Rhodobacter ruber sp. nov., isolated from a freshwater pond


 Strain CCP-1T, isolated from a freshwater pond in Taiwan, is characterized using a polyphasic taxonomy approach. Cells of strain CCP-1T are Gram-stain-negative, aerobic, non-motile, rod-shaped and form dark red colored colonies. Growth occurs at 20–40 oC, at pH 6.5-9 and with 0-0.5% NaCl. Strain CCP-1T contains bacteriochlorophyll a, and shows optimum growth under anaerobic condition by photoheterotrophy, but not by photoautotrophy. 16S rRNA gene sequence similarity indicates that strain CCP-1T is closely related to species within the genus Rhodobacter (93.9–96.2% sequence similarity), Haematobacter (96.3%) and Xinfangfangia (95.5–96.2%). Phylogenetic analyses based on 16S rRNA gene sequences and based on up-to-date bacterial core gene set (92 protein clusters) reveal that strain CCP-1T is affiliated with species in the genus Rhodobacter. The average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization identity between strain CCP-1T and Rhodobacter species are 71.3–76.3%, 70.4–77.9% and 21.4–23.2%, respectively, supporting that strain CCP-1T is a novel species of the genus Rhodobacter. The DNA G + C content is 66.2%. The predominant fatty acid is C18:1ω7c and the major isoprenoid quinone is Q-10. The polar lipids have phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, two uncharacterized aminophospholipids and two uncharacterized phospholipids. On the basis of phenotypic and genotypic properties and phylogenetic inference, strain CCP-1T should represent a novel species of the genus Rhodobacter, for which the name Rhodobacter ruber sp. nov. is proposed. The type strain is CCP-1T (= BCRC 81189T = LMG 31335T).


Introduction
The genus Rhodobacter (type species, Rhodobacter capsulatus) proposed by Imhoff et al. (1984) and emended by Srinivas et al. (2007), Wang et al. (2014) and Suresh et al. (2019), belongs to the family Rhodobacteraceae of the order Rhodobacterales in the class Alphaproteobacteria (Imhoff 2005;Pujalte et al. 2014). The genus Rhodobacter comprises 16 species with validly published names so far stated on the List of Prokaryotic names with Standing in Nomenclature (https://lpsn.dsmz.de/genus/rhodobacter).
Species of the genus Rhodobacter had been isolated from various environmental sources including freshwater, eutrophic freshwater, stagnant water, polluted water, alkaline water, hot spring, stream mud, lagoon sediment, estuarine water and marine water (Arunasri et al. 2008;Eckersley and Dow 1980;Gandham et al. 2018;Imhoff et al. 1985;Khan et al. 2019;Raj et al. 2013;Ramana et al. 2008;Sheu et al. 2020;Srinivas et al. 2007;Subhash and Lee 2016;Suresh et al. 2017Suresh et al. , 2020Venkata Ramana et al. 2009;Xian et al. 2020). Members of the genus Rhodobacter are characterized as Gram-stain-negative, motile or non-motile and ovoid to rod shaped. Chemotaxonomically, members of the genus are characterized by C 18:1 ω7c as the predominant fatty acid, ubiquinone 10 (Q-10) as the major respiratory quinone, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine as the main polar lipids, and the DNA G + C content between 62 to 73 mol% (Imhoff 2005;Pujalte et al. 2014;Suresh et al. 2019;Wang et al. 2014). The present study was carried out to clarify the taxonomic position of a putative novel species belonging to the genus Rhodobacter, designated CCP-1 T , by a polyphasic taxonomic approach.

Bacterial strains and culture conditions
During the characterization of microorganisms present in the freshwater sample of a pond in a crocodile farm (GPS location: 22°33'45'' N 120°32'07'' E) in the Chaozhou Township of Pingtung County, Taiwan, a water sample (25 o C, pH 6.5, 0% NaCl) was collected on 4 April 2016. The water sample was spread on R2A agar (BD Difco) plates by the standard dilution plating method. After incubation of the plates at 25 o C for 3 days, a novel dark red-pigmented bacterium, designated CCP-1 T , was selected and puri ed as single colonies and subjected for detailed taxonomy analyses. Sub-cultivation was performed on R2A agar at 25 o C for 48-72 h. The isolate was preserved in R2A broth with 20% (v/v) glycerol at -80 o C and also by lyophilization before storing at -80 o C.

