Tabrizicola Oligotrophica Sp. Nov. and Rhodobacter Tardus Sp. Nov., Two New Species of Bacteria Belonging to the Family Rhodobacteraceae

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 Li 2, Shih-Yi Sheu 3, Che-Chia Yang 3 and Wen-Ming Chen 2,*

Abstract Two Gram-stain- negative, aerobic, non-motile bacteria, designated KMS-5T and CYK-10T, were isolated from freshwater environments. 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 chemotaxonomic 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.

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. Reasoner's 2A (R2A) agar (BD Difco) at 25 °C for 3 days. After the optimum growth temperature had been determined, both Activities of catalase, oxidase, DNase, urease, lipase (corn strains were routinely sub-cultured under the same conditions oil), hydrolysis of starch, casein, gelatin, lecithin and Tweens and preserved in R2A broth with 20 % (v/v) glycerol at −80 °C 20, 40, 60 and 80 were determined using standard methods and also by lyophilization before storing at −80 °C. In addi- [31]. Chitin hydrolysis was assessed on chitinase-detection tion, three phylogenetic related strains, T. aquatica RCRI19T agar [32] and visualized by the formation of clear zones (=KCTC 23724T), T. sediminis DRYC-M-16T (=KCTC 72105T) around the colonies. Hydrolysis of carboxymethyl cellulose and T. alkalilacus DJCT (=KCTC 62173T), were obtained from (CM-cellulose) was tested by following the method described their culture collections, and used as reference strains and by Bowman using R2A agar as the basal medium [33]. Utiliza- evaluated together with strain KMS-5T under identical experi- tion of carbon sources was investigated in a basal medium −1 mental conditions. containing (l ): 0.4 g KH2PO4, 0.53 g Na2HPO4, 0.3 g NH4Cl,

6267 Sheu et al., Int. J. Syst. Evol. Microbiol. 2020;70:6266–6283

0.3 g NaCl, 0.1 g MgCl2·6H2O, 0.11 g CaCl2 and 1 ml trace (3.4 %) (Table S2). The major cellular fatty acid of this novel element solution, pH 7.0 [34]. Substrates were added at a strain was C18 : 1 ω7c, which is consistent with those reported concentration of 0.1 % (w/v) and the tubes incubated under for the genus Rhodobacter [4, 12, 22–24]. aerobic conditions at 25 or 30 °C. Occurrence of bacterial Polar lipids were extracted and analysed by two-dimensional growth was checked for 21 days at 1 day intervals. Additional TLC according to Embley and Wait [37]. Molybdophosphoric biochemical tests were performed using API ZYM and API acid was used for the detection of total polar lipids, ninhydrin 20NE kits (bioMérieux) according to the manufacturer's for amino lipids, Zinzadze reagent for phospholipids, Dragen- recommendations. dorff reagent for choline-containing lipids and α-naphthol Sensitivity to antibiotics of strains KMS-5T and CYK-10T reagent for glycolipids. Strain KMS-5T exhibited a complex was tested by the disc diffusion method after spreading cell polar lipid profile consisting of phosphatidylethanolamine suspensions (0.5 McFarland) on R2A agar plates as described (PE), phosphatidylglycerol (PG), phosphatidylcholine (PC), by Nokhal and Schlegel [35]. The discs (Oxoid) contained two uncharacterized aminophospholipids (APL1 and APL2), the following antibiotics: ampicillin (10 µg), chloramphenicol one uncharacterized phospholipid (PL) and one uncharacter- (30 µg), gentamicin (10 µg), kanamycin (30 µg), nalidixic acid ized aminolipid (AL) (Fig. S6). The predominant and known (30 µg), novobiocin (30 µg), rifampicin (5 µg), penicillin G polar lipids were PE, PG and PC, consistent with previous (10 U), streptomycin (10 µg), sulfamethoxazole/trimethoprim descriptions of species of Tabrizicola [5, 7, 8], but a difference (23.75 and 1.25 µg, respectively) and tetracycline (30 µg). Both being that diphosphatidylglycerol (DPG) was not detected strains KMS-5T and CYK-10T were sensitive to rifampicin, in strain KMS-5T. PE, PG and PC were major and known gentamicin, tetracycline, novobiocin, ampicillin, kanamycin, polar lipids of strain CYK-10T, and several uncharacterized chloramphenicol, nalidixic acid, penicillin G and strepto- aminophospholipids (APL1 and APL2), phospholipids (PL1 mycin. However, strain KMS-5T was resistant to sulfameth- and PL2), aminolipids (AL1 and AL2) and lipids (L1 and L2) oxazole/trimethoprim, whereas strain CYK-10T was sensitive. were detected as minor lipids in strain CYK-10T (Fig. S7). The major polar lipids were PE, PG and PC, which is the same as Cells of strain KMS-5T were aerobic, non-motile and rod- most members of the genus Rhodobacter [4, 12, 13, 20]. shaped (Fig. S3). Cells of strain CYK-10T were aerobic, non- motile and ovoid to rod-shaped (Fig. S4). Strain CYK-10T Isoprenoid quinones were extracted and purified according to exhibited a maximum of 755 nm in the absorption spectrum the method of Collins and were analysed by HPLC [38]. The (Fig. S5), which indicated the presence of bacteriochlorophyll major respiratory quinone of strain KMS-5T was Q-10 (Fig. a. Detailed results from the phenotypic and biochemical S8), which is consistent with the other four members of the analyses of strains KMS-5T and CYK-10T are provided in the genus Tabrizicola [5, 7–10]. Strain CYK-10T had Q-10 as the species descriptions and Tables 1 and 2. major respiratory quinone (Fig. S8), in line with members of the genus Rhodobacter [4, 11].

CHEMOTAXONOMY The fatty acid profiles of the bacterial cells were determined, 16S rRNA GENE PHYLOGENY and the fatty acid methyl esters were prepared and separated Genomic DNA was extracted using a bacterial genomic DNA according to the instructions of the Microbial Identification purification kit, and primers 27F and 1541R were used for System (midi), analysed by GC (Hewlett-Packard 5890 Series amplification of bacterial 16S rRNA genes by PCR [39, 40]. II) and identified by midi version 6.0 and the RTSBA6.00 The PCR product was purified using a plus PCR clean up kit, databases [36]. The predominant cellular fatty acids (>70 % of and then sequenced using primers 27F, 1541R, 520F and 800R T the total fatty acids) of strain KMS-5 were C18 : 1 ω7c (75.3 %) [39, 40] and the BigDye Terminator Cycle Sequencing kit (Table S1). The fatty acid profile of strain KMS-5T was similar (Applied Biosystems) with an ABI Prism 3730xl automated to those of the other Tabrizicola species, although there were DNA analyser (Applied Biosystems). The sequenced lengths differences in the proportions of some components such of the 16S rRNA genes of strains KMS-5T and CYK-10T were as C16 : 0, C18 : 0 and C18 : 1 ω7c 11-methyl. In addition, summed 1438 bp and 1441 bp, respectively. These gene sequences were feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) was only detected in compared to those in EzBioCloud [41]. Multiple sequence strain KMS-5T and T. alkalilacus DJCT, and summed feature alignments were performed with clustal x version 2.0 [42]

7 (C19 : 1 ω6c and/or C19 : 0 cycloω10c) was only detected in and BioEdit software [43]. Phylogenetic trees were recon- T. aquatica RCRI19T and T. alkalilacus DJCT. However, their structed by the neighbour-joining [44], maximum-likelihood major fatty acid was C18 : 1 ω7c, which constitutes 69–75 % of [45] and maximum-parsimony [46] methods using mega X the total fatty acids, and they all had C10 : 0 3-OH and C18 : 0 software [47]. In each case bootstrap values were calculated 3-OH as the predominant hydroxyl fatty acids. based on 1000 replications. The major cellular fatty acid (>60 %) of strain CYK-10T The 16S rRNA gene sequence analysis indicated that strains T T T was C18 : 1 ω7c. The cellular fatty acids of strain CYK-10 KMS-5 and CYK-10 both belonged to the family Rhodobac- were composed of C18 : 1 ω7c (64.8 %), C16 : 0 (8.0 %), C18 : 1 ω7c teraceae within the order Rhodobacterales, in class Alphapro- 11-methyl (5.9 %), C10 : 0 3-OH (5.8 %), summed feature 3 teobacteria. Phylogenetic analyses based on 16S rRNA gene T (C16 : 1 ω7c and/or C16 : 1 ω6c; 4.6 %), C18 : 0 3-OH (4.4 %) and C18 : 0 sequences revealed that strain KMS-5 was related to the

6268 Sheu et al., Int. J. Syst. Evol. Microbiol. 2020;70:6266–6283

Table 1. Differential characteristics ofTabrizicola oligotrophica KMS-5T and phylogenetically closely related Tabrizicola species Strains: 1, KMS-5T; 2, Tabrizicola aquatica RCRI19T; 3, Tabrizicola sediminis DRYC-M-16 T; 4, Tabrizicola alkalilacus DJCT; 5, Tabrizicola fusiformis SY72T. Data are from this study except those for T. fusiformis SY72T taken from Ko et al. [7], and for the DNA G+C contents of T. aquatica RCRI19T (NCBI database, NZ_PJON00000000), T. sediminis DRYC-M-16 T (Z_RPEM00000000), T. alkalilacus DJCT (Z_QWEY00000000) and T. fusiformis SY72T (NZ_ JABUHN000000000). +, Positive reaction; −, negative reaction. All strains are aerobic, non-motile and positive for oxidase, catalase, C8 esterase lipase, valine arylamidase and α-glucosidase, and activities and utilization of glucose, fructose, galactose and mannose as carbon sources. All strains are negative for: Gram staining; nitrate reduction; indole production; trypsin, α-chymotrypsin, β-glucuronidase, α-mannosidase and α-fucosidase activities and hydrolysis of gelatin.

