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TAXONOMIC DESCRIPTION Sheu et al., Int. J. Syst. Evol. Microbiol. 2020;70:6266–6283 DOI 10.1099/ijsem.0.004526 Tabrizicola oligotrophica sp. nov. and Rhodobacter tardus sp. nov., two new species of bacteria belonging to the family Rhodobacteraceae Ceshing Sheu1, Zhi- Hao Li2, Shih- Yi Sheu3, Che- Chia Yang3 and Wen- Ming Chen2,* Abstract Two Gram-stain- negative, aerobic, non- motile bacteria, designated KMS-5T and CYK-10T, were isolated from freshwater envi- ronments. 16S rRNA gene sequence similarity results indicated that these two novel strains belong to the family Rhodobacte- raceae. Strain KMS-5T is closely related to species within the genus Tabrizicola (96.1–96.8 % sequence similarity) and Cypionkella (96.5–97.0 %). Strain CYK-10T is closest to Rhodobacter thermarum YIM 73036T with 96.6 % sequence similarity. Phylogenetic analyses based on 16S rRNA gene sequences and an up- to- date bacterial core gene set showed that strain KMS-5T is affiliated with species in the genus Tabrizicola and strain CYK-10T is placed in a distinct clade with Rhodobacter blasticus ATCC 33485T, Rhodobacter thermarum YIM 73036T and Rhodobacter flagellatus SYSU G03088T. These two strains shared common chemotaxo- nomic features comprising Q-10 as the major quinone, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcho- line as the principal polar lipids, and C18 : 1 ω7c as the main fatty acid. The average nucleotide identity, average amino acid identity and digital DNA–DNA hybridization values between these two novel isolates and their closest relatives were below the cut-off values of 95–96, 90 and 70 %, respectively, used for species demarcation. The obtained polyphasic taxonomic data suggested that strain KMS-5T represents a novel species within the genus Tabrizicola, for which the name Tabrizicola oligotrophica sp. nov. is proposed with KMS-5T (=BCRC 81196T=LMG 31337T) as the type strain, and strain CYK-10T should represent a novel species of the genus Rhodobacter, for which the name Rhodobacter tardus sp. nov. is proposed with CYK-10T (=BCRC 81191T=LMG 31336T) as the type strain. The family Rhodobacteraceae proposed in 2005 by Garrity from a freshwater lake [5], Tabrizicola fusiformis isolated et al. [1, 2] and recently emended by Hördt et al. [3] cur- from a waste- activated sludge [7], Tabrizicola sediminis rently contains 180 genera (https:// lpsn. dsmz. de/ family/ isolated from the sediment of saline lake [8], Tabrizicola rhodobacteraceae) and is one of the major groups of the alkalilacus isolated from an alkaline lake [9] and Tabrizicola order Rhodobacterales in the class Alphaproteobacteria [4]. piscis isolated from the intestinal tract of a freshwater fish The genus Tabrizicola (type species, Tabrizicola aquatica) [10]. Cells are Gram- stain- negative, aerobic, chemoor- proposed by Tarhriz et al. [5] and recently emended by ganotrophic, rod- shaped, non- motile, catalase- positive Tarhriz et al. [6], belongs to the family Rhodobacteraceae. and oxidase- positive. Some species of this genus produce The genus Tabrizicola presently comprises five species with bacteriochlorophyll a under aerobic, heterotrophic condi- validly published names: Tabrizicola aquatica, isolated tions and possess the photosynthesis-related genes pufLM. Author affiliations: 1Department of Applied Chemistry, Chaoyang University of Technology, No.168, Jifong E. Rd., Wufeng, Taichung, Taiwan, ROC; 2Laboratory of Microbiology, Department of Seafood Science, National Kaohsiung University of Science and Technology, Kaohsiung City 811, No. 142, Hai- Chuan Rd. Nan- Tzu, Kaohsiung City 811, Taiwan, ROC; 3Department of Marine Biotechnology, National Kaohsiung University of Science and Technology, No. 142, Hai- Chuan Rd. Nan- Tzu, Kaohsiung City 811, Taiwan, ROC. *Correspondence: Wen- Ming Chen, p62365@ ms28. hinet. net Keywords: Alphaproteobacteria; polyphasic taxonomy; Rhodobacter tardus sp. nov.; Rhodobacteraceae; Rhodobacterales; Tabrizicola oligotrophica sp. nov.. Abbreviations: AAI, average amino acid identity; AL, uncharacterized aminolipid; ANI, average nucleotide identity; APL, uncharacterized aminophospholipid; dDDH, digital DNA–DNA hybridization; DPG, diphosphatidylglycerol; L, uncharacterized lipid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PL, uncharacterized phospholipid; POCP, percentage of conserved proteins; Q-10, ubiquinone-10; UBCG, up- to- date bacterial core gene set. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and the whole genome of Tabrizicola oligotrophica KMS-5T are MK215682 and NZ_JAAIVJ000000000, respectively. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and the whole genome of Rhodobacter tardus CYK-10T are MK209068 and NZ_JAABNR000000000, respectively. Eleven supplementary figures and five supplementary tables are available with the online version of this article. 004526 © 2020 The Authors 6266 Sheu et al., Int. J. Syst. Evol. Microbiol. 