Morphological, physiological, and biochemical characteristics
The morphology of bacterial cells was observed by phase-contrast microscopy (DM 2000; Leica) and transmission electron microscopy (H-7500; Hitachi) using cells grown on R2A agar for 3 days at 25 o C. Cellular motility was tested by the hanging drop method ). The Gram Stain Set S kit (BD Difco) and the Ryu non-staining KOH method (Powers 1995) were used to perform the Gram reaction.
Poly-β-hydroxybutyrate granule accumulation was examined under light microscopy after staining of the cells with Sudan black (Schlegel et al. 1970). Colony morphology was observed on R2A agar under a stereoscopic microscope (SMZ 800; Nikon).
The physiological characteristics of strain CCP-1 T and the ve reference strains were examined by growing bacteria at various pH values, temperatures and NaCl concentrations. The pH range for bacterial growth was estimated by measuring the optical densities (wavelength 600 nm) of R2A broth cultures. The pH of the medium was adjusted prior to sterilization to pH 4-9 (at intervals of 0.5 pH unit) using the following biological buffers (Breznak and Costilow 2007): citrate/Na 2 HPO 4 (pH 4-5.5); phosphate (pH 6-7.5); and Tris (pH 8-9). The temperature range for growth was determined on R2A agar at 4, 10,15,20,25,30,35,37,40,45 and 50 o C. To investigate the tolerance to NaCl, R2A broth was prepared according to the formula of the BD Difco medium with NaCl concentration adjusted to 0, 0.5% and 1-5%, w/v (at intervals of 1%). Growth under anaerobic conditions was determined after incubating strain CCP-1 T on R2A agar and on R2A agar supplemented with nitrate (0.1% KNO 3 ) in anaerobic jars by using AnaeroGen anaerobic system envelopes (Oxoid) at 25 o C for 15 days. Bacterial growth was studied on R2A, nutrient, Luria-Bertani and trypticase soy agars (all from Difco) under aerobic condition at 25 o C for 15 days. Photoheterotrophic growth under anaerobic conditions was determined after incubation in an Oxoid AnaeroGen system or in 30 ml tubes with a rubber septum under a stream of nitrogen gas in light using minimal medium containing yeast extract (0.3%, w/v), tryptone (0.3%, w/v) or sodium acetate (0.3%, w/v). Photoautotrophic growth was examined under the same conditions but using medium with thiosulfate (0.1%, w/v) or sodium bicarbonate (0.1%, w/v) as previously described by Pfennig and Trüper (1974). For photosynthetic pigment analysis, cell mass from 30 ml culture was extracted and the absorption spectrum of the extract was recorded as described by Biebl et al. (2005).
Activities of catalase, oxidase, DNase, urease, lipase (corn oil), and hydrolysis of starch, casein and lecithin were determined using the methods of Tindall et al. (2007). Chitin hydrolysis was assessed on chitinase-detection agar (Wen et al. 2002) and visualized by the formation of clear zones around the colonies. Hydrolysis of carboxymethyl cellulose (CM-cellulose) was tested as described by Bowman (2000) using R2A agar as the basal medium. Utilization of carbon sources was investigated in a basal medium containing (l − 1 ): 0.4 g KH 2 PO 4 , 0.53 g Na 2 HPO 4 , 0.3 g NH 4 Cl, 0.3 g NaCl, 0.1 g MgCl 2 ·6H 2 O, 0.11 g CaCl 2 and 1 ml trace element solution, pH 7.0 (Chang et al. 2004). Positive control tubes were prepared with 2 g yeast extract l − 1 , while the basal medium was used as a negative control. Substrates were added at a concentration of 0.1% (w/v or v/v). Incubation was prolonged for 15 days at 25 o C under aerobic condition by means of duplicate experiments, and bacterial growth was examined every two days. Additional biochemical tests were performed using API ZYM and API 20NE kits (bioMérieux). Both commercial phenotypic tests were performed according to the manufacturers' recommendations.

