxidasepositive. Growth of strain SYSU M41301 T was observed at 16-45 °C (optimum, 28 °C), pH 6.0-8.0 (optimum, pH 7.0) and in the presence of 0-5.0 % (w/v) NaCl (optimum 0.5%). The phenotypic characteristics determined by the API ZYM (bioMérieux), API 20NE (bioMérieux) and GEN III Micro-Plate system (Biolog) are provided in the Table S1. Detailed phenotypic characteristics were listed in species description, while the comparative analyses with the nearest similarity Optimum temperature for growth (°C) 28 28 28 Growth pH 6.0-8.  Table 1.
Genomic DNA of strain SYSU M41301 T was extracted as previously described [14]. Amplification of the 16S rRNA gene, purification, cloning, sequencing and assembly of the raw sequences were done as described earlier [15]. The sequences obtained were compared with the 16S rRNA gene sequences of species with validly published names in EzBioCloud server database [16]. Sequences of related species in the genus Roseomonas were retrieved from the EzBioCloud server database, and multiple alignments performed by using Clustal W in mega version 7.0 software package [17]. Phylogenetic trees were generated with maximum-likelihood [18], neighbourjoining [19] and maximum-parsimony [20] methods by using mega version 7.0 software package [21]. Distance matrix for the neighbour-joining method was generated according to Kimura two-parameter model. Stability of the phylogenetic trees were evaluated by bootstrap analysis [22]. Elioraea tepidiphila TU-7T T was used as outgroup.
Whole genome sequencing of strain SYSU M41301 T was performed using a paired-end sequencing method with HiSeq X platform (Illumina, San Diego, CA, USA) at Genewiz Company (Guangzhou, China). Reads of each data sets were filtered, and high quality paired-end reads were assembled using SPADES [23]. The COG, KEGG databases were used to annotate the genome sequence. Average nucleotide identity (ANI) values were calculated using JSpecies [24]. The G+C content of the genomic DNA was determined from the genome sequence. Phylogenomic tree was constructed as described by Salam et al. [25]. Marker genes were extracted from 21 genomes of strains SYSU M41301 T , some type species available for genus Roseomonas and one outgroup using AMPHORA2 [26]. A total of 31 conserved marker genes (dnaG, frr, infC, nusA, pgk, pyrG, rplA, rplB, rplC, rplD, rplE, rplF, rplK, rplL, rplM, rplN, rplP, rplS, rplT, rpmA, rpoB, rpsB, rpsC, rpsE, rpsI, rpsJ, rpsK, rpsM, rpsS, smpB, tsf) that are known to present universally were selected. Sequences of each of the marker genes were aligned separately by using muscle [27]. Cleaned alignments were concatenated by using perl script (https:// github. com/ nylander/ catfasta2phyml). Poorly aligned regions were removed from the datasets using Gblocks [28] resulting in a final set of amino acid position that were used in generating the phylogenetic tree with RAxML method [29].
Strain SYSU M41301 T showed distant relationships with members of the genus Roseomonas: Roseomonas stagni KCTC 22213 T (97.9 % sequence identity) and Roseomonas riguiloci KCTC 23339 T (96.4%). Strain SYSU M41301 T formed a distinct clade within the genus Roseomonas in the neighbour-joining tree (Fig. 1). This relationship was also supported by the maximum-likelihood (Fig. S2) and maximum-parsimony trees (Fig. S3). Phylogenomic tree (Fig. 2) based on the concatenated alignment of 31 marker genes provides further evidence for the distinct lineage of the strains SYSU M41301 T . The assembled genome of strain SYSU M41301 T has been deposited in the GenBank database under the accession numbers JAERQN000000000, and the raw data of the genome has been deposited under the accession number PRJNA690213. Based on the genome information, strain SYSU M41301 T had a genome size of 6 732 422 bp, which were retrieved from 29 contigs. The largest length of the contig was 1 574 521 bp, with N 50 length of 437 051 bp. The G+C content based on genomic DNA was calculated at 72.0 mol%. For strain SYSU M41301 T , a total of 4596 genes were predicted, which includes 4488 protein-coding genes and 108 RNA genes. A total of 86.5 % of the genes were assigned a putative function while the remaining protein-coding genes were annotated as hypothetical proteins. COGs (Clusters of Orthologous Groups) categories distributions for the genes are presented in the Table S2. The results of anabolic and catabolic metabolism inferring from Table S2 were consistent with the physiological characterization of strain SYSU M41301 T . The most abundantly represented CDSs (sequence coding for amino acids in protein) in strain SYSU M41301 T were amino acid transport and metabolism (COG category E), followed by inorganic ion transport and metabolism (COG category P) and energy production and conversion (COG category C). Strain M41301 T showed positive results for ecological functions and utilization of different carbon sources, so the KEGG database was further used to annotate the genome sequence to identify potential functions (Table S3). Based on the results of this analysis, narG, narZ, nxrA, nirB, nasA, nirA, cysNC, cysH, cysJ were encoded in the genome of SYSU M41301 T , which suggested the strain played an important role in the aquatic ecosystem and participated in driving the nitrogen cycle and sulphur cycle. In addition, some carbon sources metabolism related genes including maeB, ppdK, tpiA, pgm, pgi, glgA and other genes have also been found, which implied that strain SYSU M41301 T might participate the biogeochemical cycle in the Pearl River estuary.

Roseomonas alkaliterrae YIM 78007 T (JACIJE000000000)
Roseomonas Biomass for study of chemotaxonomic features of strain SYSU M41301 T was obtained from cultures grown in R 2 A agar for 7 days (late logarithmic phase of growth). Respiratory quinones were extracted from lyophilized cells [30], and the extract analysed by using a high-performance liquid chromatography (HPLC) [31]. The polar lipids were determined by using a two-dimensional thin-layer chromatography (TLC) procedure on silica gel G 60 plates (Merck) [32,33]. The staining reagents phosphomolybdate was used for detecting the total lipids, ninhydrin for free-amino group containing lipids, molybdenum blue for phospholipids and α-naphthol for sugar-group containing lipids. Cellular fatty acids were extracted, methylated and identified by using gas chromatography (Agilent Technologies 7890B GC System), following the instructions of the Sherlock Microbial Identification System (MIDI) version 6.1 and the TSBA6 database [34].
The phylogenetic analysis, morphological and chemotaxonomical characteristics support the characterization of strain SYSU M41301 T as a member of the genus Roseomonas. The strain can be considered to represent a distinct lineage within the genus Roseomonas from its low 16S rRNA gene sequence identities with other members of the genus Roseomonas (<98.0 %). In addition, the ANI values between the novel isolate and species in the genus Roseomonas were lower than 90.0 %, which are strong evidence for assigning novel isolate into genus Roseomonas (Table S5). Besides, strain SYSU M41301 T could be differentiated from the nearest species in the genus Roseomonas by the features summarized in Table 1. Based on the differentiating characteristics, strain SYSU M41301 T represents a novel species of the genus Roseomonas, for which the name Roseomonas ponticola sp. nov. is proposed.
The type strain SYSU M41301 T (=KCTC 72726 T =CGMCC 1.18613 T ) was isolated from water sample collected from the Pearl River, Guangdong, China. The 16S rRNA gene of strain SYSU M41301 T was submitted to GenBank with accession number MW240957. The raw data and assembly genome with accession number PRJNA690213 and JAERQN000000000, respectively.