The genus Phreatobacter, belonging to the class Alphaproteobacteria, was first proposed with Phreatobacter oligotrophus by Tóth et al. (2014). At the time of writing, the genus consists of three validly published species (http://www.bacterio.net), Phreatobacter oligotrophus, Phreatobacter stygius, and Phreatobacter cathodiphilus, which were isolated from ultrapure water in a Hungarian power plant (Tóth et al., 2014), pieces of wood in a lava cave (Lee et al., 2017b) and cathode of a microbial fuel cell (Kim et al., 2018), respectively. All three Phreatobacter species were isolated in an oligotrophic artificial environment. Members of the genus Phreatobacter is characterized as being Gram-stain-negative, motile by flagellum, oxidase- and catalase-positive, aerobic and rod-shaped. They contains C_18:1 ω7c and C_18:1 ω7c 11-methyl as the predominant fatty acids, diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine as the common polar lipids and ubiquinone 10 (Q-10) as the major respiratory quinone (Tóth et al., 2014; Lee et al., 2017b; Kim et al., 2018). On the basis of the polyphasic taxonomy data in this study, we characterized the taxonomic position a bacterial strain NMCR1094^T as the type strain of a novel species belonging to the genus Phreatobacter, for which the name Phreatobacter aquaticus sp. nov. is proposed.

Microorganisms

A sample was collected from freshwater in Oship stream, Yeongdeok-gun, Gyeongsangbuk-do, Republic of Korea (36° 24' 41.3" N, 129° 21' 51.0" E) in March 2018. Strain NMCR1094^T was isolated using standard dilution plating method on an oligotrophic medium consisting of R2A agar (BD Difco) diluted 1:10 (v/v) with distilled water (referred to as 1/10 R2A) after 1 month of incubation at 20°C. The plates were then incubated at 25°C for 5 days. Colonies grown on 1/10 R2A plates were purified after 5 days by subculturing on new R2A plates. A novel bacterial strain, NMCR1094^T, was routinely cultured on R2A agar and preserved as glycerol suspension (20% in distilled water, v/v) at -80°C.

For the comparisons of phenotypic properties and chemotaxonomic and genomic investigations, P. cathodiphilus KACC 18497^T was used as a reference strain, which was obtained from the Korean Agricultural Culture Collection (KACC). Phreatobacter oligotrophus KCTC 52181^T and P. stygius KCTC 52518^T, obtained from the Korean Collection for Type Cultures (KCTC), were also used as reference strains.

16S rRNA gene-based phylogenetic analysis

For 16S rRNA sequence analysis, the genomic DNA of strain NMCR1094^T was extracted using the DNeasy Blood & Tissue kit (Qiagen) according to the manufacturer’s instructions. The 16S rRNA gene of strain NMCR1094^T was amplified using bacteria universal primers (800R, 1492R, 27F, and 518F) (Lane, 1991) and directly sequenced by using the Sanger method. The nearly complete 16S rRNA gene sequence was compared with those in the EzBioCloud database (Yoon et al., 2017). To determine the phylogenetic position of strain NMCR1094^T, the 16S rRNA gene sequences aligned using the SILVA aligner (Pruesse et al., 2012) were transferred to mega X program (Kumar et al., 2018) and subjected to phylogenetic analyses. Phylogenetic trees of the 16S rRNA gene sequences were reconstructed using the neighbor-joining (Saitou and Nei, 1987), maximum-likelihood (Felsenstein, 1981) and maximum-parsimony (Fitch, 1971) methods using the Jukes-Cantor distance (Jukes and Cantor, 1969). The robustness of the tree topologies was assessed by bootstrap analyses based on 1000 replications of the sequences (Felsenstein, 1985).