Determination of cellular fatty acids, polar lipids and isoprenoid quinones
The fatty acid pro les of strain CCP-1 T and the ve phylogenetic related strains were analyzed on cells grown on R2A agar at 25 o C for 3 days. Fatty acid methyl esters were extracted and separated according to the standard protocol of MIDI (Sherlock Microbial Identi cation System, version 6.0), analyzed by GC (Hewlett-Packard 5890 Series II) and identi ed by using the RTSBA6.00 database of the microbial identi cation system (Sasser 1990).
The polar lipids were extracted and analyzed by two-dimensional TLC according to Embley and Wait (1994). Ethanolic molybdophosphoric acid (10%) was used for the detection of the total polar lipids, ninhydrin for amino lipids, the α-naphthol reagent for glycolipids and the Zinzadze reagent for phospholipids. The isoprenoid quinones were extracted and puri ed according to the method of Collins (1994) and analyzed by HPLC with a Spherisorb ODS column using methanol/1-chlorobutane (100:10, v/v) as mobile phase (1.5 ml min − 1 ).

16S rRNA gene sequencing and phylogenetic analysis
Genomic DNA was isolated using a bacterial genomic DNA puri cation kit (DP02-150, GeneMark). The 16S rRNA gene was ampli ed using the universal primer set (27F and 1541R) (Weisburg et al. 1991;Anzai et al. 1997). PCR products were puri ed using a plus PCR clean up kit (DP04P, GeneMark), and then sequenced using four primers (27F, 520F, 800R and 1541R) (Weisburg et al. 1991;Anzai et al. 1997), the BigDye Terminator Cycle Sequencing kit (Applied Biosystems) with an ABI Prism 3730xl automated DNA analyzer (Applied Biosystems). The novel sequence was compared to those available from the EzBioCloud (Yoon et al. 2017).
Whole genome analysis, average nucleotide identity and average amino acid identity calculations, digital DNA-DNA hybridization scores, UBCG phylogenetic tree construction and genome comparative analysis A whole genome sequence was prepared by the Genomics BioSci & Tech. Co., Ltd. (Taipei, Taiwan, ROC) using the Illumina NextSeq sequencer platform and using MultiQC v1.2 for evaluating read quality (Ewels et al. 2016). The whole genome was assembled using SPAdes (version 3.10.1) (Bankevich et al. 2012), and gene prediction and annotation by Prokka pipeline (Seemann 2014). The protein encoding genes were classi ed into functional categories based on eggNOG (evolutionary genealogy of genes: Nonsupervised Orthologous Groups)-Mapper using precomputed cluster and phylogenies from the eggNOG database as described by Huerta-Cepas et al. (2016. The estimated genome-sequence-based digital DNA-DNA hybridization (dDDH) values were calculated as described by Meier-Kolthoff et al. (2013). Average nucleotide identity (ANI) calculations were performed by OrthoANI analysis . Average amino acid identity (AAI) calculations were performed (http://enve-omics.ce.gatech.edu/). An up-to-date bacterial core gene set (UBCG, concatenated alignment of 92 core genes) and pipeline was utilized for phylogenetic tree construction as described by Na et al. (2018).
For genome comparative analysis, the genome sequences of strain CCP-1 T and genome sequences from the genus Rhodobacter including the type strain of type species of the genus, Rhodobacter capsulatus ATCC 11166 T , and four type strains isolated from various environments, Rhodobacter tardus CYK-10 T , Rhodobacter agellatus SYSU G03088 T , Rhodobacter blasticus ATCC 33485 T and Rhodobacter thermarum YIM 73036 T , were annotated by the NCBI Prokaryotic Genome Annotation Pipeline and also submitted to Rapid Annotation of microbial genomes using Subsystem Technology (RAST) as described by Overbeek et al. (2014). Comparative gene content analyses were performed by EDGAR 2.0, an enhanced software platform as described by Blom et al. (2016).