Characteristic 1 2 3 4 5

Isolation source Freshwater ditch Freshwater lake Sediment of saline lake Alkaline lake Industrial wastewater treatment plant

Colony pigmentation Translucent white Cream Pink Cream Translucent white

Temperature range for growth (°C) (optimum) 15–35 (30) 15–45 (40) 4–35 (20–25) 15–37 (25–30) 15–40 (30–37)

pH range for growth (optimum) 5.5–8.5 (6–8) 6–9 (7) 7–9 (7.0–7.5) 6–10 (7–8) 6–8 (6–7)

NaCl range for growth (%) (optimum) 0–0.5 (0) 0–3 (0–1) 1–2 (1.5) 0–3 (0–1) 0–1 (0)

Glucose fermentation − − − + −

Hydrolysis of:

Urea − − − + +

Aesculin − + + + +

Enzymatic activities:

Arginine dihydrolase − − − + +

Alkaline phosphatase + − + + +

C4 Esterase + − + + +

C14 Lipase − − − − +

Leucine arylamidase + + + + −

Cystine arylamidase − − − + −

Acid phosphatase + + + − +

Naphthol-AS-BI-phosphophydrolase + − + − −

α-Galactosidase − + + − −

β-Galactosidase − + + + −

β-Glucosidase − + + + +

N-Acetyl-β-glucosaminidase − − − − +

Utilization of:

Sucrose + + + + −

Arabinose + + − + +

Maltose − + + + +

Trehalose − − + + −

Cellobiose − + + + +

Raffinose − − + + −

Mannitol + + − + +

Sorbitol + + − + −

Glycerol − − + − +

DNA G+C content (mol%) 65.5 66.4 63.0 62.9 65.1

6269 Sheu et al., Int. J. Syst. Evol. Microbiol. 2020;70:6266–6283

Table 2. Differential characteristics ofRhodobacter tardus CYK-10T and the type strains of related Rhodobacter species Strains: 1, CYK-10T; 2, Rhodobacter thermarum YIM 73036T; 3, Rhodobacter flagellatus SYSU G03088T; 4, Rhodobacter blasticus ATCC 33485T; 5, Rhodobacter capsulatus ATCC 11166T. Data for R. thermarum YIM 73036T from Khan et al. [22], R. flagellatus SYSU G03088T and R. blasticus ATCC 33485T from Xian et al. [23] and R. capsulatus ATCC 11166T from Wang et al. [12] and from Subhash and Lee [19]. +, Positive reaction; –, negative reaction.

Characteristic 1 2 3 4 5

Isolation source Freshwater pond Sediment of a hot spring Hot spring Eutrophic freshwater Stagnant water

Motility − − + − +

Temperature range for growth (°C) (optimum) 20–35 (25) 20–55 (37–45) 28–50 (45) 28–37 (30) 15–40 (30)

pH range for growth (optimum) 6.0–7.5 (7) 6–8 (7.0–7.5) 7–8 (7) 7–8 (7) 6–8 (6–7)

NaCl range for growth (%) 0–0.5 0.5–3.5 0–1 0–2 0–2

Enzymatic activities (API ZYM):

Alkaline phosphatase + + + − +

C4 Esterase + + + + −

C8 Esterase lipase + + + + −

C14 Lipase − + − − −

Valine arylamidase − + + + −

Cystine arylamidase − + + + −

Trypsin − + + − −

α-Chymotrypsin − − + − −

Acid phosphatase + − + − +

Naphthol-AS-BI-phosphohydrolase + + + − −

α-Galactosidase + − − − −

β-Galactosidase + + + − −

β-Glucosidase + + + − − genera Tabrizicola (96.1–96.8 % sequence similarity), Cypi- Rhodobacter capsulatus ATCC 11166T. Phylogenetic analysis onkella (96.5–97.0 %), Gemmobacter (<95.9 %), Rhodobacter based on 16S rRNA gene sequence indicated that strain (<95.9 %), Haematobacter (<95.5 %) and Pseudorhodobacter CYK-10T formed a separate phylogenetic branch clustered (<95.5 %). Phylogenetic analysis based on 16S rRNA gene with Rhodobacter blasticus ATCC 33485T, R. thermarum YIM sequences revealed that strain KMS-5T formed a mono- 73036T and R. flagellatus SYSU G03088T in the neighbour- phyletic clade with species of the genus Tabrizicola in the joining tree (Fig. 1). neighbour-joining tree (Fig. 1). The overall topologies of the maximum-likelihood and maximum-parsimony trees were similar (Figs S9 and S10). However, although strain KMS-5T GENOME FEATURES had the highest similarity to Cypionkella collinsensis 4 T-34T To further investigate the taxonomic rank of strain KMS-5T (97.0 %) and Cypionkella psychrotolerans PAMC 27389T and strain CYK-10T, two whole genome sequences were (97.0 %), it is obvious from the phylogenetic tree that they prepared by the Genomics BioSci and Tech. Co., Ltd. (Taipei, belong to different genera. Strain KMS-5T formed a separate Taiwan, ROC) using the Illumina NextSeq sequencer plat- phylogenetic branch cluster with T. alkalilacus DJCT and form; and read quality was evaluated in MultiQC version T. sediminis DRYC-M-16T within the genus Tabrizicola. 1.2 [48]. The whole genome was assembled using SPAdes In addition, sequence similarity calculations revealed that (version 3.10.1) [49]. For strain KMS-5T, 62 contigs were strain CYK-10T was related to the genera Rhodobacter (93.1– obtained, with an average coverage of 190× and an N50 size 96.6 % sequence similarity) and Tabrizicola (94.6–95.7 %). of 252 520 bp. The estimated genome size was 3.85 Mb, with The closest relative of strain CYK-10T was Rhodobacter an average G+C content of 65.5 mol%. Gene prediction and thermarum YIM 73036T (96.6 %), followed by Rhodobacter annotation by Prokka pipeline [50] resulted in the identifica- flagellatus SYSU G03088T (95.8 %), whereas it shared 95.6 % tion of 3638 protein-encoding genes, three rRNA genes and similarity to the type strain of the type species of the genus, 46 tRNA genes. The estimated genome size of CYK-10T was

6270 Sheu et al., Int. J. Syst. Evol. Microbiol. 2020;70:6266–6283

T 100 Deﬂuviimonas albacai 42 (KC222646) 99 Frigidibacter albus SP32T (KF944301) T 100 Haematobacter massiliensis CCUG 47968 (DQ342309) 99 Haematobacter missouriensis CCUG 52307T (DQ342315) Rhodobacter capsulatus DSM 1710T (D13474) 99 Rhodobacter sediminis N1T (LT009496) 96 Rhodobacter viridis JA737T (HE572577) 0.01 90 Rhodobacter azollae JA912T (LN810641) 78 T 98 Rhodobacter lacus JA826 (LN835251) 89 94 Rhodobacter maris JA276T (AM745438) 86 Rhodobacter aestuarii JA296T (AM748926) Rhodobacter tardus CYK-10T (MK209068) 82 Rhodobacter thermarum YIM 73036T (KY608089) 98 96 Rhodobacter ﬂagellatus SYSU G03088T (MN174111) 73 Rhodobacter blasticus NCIMB 11576T (D13478) T 100 Tabrizicola alkalilacus DJC (MF162183) Tabrizicola sediminis DRYC-M-16T (MK693145) Tabrizicola oligotrophica KMS-5T (MK215682) Tabrizicola piscis K13M18T (MK285603) T 93 Tabrizicola aquatica RCRI19 (HQ392507) 73 Tabrizicola fusiformis SY72T (MF543060) T 90 Cypionkella collinsensis 4-T-34 (KM978076) 93 Cypionkella psychrotolerans PAMC 27389T (KT163920) Cypionkella aquatica DC2N1-10T (KT985057) Cypionkella sinensis Y1R2-4T (KT985055) T 99 Pseudorhodobacter aquimaris HDW-19 (GU086365) 99 Pseudorhodobacter ponti HWR-46T (KX771233) Pseudorhodobacter wandonensis KCTC 23672T (JN247434) 88 T 76 79 Pseudorhodobacter antarcticus CGMCC 1.10836 (FJ196030) Pseudorhodobacter ferrugineus IAM 12616T (D88522) Rhodobacter sediminicola JA983T (LR596790) 90 78 Rhodobacter alkalitolerans JA916T (LN810645) Rhodobacter megalophilus DSM 18937T (AM421024) Gemmobacter tilapiae Ruye-53T (HQ111526) Gemmobacter aquatilis DSM 3857T (FR733676) Gemmobacter megaterium DSM 26375T (JN620361) Pararhodobacter aggregans D1-19T (AM403160) Pararhodobacter zhoushanesis ZQ420T (MH087458) Falsirhodobacter halotolerans JA744T (HE662814) Yoonia rosea Fg36T (AY682199)

Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the positions of Tabrizicola oligotrophica KMS-5T, Rhodobacter tardus CYK-10T and closely related taxa within the family Rhodobacteraceae. Numbers at nodes are bootstrap percentages (>70 %) based on the neighbour-joining (above nodes) and maximum-parsimony (below nodes) tree-making algorithms. Filled circles indicate nodes that are also found with the maximum-likelihood and maximum-parsimony algorithms. Yoonia rosea Fg36T was used as an outgroup. Bar, 0.01 substitutions per nucleotide position.

6271 Sheu et al., Int. J. Syst. Evol. Microbiol. 2020;70:6266–6283

T 92 Cereibacter sphaeroides NBRC 12203 92 Cereibacter johrii JA192T 91 Cereibacter azotoformans KA25T 88 Cereibacter ovatus JA234T Cereibacter changlensis DSM 18774T 39 T 92 Haematobacter missouriensis CCUG 52307 32 Haematobacter massiliensis CCUG 47968T 92 0.05 Frigidibacter albus SP32T 25 Gemmobacter megaterium DSM 26375T Gemmobacter aquatilis DSM 3857T

T 92 Tabrizicola piscis K13M18 Tabrizicola aquatica RCRI19T 64 36 Tabrizicola oligotrophica KMS-5T 92 Tabrizicola fusiformis SY72T 90 Tabrizicola sediminis DRYC-M-16T 92 Tabrizicola alkalilacus DJCT Cypionkellapsychrotolerans PAMC 27389T Pseudorhodobacter aquimaris KCTC 23043T Pseudorhodobacter wandonensis KCTC 23672T 92 T 92 Pseudorhodobacter ferrugineus DSM 5888 92 Pseudorhodobacter antarcticus KCTC 23700T Yoonia rosea DSM 29591T

Fig. 2. Phylogenetic tree inferred using UBCGs (concatenated alignment of 92 core genes) showing the position of Tabrizicola oligotrophica KMS-5T and closely related taxa within the family Rhodobacteraceae. The number of single gene trees supporting a branch in a UBCG tree is calculated and designated the gene support index (GSI). The GSIs are given at branching points. Yoonia rosea DSM 29591T was used as an outgroup. Bar, 0.05 substitutions per position.

4.59 Mb, with 4240 protein-encoding genes, three rRNA supported that these two novel strains KMS-5T and CYK-10T genes and 50 tRNA genes. The genome was sequenced at should be assigned to novel species of the genera Tabrizicola approximately above 185× coverage, having 114 contigs in and Rhodobacter, respectively. the genome assembly, with an N50 of 193 916 bp. The DNA Average nucleotide identity (ANI) calculations were G+C content calculated from the genome was 66 mol%. The performed by OrthoANI analysis [52], which gave sequences of the 16S rRNA gene from these two genomes OrthoANI values of 76.0, 74.9, 75.3, 75.3 and 77.9 % when and those of PCR-determined sequences are very close but strain KMS-5T was compared to T. aquatica RCRI19T, not identical with one nucleotide difference. However, the T. sediminis DRYC-M-16T, T. alkalilacus DJCT, T. piscis original sequences determined by PCR have been corrected K13M18T and T. fusiformis SY72T. These values were lower to the sequences from these two genomes for the 16S rRNA than the 95–96 % cut-off values previously proposed for gene analysis. species delimitation [53]. Average amino acid identity (AAI) In order to further explore the relationships among strain calculations were performed (http://enve-omics.ce.gatech. KMS-5T, strain CYK-10T and related genera within the edu/), which gave AAI values of 74.0, 73.3, 73.6, 73.8 and family Rhodobacteraceae, an up-to- date bacterial core gene 76.5 % when compared to T. aquatica RCRI19T, T. sediminis set (UBCG) and pipeline was utilized for phylogenetic tree DRYC-M-16T, T. alkalilacus DJCT, T. piscis K13M18T and reconstruction [51]. The phylogenetic tree based on the T. fusiformis SY72T. These data were below the threshold coding sequences of 92 protein clusters (Fig. 2) showed that of 90 % for species boundary and above the threshold of strain KMS-5T formed a distinct phylogenetic lineage within 60 % for genus boundary [54]. The percentage of conserved the genus Tabrizicola. The phylogenetic analysis and clustering proteins (POCP) values were also calculated as described based on the UBCG pipeline also showed the affiliation of by Qin et al. [55]. The POCP values between strain KMS-5T strain CYK-10T to the genus Rhodobacter (Fig. 3). These data and T. aquatica RCRI19T, T. sediminis DRYC-M-16T,

6272 Sheu et al., Int. J. Syst. Evol. Microbiol. 2020;70:6266–6283

T 0.05 Rhodobacter veldkampii DSM 11550 Rhodobacter vinaykumarii JA123T

38 T 73 Rhodobacter aestuarii JA296 Rhodobacter maris JA276T 86 Rhodobacter viridis JA737T 87 Rhodobacter capsulatus DSM 1710T Rhodobacter tardus CYK-10T 49 Rhodobacter blasticus DSM 2131T

T 51 Rhodobacter ﬂagellatus SYSU G03088 86 Rhodobacter thermarum YIM 73036T

T 84 Rhodobacter sediminicola JA983 T 46 Cereibacterazotoformans KA25 76 T 81 Rhodobacter megalophilus DSM 18937 76 Cereibacter sphaeroides NBRC 12203T 90 Cereibacter johrii JA192T

T 76 Cereibacter ovatus JA234 Cereibacter changlensis DSM 18774T Yoonia rosea DSM 29591T

Fig. 3. Phylogenetic tree inferred using UBCGs (concatenated alignment of 92 core genes) showing the position of Rhodobacter tardus CYK-10T and type strains of species of the genus Rhodobacter. The number of single gene trees supporting a branch in a UBCG tree is calculated and designated the gene support index (GSI). The GSIs are given at branching points. Yoonia rosea DSM 29591T was used as an outgroup. Bar, 0.05 substitutions per position.

T. alkalilacus DJCT, T. piscis K13M18T and T. fusiformis RCRI19T, T. sediminis DRYC-M-16T, T. alkalilacus DJCT, SY72T were 62.0, 58.6, 55.4, 69.4 and 65.5 %, respectively. T. piscis K13M18T and T. fusiformis SY72T had an interesting All values are higher than 50 % which is considered as the pattern, all strains had some genes in common and some boundary to separate genera [55]. The estimated genome- genes differed among them (Table 3). The most obvious sequence-based digital DNA–DNA hybridization (dDDH) differences are that strain KMS-5T and T. fusiformis SY72T values were calculated as described by Meier-Kolthoff et have no genes related to photosynthesis such as chlorophyll al. [56]. The dDDH values between strain KMS-5T and biosynthesis, bacterial light-harvesting proteins and photo- T. aquatica RCRI19T, T. sediminis DRYC-M-16T, T. alkali- system II-type photosynthetic reaction centre including lacus DJCT, T. piscis K13M18T and T. fusiformis SY72T were light-harvesting LHI, alpha subunit and beta subunit, 21.9, 22.8, 22.5, 22.4 and 20.9 %, respectively, which is below photosynthetic reaction centre L subunit, M subunit and the threshold of 70 % for species delineation [57]. These data H subunit, protein PufQ (involved in assembly of B875 and T confirmed that strain KMS-5 represents a distinct species B800-850 pigment-protein complexes), putative photo- of the genus Tabrizicola. Furthermore, these genome relat- synthetic complex assembly protein, protoporphyrin IX T edness values between strain CYK-10 and Rhodobacter Mg-chelatase subunit H (EC 6.6.1.1), Mg protoporphyrin species with available genomes were 70.4–74.8 % (for ANI), O-methyltransferase (EC 2.1.1.11), Mg protoporphyrin 68.0–73.3 % (for AAI) and 22.0–23.2 % (for dDDH), repre- IX monomethyl ester oxidative cyclase (aerobic) (EC T senting that strain CYK-10 is a novel species separated 1.14.13.81), light-independent protochlorophyllide reduc- from pre-existing Rhodobacter species (Table S5). tase iron-sulphur ATP-binding protein ChlL (EC 1.18.-.-), For gene function prediction, the obtained genome chlorophyll a synthase ChlG (EC 2.5.1.62), geranylgeranyl sequences of strain KMS-5T and strain CYK-10T were hydrogenase BchP, geranylgeranyl reductase (EC 1.3.1.83), annotated by the NCBI Prokaryotic Genome Annota- chlorophyllide reductase subunit BchZ (EC 1.18.-.-), tion Pipeline and also submitted to Rapid Annotation of 2-vinyl bacteriochlorophyllide hydratase BchF (EC 4.2.1.-) microbial genomes using Subsystem Technology (rast) and 2-desacetyl-2-hydroxyethyl bacteriochlorophyllide for further comparative analyses [58, 59]. An overview A dehydrogenase BchC. However, T. aquatica RCRI19T, of genome characteristics of these strains is given in T. sediminis DRYC-M-16T, T. alkalilacus DJCT and T. piscis Table S3. The gene content of strain KMS-5T, T. aquatica K13M18T possess these photosynthesis-related genes