Whole genome sequence analysis

The whole genome sequence of strain NMCR1094^T was reported by Baek and Choi (2019) using the PacBio RS II platform (Pacific Biosciences) using one single-molecule real-time (SMRT) cell at the DNA Link. The whole genomes of strain NMRC1094^T was obtained using the PacBio RS II platform (Pacific Biosciences) using one single-molecule real-time (SMRT) cell at the DNA Link. The de novo assembly was performed using the Hierarchical Genome Assembly Process 3 (HGAP3) within PacBio SMRT analysis 2.3.0 (Chin et al., 2013). The genomes were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP v4.8) (Tatusova et al., 2016). The genomic DNA G + C content was calculated directly from the genome sequence. Potential contamination in genome assembles was checked by the Contamination Estimator by 16S (ContEst16S) (Lee et al., 2017a).

Levels of genome-based relatedness between strain NMCR1094^T and members of the genus Phreatobacter were estimated by determination of average nucleotide identity (ANI) values using the OrthoANI algorithm in the EzBioCloud service (http://www.ezbiocloud.net/tools) (Lee et al., 2016) and the digital DNA-DNA hybridization (dDDH) values using the Genome-to-Genome Distance Calculator (GGDC) (http://ggdc.dsmz.de/distcalc2.php) described by Meier-Kolthoff et al. (2013). The genomes of P. sygius KCTC 52518^T (GCA_005144885, GenBank assembly accession), P. oligotrophus DSM 25521^T (GCA_003046185) and P. cathodiphilus S-12^T (GCA_003008515) were downloaded from GenBank for comparison. To infer a genome-based phylogenetic tree, the universal core gene set was extracted using the up-to-date bacterial core gene set (UBCG) pipeline (Na et al., 2018) and subjected to FastTree (Price et al., 2009) to reconstruct a phylogenetic tree based on amino acid alignment.

Morphological, physiological and biochemical characterization

Cellular morphology, cell size and flagellation were observed using phase-contrast (Axio Imager A2; Carl Zeiss) and transmission electron microscopy (TEM) (CM200; Philips), after cells were grown at 25°C in R2A broth for 5 days. Specimens for TEM analysis were prepared by mounting negatively stained cells with 2.0% (w/v) uranyl acetate on a carbon-coated copper grid. Gram reaction was determined using the Gram staining kit (bioMérieux) according to the manufacturer’s instructions. Growth under anaerobic conditions was examined for 2 weeks on R2A agar plates, using the anaerobic gas pack system (MGC). The temperature range and optimum for bacterial growth were determined in R2A broth (MB Cell) at temperature ranging from 4 to 50°C (4, 10, 15, 20, 25, 30, 35, 40, 45 and 50°C). The pH range for growth was investigated using R2A broth with pH adjusted to 5.0 to 10.0 (at intervals of 0.5 pH units). The buffers MES (pH 5.0–6.0), MOPS (pH 6.5–7.0), HEPES (pH 7.5–8.0), Tris (pH 8.5–9.0) and CHES (pH 9.5–10.0) were used at a final concentration of 0.05 M to maintain the corresponding pH. Growth at various concentrations of NaCl (0–3%, in increments of 0.5%) was investigated using R2A broth. The increase in turbidity of each culture was monitored daily using a spectrophotometer (NovaspecPro) to measure the optical density at 600 nm (OD600) for up to 10 days. Oxidase and catalase activities were measured using oxidase reagent (bioMérieux) and 3% (v/v) H₂O₂, respectively. Hydrolysis of various substrates were evaluated after 10 days and were determined using R2A agar supplemented the following macromolecules: casein (1% skimmed milk, w/v), colloidal chitin (1%, w/v), CM-cellulose (1%, w/v), hypoxanthine (0.5%, w/v), starch (1%, w/v), Tween 80 (1%, v/v), L-tyrosine (0.5%, w/v) and xanthine (0.5%, w/v) and confirmed by the formation of distinct zones around the colonies after adding appropriate solutions or observing directly (Tindall et al., 2007). The DNase test was conducted on DNase test agar (Difco). Other physiological properties, enzymatic activities and carbon source assimilation tests were performed using API 20NE, API ZYM and API 32GN kits, respectively, according to the manufacturers’ manuals (bioMérieux).