2020;70:6266–6283 Chemotaxonomically, cells possess ubiquinone-10 (Q-10) MORPHOLOGY AND PHYSIOLOGY as the major respiratory quinone, C ω7c and C ω7c 18 : 1 18 : 1 The morphology of bacterial cells was observed by phase- 11- methyl as the predominant fatty acids, C 3- OH as the 18 : 0 contrast microscopy (Leica DM 2000) and transmission cellular hydroxy fatty acid, and DNA G+C contents between electron microscopy (H-7500, Hitachi) using cells grown on 62.9 and 65.9 mol% [5–10]. R2A agar. The Gram Stain Set S kit (BD Difco) and the Ryu The genus Rhodobacter (type species, Rhodobacter capsulatus) non- staining KOH method were used for testing the Gram proposed by Imhoff et al. [11] and emended by Wang et al. reaction. Motility was tested by the hanging drop method [25]. [12] and Suresh et al. [13], belongs to the family Rhodobacte- The Spot Test Flagella Stain (BD Difco) was used for flagellum raceae [14]. The genus Rhodobacter comprises 13 species with staining. Poly-β-hydroxybutyrate granule accumulation was validly published names so far stated on the List of Prokaryotic examined under light microscopy after staining of the cells names with Standing in Nomenclature (https:// lpsn. dsmz. de/ with Sudan black [26] and visualized by UV illumination genus/ rhodobacter). Species of the genus Rhodobacter have after directly staining growing bacteria on plates containing been isolated from various environmental sources, including Nile red [27]. Colony morphology was observed on R2A agar eutrophic freshwater, stagnant water, polluted water, alkaline under a stereoscopic microscope (SMZ 800, Nikon). water, hot spring, lagoon sediment, estuarine water and The pH range for growth was determined by measuring marine water [15–24]. Cells are Gram- stain- negative, motile the optical densities (absorbance at 600 nm) of R2A broth or non- motile and ovoid to rod- shaped. Most species are cultures. The pH of the medium was adjusted prior to steri- freshwater bacteria and do not require salt, but some species lization to pH 4.0–11.0 (at intervals of 0.5 pH unit) using the require salt to adapt to marine environments. Q-10 is the following biological buffers [28]: 100 mM citrate/Na HPO major respiratory quinone, C ω7c is the predominant fatty 2 4 18 : 1 (pH 4.0–5.5); 100 mM phosphate (pH 6.0–7.5); and 100 mM acid, phosphatidylethanolamine, phosphatidylglycerol and Tris (pH 8.0–9.0). The temperature range for growth was phosphatidylcholine are the principal polar lipids and the determined on R2A agar at 4, 10, 15, 20, 25, 30, 35, 37, 40, 45 DNA G+C content is 62.9–70.6 mol% [4, 12–14]. and 50 °C. To investigate the tolerance to NaCl, R2A broth was In the present study, we report the phenotypic and genomic prepared according to the formula of the BD Difco medium characteristics of strains KMS-5T and CYK-10T, which were with NaCl concentration adjusted to 0, 0.5 and 1–5 %, w/v isolated from a freshwater ditch and a freshwater pond, (at intervals of 1 %). Growth under anaerobic conditions was respectively. Based on the taxonomic distinctiveness of the determined after incubating bacterial cells on R2A agar and T two strains, strain KMS-5 belongs to the genus Tabrizicola on R2A agar supplemented with nitrate (0.1 % KNO3) in the and strain CYK-10T belongs to the genus Rhodobacter. Oxoid AnaeroGen system. Bacterial growth was studied on R2A, nutrient, Luria–Bertani and trypticase soy agars (all from Difco) under aerobic conditions at 25 or 30 °C. Photo- HOME HABITAT AND ISOLATION OF THE heterotrophic growth under anaerobic conditions was deter- BACTERIAL STRAINS mined after incubation in an Oxoid AnaeroGen system or in T 30 ml tubes with a rubber septum under a stream of nitrogen Strain CYK-10 was isolated from a freshwater pond in gas in light using minimal medium containing yeast extract ′ ″ ′ ″ Hinoki Village (GPS location: 23° 45 34 N, 121° 26 49 E) (0.3 %, w/v), tryptone (0.3 %, w/v) or sodium acetate (0.3 %, in Chiayi County, Taiwan on 8 February 2015 (Fig. S1, avail- w/v). Photoautotrophic growth was examined under the same able in the online version of this article). The water sample conditions but using a medium with thiosulfate (0.1 %, w/v) was characterized by temperature of 25 °C, pH value of 7.5 T or sodium bicarbonate (0.1 %, w/v) as previously described and NaCl concentration of 0 %. Strain KMS-5 was isolated by Pfennig and Trüper [29]. For photosynthetic pigment from a freshwater ditch in the Shuangsi Forest Recreation analysis, cell mass from 30 ml culture was extracted and the ′ ″ ′ ″ Area (GPS location: 22° 55 59 N, 120° 35 29 E; Fig. S2) absorption spectrum of the extract was recorded as described in Meinong District of Kaohsiung County, Taiwan on 8 by Biebl et al. [30]. The absorption spectra of the pigments March 2018. The water sample was characterized by 25 °C, were examined on a spectrophotometer, and the presence of pH 7 and 0 % NaCl. Two novel strains were isolated as single bacteriochlorophyll a in bacterial strains is typically indicated colonies after plating serially diluted freshwater samples on by maxima of 367 and 755 nm in the absorption spectrum.

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