6273 Sheu et al., Int. J. Syst. Evol. Microbiol. 2020;70:6266–6283

Table 3. Comparison of the presence and absence of selected genes among Tabrizicola oligotrophica KMS-5T and five strains of the genus Tabrizicola Strains: 1, KMS-5T; 2, Tabrizicola aquatica RCRI19T; 3, Tabrizicola sediminis DRYC-M-16 T; 4, Tabrizicola alkalilacus DJCT; 5, Tabrizicola piscis K13M18T; 6, Tabrizicola fusiformis SY72T. +, Present; −, absent.

Genes putatively encoding 1 2 3 4 5 6

Photosynthesis

Chlorophyll biosynthesis − + + + + −

Bacterial light-harvesting proteins − + + + + −

Photosystem II-type photosynthetic reaction centre − + + + + −

Cell wall and capsule

CMP-N-acetylneuraminate biosynthesis − + + + + −

Legionaminic acid biosynthesis − + + + + −

Lipopolysaccharide assembly − + + + + −

Iron acquisition and metabolism

Heme, hemin uptake and utilization systems − + + + + −

Hemin transport system − + + + + −

Resistance to antibiotics and toxic compounds

Multidrug resistance efflux pumps − + + + + +

Arsenic resistance − − + + − +

Cofactors and vitamins

Biotin biosynthesis + + + + + +

Heme biosynthesis orphans + − − − − −

Coenzyme B12 biosynthesis + − + + − +

Flavodoxin + + − − + +

Molybdenum cofactor biosynthesis − + − − + −

Membrane transport

Protein secretion system, type I (for aggregation) − − + + − −

Protein and nucleoprotein secretion system, type IV (Vir-like type four + − − − − − secretion system, pVir plasmid of Campylobacter)

Protein secretion system, type VI − + − − + +

ABC transporter (periplasmic-binding-protein dependent transport system for − + + + + − α-glucosides)

ABC transporter alkylphosphonate − + + + + +

AttEFGH ABC transport system − + − − + −

Cation transporter (transport of nickel and cobalt) + − + + + +

Multi-submit cation antiporter − + + + + +

RNA metabolism

Possible RNA modification and stress response cluster − + − + − −

Metabolism of nucleoside and nucleotide

Xanthine dehydrogenase subunits + − − − − −

Protein metabolism

N-linked glycosylation in Bacteria + − − + − − Continued

6274 Sheu et al., Int. J. Syst. Evol. Microbiol. 2020;70:6266–6283

Table 3. Continued

Genes putatively encoding 1 2 3 4 5 6

G3E family of P-loop GTPase (metallocenter biosynthesis) − + + + + +

Chemotaxis

Bacterial chemotaxis + + − − − +

Regulation and cell signalling

Oxygen and light sensor PpaA-PpsR − + + + + −

Murein hydrolase regulation and cell death + − + + − +

Toxin-antitoxin replicon stabilization system + + − + − −

Phd-Doc, YdcE-YdcD toxion-antitoxin system + − − + + +

DNA metabolism

Nonhomologus end-joining in Bacteria − + + + + −

CRISPRs + − − − − −

DNA replication strays + − + + − +

Plasmid replication + − + + − +

Type I restriction-modification − + + − − +

Restriction-modification system + + + + − +

DNA ligases − + + + + −

Nitrogen metabolism

Dissimilatory nitric reductase + − − − − −

Nitrate and nitrite ammonification + − + + − +

Nitrilase + − − + − −

Denitrification + + + + − +

Stress response

Osmoregulation + + − + + +

Ectoine biosynthesis and regulation + − + − + −

Synthesis of osmoregulated periplasmic glucans − + + + + +

Dimethylarginine metabolism + + − − + +

Bacterial haemoglobins + + − − + +

Sulphur metabolism

Inorganic sulphur assimilation − − − − − +

Sulphur oxidation − + + + − −

Taurine utilization − − + + + +

Alkanesulfonate utilization − + − + + +

Utilization of glutathione as a sulphur source + − − + − −

Phosphorus metabolism

Alkylphosphonate utilization − + + + + +

Metabolism of aromatic compounds

p-Hydroxybenzoate degradation + − − − − − Continued

6275 Sheu et al., Int. J. Syst. Evol. Microbiol. 2020;70:6266–6283

Table 3. Continued

Genes putatively encoding 1 2 3 4 5 6

Biphenyl degradation − − + + − +

Chloroaromatic degradation pathway − − + + + +

N-Heterocyclic aromatic compound degradation − − + − + −

Protocatechuate branch of beta-ketoadipate pathway + − + + − +

4-Hydroxyphenylacetic acid catabolic pathway + − − − − +

Homogentisate pathway of aromatic compound degradation + − − − − +

Central meta-cleavage pathway of aromatic compound degradation + − − − − +

Aromatic amine catabolism + + − − − +

Metabolism of amino acid and derivatives

Urea carboxylase and allophanate hydrolase cluster + − + − + −

Urease subunits − + + + + +

Urea decomposition − + + + + +

Methionine degradation + − − + − −

HMG-CoA synthesis and HMG-CoA metabolism + − − − − −

Leucine, isoleucine and valine degradation + − − − − −

Carbohydrate metabolism

Maltose and maltodextrin utilization + − − − − +

Trehalose biosynthesis − − + + − −

Sucrose utilization − + + + + +

Lactose and galactose uptake and utilization − + + − + −

Lactate fermentation − − + + + −

Acetoin, butanediol metabolism − − + + + −

CO2 fixation: CO2 uptake, carboxysome + − − − − +

CO2 fixation: photorespiration (oxidative C2 cycle) − − + + − +

CO2 fixation: Calvin–Benson cycle − − − − − +

Mannitol utilization + + − + + +

Alpha-amylase locus in Streptococcus + + − − − +

d-Gluconate and ketogluconate metabolism − + + + + +

d-Galacturonate and d-glucuronate utilization − + + + + +

(Table S4). In addition, T. aquatica RCRI19T, T. sediminis 4-hydroxyphenylacetic acid catabolic pathway, homogen- DRYC-M-16T, T. alkalilacus DJCT and T. piscis K13M18T tisate degradation pathway and central meta-cleavage have several genes putatively encoding proteins associated degradation pathway. with CMP-N-acetylneuraminate biosynthesis, legionaminic T acid biosynthesis, lipopolysaccharide assembly, heme, In addition, strain KMS-5 possesses genes putatively hemin uptake and utilization systems and hemin trans- encoding for heme biosynthesis orphans, protein and port system, but strain KMS-5T and T. fusiformis SY72T nucleoprotein secretion system, type IV (Vir-like type 4 do not have them. Concerning metabolism of aromatic secretion system, pVir plasmid of Campylobacter), xanthine compounds, six strains showed some shared genes in dehydrogenase subunits, CRISPR-associated protein connection with different degradation pathways, but only Cas1 (WP_164626556), CRISPR-associated helicase Cas3 strain KMS-5T and T. fusiformis SY72T have genes for the (WP_164626569), CRISPR-associated protein Cas1 family