Chemotaxonomic analyses

For cellular fatty acid analysis, strain NMCR1094^T and the three reference strains were grown on R2A broth at 25°C to the late exponential phase. Fatty acid methyl ester (FAME) preparation was performed using a Microbial Identification System (Microbial ID; MIDI, Inc.) according to the manufacturer’s instructions. FAME extracts were analyzed by a GC (Agilent 6890) and identified them by comparing fatty-acid profiles with the TSBA 6.0 database provided with the Sherlock Software version 6.1. Respiratory quinones were extracted from freeze-dried cells using chloroform/methanol (2:1, v/v) and analyzed using high-performance liquid chromatography (HPLC; Younglin) equipped with a Spherisorb 5 μm ODS2 4.6 mm × 250 mm column (Waters). Respiratory quinones were eluted with a mixture of methanol/isopropyl ether (4:1, v/v) at a flow rate of 1 ml min^-1 and temperature of 40°C. They were detected by UV absorbance at 254 nm. Polar lipids were examined using two-dimensional thin-layer chromatography (TLC) with the proper detection reagents (Minnikin et al., 1984; Komagata and Suzuki, 1988). All polar lipids on the TLC plates were visualized by spraying molybdophosphoric acid solution (Sigma-Aldrich) and subsequent identification of lipids containing specific functional groups were performed by spraying molybdenum blue (Sigma-Adrich) reagent for phospholipids, ninhydrin solution (Sigma-Adrich) for aminolipids and and alpha-naphthol solution (Sigma-Adrich) for glycolipids.

16S rRNA gene-based phylogeny

The almost complete 16S rRNA gene sequence of strain NMCR1094^T obtained using Sanger method was 1,444 bp (MH282932). Comparison of 16S rRNA gene sequences showed that strain NMCR1094^T was closely related to P. cathodiphilus S-12^T (98.6% sequence similarity), followed by P. stygius YC6-17^T (98.6%) and P. oligotrophus PI_21^T (98.4%). All other species showed lower than 94.0% similarity. The neighbor-joining, maximum-likelihood and maximum-parsimony trees showed that strain NMCR1094^T, and the type strains of three Phreatobacter species formed a robust clade with high bootstrap values (99–100%), indicating that strain NMCR1094^T was a member of the genus Phreatobacter (Fig. 1).

Fig. 1. Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing positions of strain NMCR1094^T and other closely related members of the or Hyphomicrobiales. Bootstrap values (expressed as percentages of 1000 replications) over 70% are shown at nodes for neighbor-joining, maximum-parsimony and maximum-likelihood methods, respectively. Filled circles indicate that the corresponding nodes were recovered by all treeing methods. Open circle indicates that the corresponding node was recovered by the neighbor-joining and maximum-likelihood methods. Sphingomonas paucimobilis ATCC 29837^T (BBJS01000072) was used as an out-group. Bar, 0.02 substitutions per nucleotide position.

Genome characterization

The 5.0 Mb (4,975,052 bp) circularized genome of strain NMCR1094^T sequenced by PacBio RS II system has been reported by Baek and Choi (2019). The genome was predicted to have 4,675 coding sequences (CDSs), 48 tRNA genes, and 6 rRNA genes (Supplementary data Table S1). Contamination was not confirmed when comparing two 16S rRNA gene fragments with ContEst16S results. The Phreatobacter sp. NMCR1094 genome contains a formate dehydrogenase gene cluster (Baek and Choi, 2019). The genes fdhF, fdsB, fdsG, and fdhD are predicted to encode subunits of formate dehydrogenase (FDH), which catalyzes the final step in the pathway involved in the reversible conversion of formate to CO₂. mobB, moeA, and mobA are predicted to encode proteins for the synthesis of a molybdenum cofactor essential for the activity of most bacterial molybdoenzymes.