Results And Discussion
The novel strain CCP-1 T was isolated during the characterization of microorganisms present in the freshwater crocodile pond in Taiwan on 4 April 2016 ( Supplementary Fig. S1). For strain CCP-1 T , the 16S rRNA gene sequence (1431 nucleotides) has been deposited in GenBank under accession number LT852521. Sequence similarity calculations (over 1400 bp) revealed that the novel isolate was related to the genera Rhodobacter (93.9-96.2% sequence similarity), Haematobacter (96.3%) and Xinfangfangia (95.5-96.2%). The closest relatives of strain CCP-1 T were Haematobacter massiliensis CCUG 47968 T and Haematobacter missouriensis CCUG 52307 T (96.3%), followed by Rhodobacter tardus CYK-10 T and Xinfangfangiasoli ZQBW T (96.2%). Phylogenetic analysis based on 16S rRNA gene sequence revealed that strain CCP-1 T formed a separate phylogenetic branch cluster with Rhodobacter tardus CYK-10 T , Rhodobacter blasticus ATCC 33485 T , Rhodobacter thermarum YIM 73036 T and Rhodobacter agellatus SYSU G03088 T within the genus Rhodobacter in the neighbour-joining tree (Fig. 1). The overall topologies of the maximum-likelihood and maximum-parsimony trees were similar (Supplementary Figs. S2 and S3). However, although the novel isolate had the highest similarity to Haematobacter massiliensis CCUG 47968 T and Haematobacter missouriensis CCUG 52307 T , it is obvious from the phylogenetic tree that they belong to different genera. In addition, the novel isolate had a higher similarity to Xinfangfangiasoli ZQBW T , and two validly published Xinfangfangia species were adjacent in the phylogenetic tree. But, based on the low 16S rRNA gene sequence similarity values and the absence of photosynthesis genes and photosynthesis pigments, Xinfangfangia has been published as a different genus from Rhodobacter (Hu et al. 2018). The absence of photosynthesis genes in genome of Xinfangfangia humi CIP 111625 T was also con rmed by Rapid Annotation of microbial genomes using Subsystem Technology ( Supplementary Fig. S4B). Furthermore, the presence of photosynthesis genes ( Supplementary Fig. S4A), photoheterotrophy and bacteriochlorophyll a (described below) for this novel isolate, indicated that it might be assigned to a novel species of the Rhodobacter.
The whole genome sequence of strain CCP-1 T was prepared and assembled and deposited in GenBank under accession number NZ_ JAAATW000000000. The estimated genome size was approximately 3.96 Mb, the coverage depth was 263., the number of contings was 23 and the N50 length was 809392 bp. The genomic G+C content of the DNA was 66.2%. The sequences of the 16S rRNA gene encoded in the genome of strain CCP-1 T and that of PCR determined sequence are the same. It contained 3758 protein encoding genes, 3 rRNA genes and 48 tRNA genes. Based on the eggNOG database, the 3758 protein encoding genes were classi ed into 21 functional categories (Supplementary Table S1). Most of coding sequences in strain CCP-1 T genome are classi ed as amino acid transport and metabolism (E, 9.4 %), followed by those identi ed as having roles in general function prediction only (R, 9.4 %), functional unknown (S, 8.6 %), carbohydrate transport and metabolism (G, 6.6 %) and transcription (K, 5.0 %).
In order to further explore the relationships of strain CCP-1 T and related species in the genus Rhodobacter, an UBCG and pipeline was utilized for phylogenetic tree construction. The phylogenetic tree based on the coding sequences of 92 protein clusters showed that strain CCP-1 T formed a distinct phylogenetic lineage in the Rhodobacter (Fig. 2), which supported that strain CCP-1 T might be assigned to a novel species of the Rhodobacter.