6276 Sheu et al., Int. J. Syst. Evol. Microbiol. 2020;70:6266–6283

(WP_164626567), Cas3 family (WP_164626558), Cas4 degradation pathway and aromatic amin catabolism, and family (WP_164626563), Cas5e family (WP_164626561) and related to fermentation, e.g. acetoin and butanediol metabo- dissimilatory nitric reductase, but the other five strains do lism. The other four strains,R. thermarum YIM 73036T, not have these genes. Regarding metabolism of amino acid R. flagellatus SYSU G03088T, R. blasticus ATCC 33485T and and derivatives and metabolism of various carbohydrates, the R. capsulatus ATCC 11166T, did not have these related genes. six strains showed different patterns. However, only strain Instead, these four strains had genes putatively encoding KMS-5T has genes putatively encoding proteins associated proteins involved in cell wall and capsule synthesis such as with HMG-CoA synthesis, HMG-CoA metabolism, leucine, CMP-N-acetylneuraminate biosynthesis, legionaminic acid isoleucine and valine degradation. Only strain KMS-5T does biosynthesis and sialic acid metabolism, involved in cellular not have genes putatively encoding proteins related to urease virulence such as multidrug resistance efflux pumps, involved subunits, urea decomposition, d-gluconate and ketogluconate in membrane transport including ABC transporter alkylphos- metabolism, sucrose, d-galacturonate and d-glucuronate phonate, multi-subunit cation transporter, TRAP transporter, utilization (Table 3). Because strain KMS-5T, T. aquatica transport nickel and cobalt and involved in carbohydrate RCRI19T, T. sediminis DRYC-M-16T, T. alkalilacus DJCT, metabolism, e.g. propionyl-CoA to succinyl-CoA module. T. piscis K13M18T and T. fusiformis SY72T are isolated from The novel strain CYK-10T did not have these related genes. freshwater ditch, freshwater lake, sediment of saline lake, Additionally, R. blasticus ATCC 33485T exhibited genes alkaline lake, the intestinal tract of freshwater fish and an putatively encoding proteins with regarded to denitrification industrial wastewater treatment plant, respectively, different and organic sulphur assimilation (alkanesulfonate utiliza- metabolic abilities are likely crucial for the adaptation in their tion), but the other four strains did not have these related respective environments. genes. R. capsulatus ATCC 11166T exhibited unique genes After annotation byrast , the gene compositions of strain putatively encoding proteins for stress response, e.g. redox- CYK-10T, R. capsulatus ATCC 11166T, R. blasticus ATCC dependent regulation of nucleus processes, for respiration, + 33485T, R. thermarum YIM 73036T and R. flagellatus SYSU e.g. Na translocating NADH-quinone oxidoreductase and + G03088T also showed similar results. Most genes were Na translocating decarboxylase, for potassium metabolism, shared in all strains while some genes were not (Table 4). e.g. glutathion-regulated potassium-efflux system, for iron Five strains had genes putatively encoding proteins asso- acquisition and metabolism e.g. siderophore enterobactin ciated with photosynthesis such as photosystem II-type and ferrous iron transporter EfeUOB (low-pH- induced), for photosynthetic reaction centre (photosynthetic reaction valine and leucine degradation, HMG-CoA synthesis and centre L, M, H subunits and putative photosynthetic HMG-CoA metabolism and for fermentation of mixed acid. T complex assembly protein), bacterial light-harvesting Moreover, R. blasticus ATCC 33485 and R. capsulatus ATCC T proteins (light-harvesting LHI, alpha subunit and beta 11166 had genes putatively encoding proteins concerning subunit) and chlorophyll biosynthesis [protoporphyrin IX DNA metabolism such as CRISPR-associated protein Cas1, Mg-chelatase subunit H (EC 6.6.1.1), Mg-protoporphyrin CRISPR-associated helicase Cas3, CRISPR-associated protein O-methyltransferase (EC 2.1.1.11), Mg-protoporphyrin (Cse1 family, Cse3 family, Cse4 family and Cas5e family), IX monomethyl ester oxidative cyclase (anaerobic) (EC concerning nitrogen fixation, concerning ABC transporter 1.14.13.81), protein BchJ (reduction of C-8 vinyl of divinyl (periplasmic-binding-protein dependent transport system protochlorophyllide), light-independent protochlorophyl- for α-glucosides) and concerning stress response such as lide reductase iron-sulphur ATP-binding protein ChlL synthesis of osmoregulated periplasmic glucans. Three strains, (EC 1.18.-.-), chlorophyll a synthase ChlG (EC 2.5.1.62), R. flagellatus SYSU G03088T, R. blasticus ATCC 33485T and geranylgeranyl hydrogenase BchP/geranylgeranyl reductase R. capsulatus ATCC 11166T, possessed genes putatively

(EC 1.3.1.83), chlorophyllide reductase subunit BchZ (EC encoding proteins with respect to CO2 fixation including CO2 1.18.-.-), 2-vinyl bacteriochlorophyllide hydratase BchF uptake, carboxysome, photorespiration (oxidative C2 cycle) (EC 4.2.1.-) and 2-desacetyl-2- hydroxyethyl bacteriochlo- and the Calvin–Benson cycle. rophyllide A dehydrogenase BchC]. Furthermore, the percentage of genes that strain KMS-5T Other features are that only strain CYK-10T possessed genes shared with its related type species of the genus Tabrizicola putatively encoding proteins related to transposable elements, was estimated. On the basis of the data obtained from e.g. TniB NTP-binding protein, TniA putative transposase, edgar 2.0, an enhanced software platform for comparative Mg2+ chelatase family protein/ComM-related protein, gene content analyses [60], when strain KMS-5T, T. aquatica plasmid replication protein RepA, segregation and conden- RCRI19T, T. sediminis DRYC-M-16T, T. alkalilacus DJCT and sation protein B, protein of unknown function DUF1403, T. piscis K13M18T were analysed together, it can be found related to membrane transport e.g. protein and nucleopro- that there are 1928 genes in common, which is about 52.6 % tein secretion system (type IV), protein secretion system of the total number of genes of strain KMS-5T. For this (type VI), AttEFGH ABC transport system, copper uptake novel strain, there are 992 genes present as strain KMS-5T- system CopCD, related to stress response, e.g. flavohaemo- specific genes, accounting for about 27.1 % (Fig. S11). When globin, related to respiration, e.g. F0F1-type ATP synthase the percentage of genes that strain CYK-10T shared with and terminal cytochrome O ubiquinol oxidase, related to R. thermarum YIM 73036T, R. flagellatus SYSU G03088T, metabolism of aromatic compounds, e.g. chloroaromatic R. blasticus ATCC 33485T and R. capsulatus ATCC 11166T

6277 Sheu et al., Int. J. Syst. Evol. Microbiol. 2020;70:6266–6283

Table 4. Comparison of the presence and absence of selected genes among Rhodobacter tardus CYK-10T and four type strains of the genus Rhodobacter Strains: 1, CYK-10T; 2, Rhodobacter thermarum YIM 73036T; 3, Rhodobacter flagellatus SYSU G03088T; 4, Rhodobacter blasticus ATCC 33485T; 5, Rhodobacter capsulatus ATCC 11166T. +, Present; −, absent.

Genes putatively encoding 1 2 3 4 5

Photosynthesis

Photosystem II-type photosynthetic reaction centre :

Photosynthetic reaction centre L, M, H subunits + + + + +

Putative photosynthetic complex assembly protein + + + + +

Bacterial light-harvesting proteins :

Light-harvesting LHI, alpha subunit and beta subunit + + + + +

Chlorophyll biosynthesis + + + + +

Cofactors and vitamins

Ubiquinone biosynthesis + − + + +

Coenzyme B12 biosynthesis − − − + +

Riboflavin to FAD + − + + +

Flavodoxin − + + + +

Molybdenum cofactor biosynthesis − + + − −

Pterin carbinolamine dehydratase + + + + −

Cell wall and capsule

CMP-N-acetylneuraminate biosynthesis − + + + +

Legionaminic acid biosynthesis − + + + +

Sialic acid metabolism − + + + +

Lipopolysaccharide assembly − + + + −

Virulence, disease and defence

Multidrug resistance efflux pumps − + + + +

Arsenic resistance − + − + +

Mercuric reductase, mercury resistance operon + + + + −

Transposable elements

TniB NTP-binding protein, TniA putative transposase, Mg2+ chelatase family protein/ComM-related + − − − − protein, plasmid replication protein RepA, segregation and condensation protein B, protein of unknown function DUF1403

Membrane transport

Protein and nucleoprotein secretion system, type IV (Vir-like type four secretion system, pVir plasmid of + − − − − Campylobacter)

Protein secretion system, type VI + − − − −

AttEFGH ABC transport system + − − − −

Copper uptake system CopCD + − − − −

Tricarboxylate transport system + − + + −

ABC transporter (periplasmic-binding-protein dependent transport system for α-glucosides) − − − + +