The ANI values between strain NMBR1094^T and three Phreatobacter type strains ranged from 78.3 to 78.5% (Supplementary data Table S2), which was less than the 95% threshold (Goris et al., 2007; Richter and Rosselló-Móra, 2009; Kim et al., 2014). The dDDH values for strain NMCR1094^T versus P. oligotrophus DSM 25521^T, P. cathodiphilus S-12^T, and P. stygius KCTC 52181^T, respectively, were 20.5, 20.3, and 20.1%, falling short of the 70% threshold for species delineation (Auch et al., 2010) (Supplementary data Table S2). Furthermore, the phylogenomic tree, the genome of strain NMCR1094^T formed a clade with the genome of Phreatobacter species, confirming again that strain NMCR1094^T represents a novel species of the genus Phreatobacter (Supplementary data Fig. S1). Overall genomic characteristics, particularly ANI and dDDH values, suggest that strain NMCR1094^T represents a novel species of the genus Phreatobacter.

Morphological, physiological and biochemical analysis

The cells of strain NMCR1094^T were Gram-stain-negative, rod-shaped (0.8–0.9 µm in diameter and 2.0–2.3 µm in length) and motile by means of a polar flagellum (Supplementary data Fig. S2). The temperature range for growth is 10–40°C (optimum, 25°C). The pH range for growth is 6.5–10.0, (optimum, 7.0). Growth occurs with 0–0.5% (w/v) NaCl (optimum, 0%). Optimal conditions for culture growth were determined and then the cultures were maintained on R2A agar at 25°C. The cells were positive for catalase and oxidase activities. The phenotypic characteristics of the strain are presented in species description, Table 1, and Supplementary data Table S3. Strain NMCR 1094^T was differentiated from members of the genus Phreatobacter by indole production, activities of lipase (C14) and α-chymotrypsin, assimilation of sucrose, itaconic acid, malonate, acetate and citrate and hydrolysis of CM-cellulose and xanthine.

Table 1

Strains: 1, NMCR1094^T; 2, P. oligotrophus KCTC 52181^T; 3, P. stygius KCTC 52518^T; P. cathodiphilus KACC 18497^T. Data were obtained in this study unless otherwise stated. +, Positive; -, negative.

Characteristic	1	2	3	4
Origin of isolation	Freshwater of the stream	Ultrapure water from a power planta	Pieces of wood in a caveb	Cathodes of microbial fuel cellc
Temperature range (optimum) (°C)	10–40 (25)	20–45 (25–37)a	10–30 (30)b	10-40 (28-30)c
NaCl tolerance (%, w/v)	0–0.5	0a	0–2b	0–1c
API 20NE:
Nitrate reduction	+	-	-	+
Indole production	+	-	-	-
Esculin hydrolysis	-	+	+	+
Gelatinase	-	+	-	+
Enzyme activity of:
Trypsin	+	+	+	-
Valine arylamidase	+	-	+	-
Alkaline phosphatase, esterase (C4), esterase lipase (C8)	+	-	-	+
Lipase (C14), α-chymotrypsin	+	-	-	-
Assimilation of:
L-Arabinose	+	+	-	-
L-Proline	+	-	+	-
Valeric acid	+	-	-	+
Sucrose, itaconic acid, malonate, acetate, citrate	+	-	-	-
3-Hydroxybutyric acid	-	-	+	+
L-Alanine	-	-	+	-
Glycogen, propionic acid	-	-	-	+
Hydrolysis of:
Colloidal chitin	+	-	+	-
CM-Cellulose, xanthine	+	-	-	-
Casein, starch	-	-	+	-
L-Tyrosine	-	-	-	+
DNA G + C contentd	65.8	68.2	66.7	69.3
Polar lipidse	DPG, PE, PG, PC, 6GLs, 3PLs, 1AL	DPG, PE, PG, PC, 1AL, 1PL, 8Lsa	DPG, PE, PC, 2GLs, 1Lb	DPG, PE, PG, PC, 1Lc

* Data from ^a Tóth et al. (2014); ^b Lee et al. (2017b); ^c Kim et al. (2018).

^d The G + C content was calculated on genome.