Following the proposed minimal standards for the use of genome data for the taxonomy of prokaryotes , both dDDH and ANI between strain CCP-1 T and other related Rhodobacter species with whole genome sequence publicly available were determined. Genomic comparison with ANI and dDDH calculations between strain CCP-1 T and other related Rhodobacter species indicated that 71.3-76.3% of ANI and 21.4-23.2% of dDDH, respectively (Supplementary Table S2), were su cient for the threshold of species-level differentiation, 95-96% ANI (Richter and Rosselló-Móra 2009) and 70% dDDH (Goris et al. 2007). In addition, AAI calculations were performed, which gave AAI values of 70.4-77.9% when strain CCP-1 T compared to the related strains (Supplementary Table S2). The calculated AAI values were clearly lower than the AAI threshold (about 90%) for species demarcation and higher than the threshold (about 60%) for genus boundary proposed by Rodriguez-R and Konstantinidis (2014). These data warranted the status of strain CCP-1 T as a separate species in the genus Rhodobacter.
Additionally, concerning iron acquisition and metabolism, sulfur, potassium, nitrogen, nucleoside and nucleotide, amino acid and derivatives, protein, DNA, lipid, aromatic compound and carbohydrate metabolism, all strains showed highly diverse distribution pattern among them. Furthermore, the percentage of genes of strain CCP-1 T shared with the type species of the genus was estimated. Strain CCP-1 T showed 1935 genes (51.5%) shared with Rhodobacter capsulatus ATCC 11166 T while the rest of 1823 genes (48.5%) were different ( Supplementary Fig. S5A). When strain CCP-1 T and another four strains were analyzed together, it can be found that there are 1906 genes in common, which is about 50.7% of the total number of genes of strain CCP-1 T . For strain CCP-1 T , there are 879 genes present as strain CCP-1 T speci c genes, accounting for about 23.4% (Supplementary Fig. S5B). Because strain CCP-1 T , Rhodobacter tardus CYK-10 T , Rhodobacter agellatus SYSU G03088 T , Rhodobacter blasticus ATCC 33485 T , Rhodobacter thermarum YIM 73036 T and Rhodobacter capsulatus ATCC 11166 T are isolated from freshwater crocodile pond, freshwater lotus pond, hot spring, eutrophic freshwater, sediment of a hot spring and stagnant water, respectively. In order to adapt to complex microbial ecosystem, they may develop different cellular regulation, toxic resistance, stress response and metabolic activities.
Cells of strain CCP-1 T are non-motile and rod-shaped ( Supplementary Fig. S6). The presence of bacteriochlorophyll a in strain CCP-1 T is typically indicated by maximum of 765 nm in the absorption spectrum ( Supplementary Fig. S7). Strain CCP-1 T was resistant to sulfamethoxazole plus trimethoprim, and sensitive to chloramphenicol, kanamycin, nalidixic acid, novobiocin, rifampicin, streptomycin, tetracycline, gentamicin, ampicillin and penicillin G. Detailed results from the phenotypic and biochemical analyses of strain CCP-1 T are provided in the species description, Table 2 and Supplementary Table S4.
Phenotypic examination revealed many common traits between the novel strain and the ve reference strains. However, strain CCP-1 T could be clearly differentiated from these ve closest relatives by different properties listed in Table 2. Based on the phylogenetic, phenotypic, chemotaxonomic and biochemical data, it can be concluded that strain CCP-1 T represents a novel member of the genus Rhodobacter, for which the name Rhodobacter ruber sp. nov. is proposed.
Description of Rhodobacter ruber sp. nov.
Supplementary Values are percentages of the total fatty acids; fatty acids that make up < 1 % of the total are not shown or indicated by "-". Major fatty acids (> 50%) are indicated in bold type.
For unsaturated fatty acids, the position of the double bond is located by counting from the methyl (w) end of the carbon chain. cis isomer is indicated by the su x c. *Summed features are groups of two or three fatty acids that cannot be separated by GLC using the MIDI system. Summed feature 3 comprises