ABC transporter alkylphosphonate − + + + +

Multi-subunit cation transporter − + + + + Continued

6278 Sheu et al., Int. J. Syst. Evol. Microbiol. 2020;70:6266–6283

Table 4. Continued

Genes putatively encoding 1 2 3 4 5

TRAP transporter − + + + +

Transport nickel and cobalt − + + + +

Chemotaxis

Bacterial chemotaxis + + + − −

Regulation and cell signalling

Global two-component regulator PrrBA in Proteobacteria + − − − +

Murein hydrolase regulation and cell death + − − − +

Stress response

Osmoregulation − − + + −

Synthesis of osmoregulated periplasmic glucans − − − + +

Redox-dependent regulation of nucleus processes − − − − +

NADPH:quinone oxidoreductase 2 + − − + +

Rubrerythrin + − − + +

Cluster containing glutathione synthetase + + − − −

Flavohaemoglobin + − − − −

Bacterial haemoglobin + + + − −

Respiration

F0F1-type ATP synthase + − − − −

Terminal cytochrome O ubiquinol oxidase + − − − −

Terminal cytochrome oxidase, terminal cytochrome d ubiquinol oxidase + + − − +

NiFe hydrogenase maturation + − − + +

Carbon monoxide oxidation + + + + −

Na+ translocating NADH-quinone oxidoreductase − − − − +

Na+ translocating decarboxylase − − − − +

Potassium metabolism

Hyperosmotic potassium uptake − + + + −

Glutathion-regulated potassium-efflux system − − − − +

Iron acquisition and metabolism

Siderophore enterobactin − − − − +

Heme, hemin uptake and utilization system + − − + +

Ferrous iron transporter EfeUOB, low-pH-induced − − − − +

Metabolism of nucleoside and nucleotide

Xanthine dehydrogenase subunits + + + − −

Protein metabolism

Universal GTPase + + + − +

Translational elongation factors bacterial + + + − +

N-linked glycosylation in Bacteria + − − + + Continued

6279 Sheu et al., Int. J. Syst. Evol. Microbiol. 2020;70:6266–6283

Table 4. Continued

Genes putatively encoding 1 2 3 4 5

DNA metabolism

CRISPR-associated protein Cas1, CRISPR-associated helicase Cas3, CRISPR-associated protein (Cse1 − − − + + family, Cse3 family, Cse4 family and Cas5e family)

Type I restriction-modification, restriction-modification system + + + + −

Nitrogen metabolism

Nitrogen fixation − − − + +

Nitrate and nitrite ammonification − + + − −

Nitrilase + − + − −

Denitrification − − − + −

Metabolism of aromatic compounds

Chloroaromatic degradation pathway + − − − −

N-heterocyclic aromatic compound degradation + − + − −

Homogentisate pathway of aromatic compound degradation − + + − −

Central meta-cleavage pathway of aromatic compound degradation − + + − −

Aromatic amin catabolism + − − − −

Metabolism of amino acid and derivatives

Valine, leucine degradation, HMG-CoA synthesis and HMG-CoA metabolism − − − − +

Indole-pyruvate oxidoreductase complex + + − − −

Sulphur metabolism

Sulphur oxidation − + + − −

Galactosylceramide and sulfatide metabolism + + + − −

Organic sulphur assimilation: taurine utilization − + − + −

Organic sulphur assimilation: alkanesulfonate utilization − − − + −

Carbohydrate metabolism

Di- and oligosaccharide: sucrose, melibose, maltose, maltodextrin, lactose and galactose utilization + + + − −

Organic acid: 2-methylcitrate to 2-methylaconitate metabolism cluster − + + + −

Organic acid: propionyl-CoA to succinyl-CoA module − + + + +

Organic acid: malonate decarboxylase − − − − +

Fermentation of lactate − − − + +

Fermentation of mixed acid − − − − +

Fermentation: acetoin and butanediol metabolism + − − − −

CO2 fixation: CO2 uptake, carboxysome, photorespiration (oxidative C2 cycle), Calvin-Benson cycle − − + + +

Sugar alcohol: mannitol utilization and inositol catabolism + + + + −

Monosaccharide: d-galactonate catabolism, d-gluconate and ketogluconate metabolism + + + + −

was analysed, strain CYK-10T shared 1618 genes (38.5 %) In summary, genomic information could provide the basics with its four related strains and there were 1506 genes to understand how these strains metabolize various nutrients, (35.8 %) different, which present as strain CYK-10T-specific their resistance to toxic compounds and their adaptability genes (Fig. S11). to environmental changes. These capabilities may confer the

6280 Sheu et al., Int. J. Syst. Evol. Microbiol. 2020;70:6266–6283

competitive ecological advantage for Tabrizicola or Rhodo- a is absent. Positive for poly-β-hydroxybutyrate accumula- bacter strains to adapt to diverse environments in the complex tion. Positive for oxidase and catalase activities. Negative for microbial ecosystem. hydrolysis of casein, starch, chitin, CM-cellulose, lecithin, DNA, corn oil and Tweens 20, 40, 60 and 80. Negative for nitrate reduction, indole production, glucose fermentation, TAXONOMIC CONCLUSION arginine dihydrolase and β-galactosidase (PNPG) activities, Phenotypic examination revealed many common traits urea, aesculin and gelatin hydrolysis. Positive for alkaline between the novel strain KMS-5T and the four phylogenetically phosphatase, C4 esterase, C8 esterase lipase, leucine arylami- closely related Tabrizicola species. However, strain KMS-5T dase, valine arylamidase, acid phosphatase, naphthol-AS- BI- could be clearly differentiated from these four closest rela- phosphohydrolase and α-glucosidase activities and negative tives by its inability to grow with higher NaCl concentration for C14 lipase, cystine arylamidase, trypsin, α-chymotrypsin, (>0.5 %), by its inability to hydrolyse aesculin, by the absence α-galactosidase, β-galactosidase (ONPG), β-glucuronidase, of β-glucosidase activity and by the inability to utilize maltose β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase and cellobiose as carbon sources (Table 1). However, the novel and α-fucosidase activities. Growth under aerobic conditions strain CYK-10T could also be clearly distinguished from the is positive on Tween 20, Tween 40, Tween 60, Tween 80, four closest Rhodobacter species by its lower optimal growth d-glucose, d-fructose, d-galactose, d-mannose, l-arabinose, temperature (25 °C) and by its inability to grow at higher sucrose, d-mannitol, d-sorbitol, succinate, caprate, acetate, temperature (>35 °C), at more alkaline condition (pH >7.5) l-proline, l-asparagine and l-ornithine, but negative on and with higher NaCl concentration (>0.5 %) (Table 2). In dextrin, maltose, trehalose, cellobiose, l-rhamnose, raffinose, T addition, some features of strain CYK-10 such as the absence N-acetyl-d-glucosamine, glycerol, adonitol, adipate, malate, of motility and the presence of C4 esterase, C8 esterase citrate, gluconate, l-serine, l-histidine, l-leucine, l-glutamic lipase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, acid and l-aspartic acid. The major fatty acid is C ω7c. β-galactosidase and β-glucosidase activities, distinguish this 18 : 1 The predominant hydroxy fatty acids are 18C : 0 3-OH and novel strain from the type strain of the type species, R. capsu- C 3-OH. The polar lipid profile consists of phosphatidy- T 10 : 0 latus ATCC 11166 . lethanolamine, phosphatidylglycerol, phosphatidylcholine, On the basis of the data obtained from 16S rRNA gene two uncharacterized aminophospholipids, one uncharacter- sequence and whole genome sequence comparisons, strain ized phospholipid and one uncharacterized aminolipid. The KMS-5T and strain CYK-10T occupy distinct positions within major isoprenoid quinone is Q-10. The DNA G+C content the genera Tabrizicola and Rhodobacter, respectively. The is 65.5 mol%. phylogenetic insights are supported by the unique combina- The type strain is KMS-5T (=BCRC 81196T=LMG 31337T), tion of chemotaxonomic and biochemical characteristics isolated from a freshwater ditch of the Shuangsi Forest of these two novel strains. It is clear from the phylogenetic Recreation Area in Meinong District of Kaohsiung County, T and phenotypic data that strain KMS-5 represents a novel Taiwan. The GenBank/EMBL/DDBJ accession numbers member of the genus Tabrizicola. The name Tabrizicola for the 16S rRNA gene sequence and the whole genome oligotrophica sp. nov. is proposed for this species. Strain T T of Tabrizicola oligotrophica KMS-5 are MK215682 and CYK-10 is concluded to represent a novel species in the NZ_JAAIVJ000000000, respectively. genus Rhodobacter, for which the name Rhodobacter tardus sp. nov. is proposed. DESCRIPTION OF RHODOBACTER TARDUS S P. DESCRIPTION OF TABRIZICOLA NOV. OLIGOTROPHICA SP. NOV. Rhodobacter tardus (tar′dus. L. masc. adj. tardus slow, refer- Tabrizicola oligotrophica (o.li.go.tro'phi.ca. Gr. adj. oligos few; ring to the slow growth of the organism) Gr. adj. trophikos nursing, tending or feeding; N.L. fem. adj. Cells are Gram-stain-negative, aerobic, non-motile and oligotrophica eating little, referring to a bacterium living on ovoid to rod-shaped and divide by binary fission, some- low-nutrient media). times forming chains. Cells grow well on R2A agar, but not Cells are Gram-stain- negative, aerobic, non-motile, rod- on nutrient agar, trypticase soy agar or Luria–Bertani agar. shaped and chemo-heterotrophic. Cells grow well on R2A Cells are approximately 0.6–0.8 µm wide and 1.4–2.2 µm long agar, but not on Luria–Bertani agar, nutrient agar or tryp- after 3 days incubation on R2A agar at 25 °C. Colonies are ticase soy agar. After 48 h incubation on R2A agar at 30 °C, white coloured, convex, round and smooth with entire edges. cells are 0.5–0.8 µm wide and 1.5–2.5 µm long. Colonies are The colony size is approximately 1.0–1.6 mm in diameter on translucent white coloured, convex, round and smooth with R2A agar after 3 days incubation at 25 °C. Growth occurs at entire edges. Colonies are 1.8–2.8 mm in diameter on R2A 20–35 °C (optimum, 25 °C), at pH 6–7.5 (optimum, pH 7) and agar after 48 h incubation at 30 °C. Growth occurs at 15–35 °C with 0–0.5 % NaCl (optimum, 0 %). Oxidase is positive and (optimum, 30 °C), at pH 5.5–8.5 (optimum, pH 6–8) and catalase is negative. Growth occurs under anaerobic condi- with 0–0.5 % NaCl (optimum, 0 %). No growth occurs under tions by photoheterotrophy, but not by photoautotrophy. anaerobic conditions by phototrophy. Bacteriochlorophyll Bacteriochlorophyll a is present. Poly-β-hydroxybutyrate

6281 Sheu et al., Int. J. Syst. Evol. Microbiol. 2020;70:6266–6283

accumulation is observed. Negative for hydrolysis of starch, isolated from Qurugöl Lake nearby Tabriz city, Iran. Antonie van casein, chitin, CM-cellulose, DNA, corn oil, lecithin and Leeuwenhoek 2013;104:1205–1215. 6. Tarhriz V, Hirose S, Fukushima SI, Hejazi MA, Imhoff JF et al. Tweens 20, 40, 60 and 80. In the API 20NE tests, positive reac- Emended description of the genus Tabrizicola and the species tions for aesculin hydrolysis and negative reactions for nitrate Tabrizicola aquatica as aerobic anoxygenic phototrophic bacteria. reduction, indole production, glucose fermentation, arginine Antonie van Leeuwenhoek 2019;112:1169–1175. dihydrolase, urease and β-galactosidase activities and gelatin 7. Ko DJ, Kim JS, Park DS, Lee DH, Heo SY et al. Tabrizicola fusiformis hydrolysis. In the API ZYM kit, alkaline phosphatase, C4 sp. nov., isolated from an industrial wastewater treatment plant. Int esterase, C8 esterase lipase, leucine arylamidase, acid phos- J Syst Evol Microbiol 2018;68:1800–1805. phatase, naphthol-AS-BI-phosphohydrolase,α-galactosidase, 8. Liu ZX, Dorji P, Liu HC, Li AH, Zhou YG. Tabrizicola sediminis sp. nov., one aerobic anoxygenic photoheterotrophic bacteria from sedi- β-galactosidase,α-glucosidase and β-glucosidase activities are ment of saline lake. Int J Syst Evol Microbiol 2019;69:2565–2570. present and C14 lipase, valine arylamidase, cystine arylami- 9. Phurbu D, Wang H, Tang Q, Lu H, Zhu H et al. Tabrizicola alkalilacus dase, trypsin, α-chymotrypsin, β-glucuronidase, N-acetyl-β- sp. nov., isolated from alkaline Lake Dajiaco on the Tibetan Plateau. glucosaminidase, α-mannosidase and α-fucosidase activities Int J Syst Evol Microbiol 2019;69:3420–3425. are absent. Growth under aerobic conditions is positive 10. Han JE, Kang W, Lee JY, Sung H, Hyun DW et al. Tabrizicola piscis on Tween 20, Tween 40, Tween 60, Tween 80, d-glucose, sp. nov., isolated from the intestinal tract of a Korean indigenous freshwater fish, Acheilognathus koreensis. Int J Syst Evol Microbiol d-galactose, d-mannose, maltose, cellobiose, dextrin, 2020;70:2305–2311. d-mannitol, acetate, gluconate, succinate and caprate, but 11. Imhoff JF, Truper HG, Pfennig N. Rearrangement of the species negative on glycerol, trehalose, l-rhamnose, l-arabinose, and genera of the phototrophic "purple nonsulfur bacteria". Int J sucrose, raffinose,N -acetyl-d-glucosamine, adonitol, Syst Bacteriol 1984;34:340–343. d-sorbitol, adipate, malate, citrate, l-proline, l-serine, 12. Wang D, Liu H, Zheng S, Wang G. Paenirhodobacter enshiensis gen. l-histidine, l-leucine, l-ornithine, l-glutamic acid, l-aspartic nov., sp. nov., a non-photosynthetic bacterium isolated from soil, and emended descriptions of the genera Rhodobacter and Haema- acid or l-asparagine. The predominant quinone is Q-10. The tobacter. Int J Syst Evol Microbiol 2014;64:551–558. major cellular fatty acid is C18 : 1 ω7c, and the major hydroxyl 13. Suresh G, Lodha TD, Indu B, Sasikala C, Ramana CV. Taxogenomics fatty acids are C10 : 0 3-OH and C18 : 0 3-OH. Phosphatidyle- resolves conflict in the genus Rhodobacter: a two and half decades thanolamine, phosphatidylglycerol, phosphatidylcholine, two pending thought to reclassify the genus Rhodobacter. Front Micro- uncharacterized aminophospholipids, two uncharacterized biol 2019;10:2480. phospholipids, two uncharacterized aminolipids and two 14. Imhoff JF. Genus Rhodobacter. In: Brenner DJ, Krieg NR, Staley JT, Garrity GM (editors). Bergey’s Manual of Systematic Bacteriology, uncharacterized lipids are present in the polar lipid profile. 2nd ed. New York: Springer; 2005. pp. 161–167. The DNA G+C content is 66 mol%. 15. Eckersley K, Dow CS. Rhodopseudomonas blastica sp. nov.: a member The type strain is CYK-10T (=BCRC 81191T=LMG 31336T), of the Rhodospirillaceae. J Gen Microbiol 1980;119:465–473. isolated from a freshwater pond in Hinoki Village in Chiayi 16. Venkata Ramana V, Sasikala C, Ramana CV. Rhodobacter maris sp. nov., a phototrophic alphaproteobacterium isolated from a marine County, Taiwan. The GenBank/EMBL/DDBJ accession habitat of India. Int J Syst Evol Microbiol 2008;58:1719–1722. numbers for the 16S rRNA gene sequence and the whole 17. Arunasri K, Venkata Ramana V, Spröer C, Sasikala C, Ramana CV. genome of Rhodobacter tardus CYK-10T are MK209068 and Rhodobacter megalophilus sp. nov., a phototroph from the Indian NZ_JAABNR000000000, respectively. Himalayas possessing a wide temperature range for growth. Int J Syst Evol Microbiol 2008;58:1792–1796. 18. Venkata Ramana V, Anil Kumar P, Srinivas TNR, Sasi- Funding information kala C, Ramana CV. Rhodobacter aestuarii sp. nov., a phototrophic The authors received no specific grant from any funding agency. alphaproteobacterium isolated from an estuarine environment. Int J Syst Evol Microbiol 2009;59:1133–1136. Conflicts of interest 19. Subhash Y, Lee SS. Rhodobacter sediminis sp. nov., isolated from The authors declare that there are no conflicts of interest. lagoon sediments. Int J Syst Evol Microbiol 2016;66:2965–2970. 20. Suresh G, Sailaja B, Ashif A, Dave BP, Sasikala C et al. Description References of Rhodobacter azollae sp. nov. and Rhodobacter lacus sp. nov. Int J 1. Garrity GM, Bell JA, Lilburn T. Family I. Rhodobacteraceae fam. nov.. Syst Evol Microbiol 2017;67:3289–3295. In: Brenner DJ, Krieg NR, Staley JT, Garrity GM (editors). Bergey’s Manual of Systematic Bacteriology, 2, 2nd ed. New York: Springer; 21. Gandham S, Lodha T, Chintalapati S, Chintalapati VR. Rhodobacter 2005. pp. 161–229. alkalitolerans sp. nov., isolated from an alkaline brown pond. Arch 2. Garrity GM, Bell JA, Lilburn T. Rhodobacteraceae fam. nov. In List of Microbiol 2018;200:1487–1492. new names and new combinations previously effectively, but not 22. Khan IU, Habib N, Xiao M, Li MM, Xian WD et al. Rhodobacter validly, published, Validation List no. 107. Int J Syst Evol Microbiol thermarum sp. nov., a novel phototrophic bacterium isolated 2006;56:1–6. from sediment of a hot spring. Antonie van Leeuwenhoek 3. Hördt A, López MG, Meier-Kolthoff JP, Schleuning M, Wein- 2019;112:867–875. hold LM et al. Analysis of 1,000+ type-strain genomes substantially 23. Xian WD, Liu ZT, Li MM, Liu L, Ming YZ et al. Rhodobacter flagellatus improves taxonomic classification of Alphaproteobacteria. Front sp. nov., a thermophilic bacterium isolated from a hot spring. Int J Microbiol 2020;11:468. Syst Evol Microbiol 2020;70:1541–1546. 4. Pujalte MJ, Lucena T, Ruvira MA, Arahal DR, Macián MC. The Family 24. Suresh G, Kumar D, Krishnaiah A, Sasikala Ch, RamanaChV. Rhodobacteraceae. In: Rosenberg E, DeLong EF, Lory S, Stackebrandt Rhodobacter sediminicola sp. nov., isolated from a fresh water E, Thompson F (editors). The Prokaryotes-Alphaproteobacteria and pond. Int J Syst Evol Microbiol 2020;70:1294–1299. Betaproteobacteria, 8, 4th ed. Berlin: Springer; 2014. pp. 439–512. 25. Beveridge TJ, Lawrence JR, Murray RGE et al. Sampling and 5. Tarhriz V, Thiel V, Nematzadeh G, Hejazi MA, Imhoff JF et al. Tabri- staining for light microscopy. In: Beveridge TJ, Breznak JA, Marzluf zicola aquatica gen. nov. sp. nov., a novel alphaproteobacterium GA, Schmidt TM, Snyder LR et al. (editors). Methods for General and

6282 Sheu et al., Int. J. Syst. Evol. Microbiol. 2020;70:6266–6283

Molecular Bacteriology, 3rd ed. Washington, DC: American Society sequences and whole-genome assemblies. Int J Syst Evol Microbiol for Microbiology; 2007. pp. 19–33. 2017;67:1613–1617. 26. Schlegel HG, Lafferty R, Krauss I. The isolation of mutants 42. Larkin MA, Blackshields G, Brown NP, Chenna R, McGet- not accumulating poly-β-hydroxybutyric acid. Arch Mikrobiol tigan PA et al. clustal W and clustal X version 2.0. Bioinformatics 1970;71:283–294. 2007;23:2947–2948. 27. Spiekermann P, Rehm BH, Kalscheuer R, Baumeister D, Stein- 43. Hall TA. BioEdit: a user-friendly biological sequence alignment büchel A. A sensitive, viable-colony staining method using Nile editor and analysis program for windows 95/98/NT. Nucleic Acids red for direct screening of bacteria that accumulate polyhydroxy- Symp Ser 1999;41:95–98. alkanoic acids and other lipid storage compounds. Arch Microbiol 1999;171:73–80. 44. Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987;4:406–425. 28. Breznak JA, Costilow RN et al. Physicochemical factors in growth. In: Beveridge TJ, Breznak JA, Marzluf GA, Schmidt TM, Snyder LR 45. Felsenstein J. Evolutionary trees from DNA sequences: a maximum et al. (editors). Methods for General and Molecular Bacteriology, 3rd likelihood approach. J Mol Evol 1981;17:368–376. ed. Washington, DC: American Society for Microbiology; 2007. pp. 46. Kluge AG, Farris JS. Quantitative Phyletics and the evolution of 309–329. anurans. Syst Zool 1969;18:1–32. 29. Pfennig N, Trüper HG. The phototrophic bacteria. In: Buchanan RE, 47. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. mega X: molecular Gibbons NE (editors). Bergey’s Manual of Systematic Bacteriology, evolutionary genetics analysis across computing platforms. Mol 8th ed. Baltimore: Williams & Wilkins; 1974. pp. 24–75. Biol Evol 2018;35:1547–1549. 30. Biebl H, Allgaier M, Tindall BJ, Koblizek M, Lünsdorf H et al. Dinoro- 48. Ewels P, Magnusson M, Lundin S, Käller M. MultiQC: summarize seobacter shibae gen. nov., sp. nov., a new aerobic phototrophic analysis results for multiple tools and samples in a single report. bacterium isolated from dinoflagellates.Int J Syst Evol Microbiol Bioinformatics 2016;32:3047–3048. 2005;55:1089–1096. 49. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M et al. 31. Tindall BJ, Sikorski J, Smibert RA, Krieg NR et al. Phenotypic SPAdes: a new genome assembly algorithm and its applications to characterization and the principles of comparative systematics. single-cell sequencing. J Comput Biol 2012;19:455–477. In: Beveridge TJ, Breznak JA, Marzluf GA, Schmidt TM, Snyder LR et al. (editors). Methods for General and Molecular Bacteriology, 3rd 50. Seemann T. Prokka: rapid prokaryotic genome annotation. Bioin- ed. Washington, DC: American Society for Microbiology; 2007. pp. formatics 2014;30:2068–2069. 330–393. 51. Na SI, Kim YO, Yoon SH, Ha SM, Baek I et al. UBCG: up-to- date 32. Wen CM, Tseng CS, Cheng CY, Li YK. Purification, characterization bacterial core gene set and pipeline for phylogenomic tree recon- and cloning of a chitinase from Bacillus sp. NCTU2. Biotechnol Appl struction. J Microbiol 2018;56:280–285. Biochem 2002;35:213–219. 52. Lee I, Ouk Kim Y, Park SC, Chun J. OrthoANI: an improved algo- 33. Bowman JP. Description of Cellulophaga algicola sp. nov., isolated rithm and software for calculating average nucleotide identity. Int J from the surfaces of Antarctic algae, and reclassification of Syst Evol Microbiol 2016;66:1100–1103. Cytophaga uliginosa (ZoBell and Upham 1944) Reichenbach 1989 53. Richter M, Rosselló-Móra R. Shifting the genomic gold standard as Cellulophaga uliginosa comb. nov. Int J Syst Evol Microbiol for the prokaryotic species definition. Proc Natl Acad Sci U S A 2000;50:1861–1868. 2009;106:19126–19131. 34. Chang SC, Wang JT, Vandamme P, Hwang JH, Chang PS et al. Chitinimonas taiwanensis gen. nov., sp. nov., a novel chitinolytic 54. Rodriguez-R LM, Konstantinidis KT. Bypassing cultivation to iden- bacterium isolated from a freshwater pond for shrimp culture. tify bacterial species. Microbe Magazine 2014;9:111–118. Syst Appl Microbiol 2004;27:43–49. 55. Qin QL, Xie BB, Zhang XY, Chen XL, Zhou BC et al. A proposed genus 35. Nokhal TH, Schlegel HG. Taxonomic study of Paracoccus denitrifi- boundary for the prokaryotes based on genomic insights. J Bacte- cans. Int J Syst Bacteriol 1983;33:26–37. riol 2014;196:2210–2215. 36. Sasser M. Identification of Bacteria by Gas Chromatography of 56. Meier-Kolthoff JP, Auch AF, Klenk HP, Göker M. Genome sequence- Cellular Fatty Acids, MIDI Technical Note 101. Newark, DE: MIDI Inc; based species delimitation with confidence intervals and improved 1990. distance functions. BMC Bioinformatics 2013;14:60. 37. Embley TM, Wait R. Structural lipids of eubacteria. In: Goodfellow 57. Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, M, O’Donnell AG (editors). Chemical Methods in Prokaryotic System- Vandamme P et al. DNA-DNA hybridization values and their rela- atics. Chichester: Wiley; 1994. pp. 121–161. tionship to whole-genome sequence similarities. Int J Syst Evol 38. Collins MD. Isoprenoid quinones. In: Goodfellow M, O’Donnell AG Microbiol 2007;57:81–91. (editors). Chemical Methods in Prokaryotic Systematics. Chichester: 58. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T et al. The RAST Wiley; 1994. pp. 265–309. server: rapid annotations using subsystems technology. BMC 39. Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S ribosomal DNA Genomics 2008;9:75. amplification for phylogenetic study. J Bacteriol 1991;173:697–703. 59. Overbeek R, Olson R, Pusch GD, Olsen GJ, Davis JJ et al. The seed 40. Anzai Y, Kudo Y, Oyaizu H. The phylogeny of the genera Chryseo- and the rapid annotation of microbial genomes using subsystems monas, Flavimonas, and Pseudomonas supports synonymy of these technology (RAST). Nucleic Acids Res 2014;42:D206–D214. three genera. Int J Syst Bacteriol 1997;47:249–251. 60. Blom J, Kreis J, Spänig S, Juhre T, Bertelli C et al. EDGAR 2.0: an 41. Yoon SH, Ha SM, Kwon S, Lim J, Kim Y et al. Introducing EzBio- enhanced software platform for comparative gene content anal- Cloud: a taxonomically United database of 16S rRNA gene yses. Nucleic Acids Res 2016;44:W22–W28.

6283