^e DPG, diphosphatidylglycerol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PC, phosphatidylcholine; GL, unidentified glycolipid; PL, unidentified phospholipid; L, unidentified lipid; AL, unidentified aminolipid.

Chemotaxonomic characterization

The major cellular fatty acids (>10% of the total) of strain NMCR1094T were identified as summed feature 8 (C_18:1 ω7c and/or C_18:1 ω6c) (51.0%) C_18:1 ω7c 11-methyl (39.0%), which were consistent with those of other members of the genus Phreatobacter despite of quantitative differences (Table 2). Strain NMCR1094^T exhibited a quinone system consisting of ubiquinones Q-10 (97.2%) and Q-9 (2.8%). The polar lipids found in strain NMCR1094^T were diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylcholine (PC), six unidentified glycolipids, three unidentified phospholipids and an unidentified aminolipid (Supplementary data Fig. S3); it shared DPG, PE and PC as the common polar lipids, typical for the genus Phreatobacter (Lee et al., 2017b). These chemotaxonomic characteristics are consistent with the basic character of the genus Phreatobacter.

Table 2

Strains: 1, NMCR1094^T; 2, P. oligotrophus KCTC 52518^T; 3, P. stygius KCTC 52181^T; 4, P. cathodiphilus KACC 18497^T. Data were taken from this study using cells grown on R2A agar at 25°C to the late exponential phase. Values are percentages of the total fatty acids. Fatty acids that represented < 1.0% in all strains are not shown. tr, Trace (< 1.0%); -, not detected.

Fatty acid	1	2	3	4
C16:0	5.1	2.2	8.8	2.7
C17:0 anteiso	-	tr	1.7	-
C18:0	2.2	2.9	2.6	1.8
C18:1 ω7c 11-methyl	39.0	18.9	31.4	14.2
C19:0 cyclo ω8c	-	-	1.4	-
C20:1 ω7c	-	1.7	1.0	-
Summed features*
2 (C14:0 3-OH and/or C16:1 iso-I)	-	tr	1.2	tr
3 (C16:1 ω7c and/or C16:1 ω6c)	2.7	2.1	3.5	2.2
8 (C18:1 ω7c and/or C18:1 ω6c)	51.0	71.2	48.4	78.7

* Summed features represent groups of two fatty acids, which cannot be separated by the MIDI system.

In conclusion, the phylogenetic evidence and similar chemotaxonomic characteristics supported that strain NMCR1094^T could be assigned to the genus Phreatobacter. Moreover, low ANI and dDDH values and several differential phenotypic characteristics (Table 1) suggest that strain NMCR1094^T represents a novel species of the genus Phreatobacter, which the name Phreatobacter aquaticus sp. nov. is proposed.

Emended description of the genus Phreatobacter (TÓTH ET AL. 2014 EMEND. LEE ET AL. 2017b)

The genus Phreatobacter is described here, as suggested by Tóth et al. (2014) and Lee et al. (2017b), with the following modifications. The G + C content of in silico DNA is 66.7–69.3%.

Description of Phreatobacter aquaticus sp. nov.

Phreatobacter aquaticus (a.qua′ti.cus. L. masc. adj. aquaticus living, growing or found in or by water, aquatic).

Cells are Gram-negative, strictly aerobic, catalase and oxidase positive, rod shaped (0.8 – 0.9 × 2.0 – 2.3 µm) and motile by means of a polar flagellum. The temperature range for growth is 10–40°C, (optimum, 25°C). The pH range for growth is 6.5–10.0 (optimum, 7.0). The range of NaCl concentration for growth is 0–0.5% (w/v), with optimum growth in 0% NaCl. Chitin, CM-cellulose, and xanthine are hydrolysed, but casein, hypoxanthine, starch, Tween 80, tyrosine, and DNA are not hydrolysed. The API 20NE strip indicates positive for nitrate reduction, indole production, and urease activity; however, glucose fermentation, arginine dihydrolase, esculin hydrolysis, gelatinase, and β-galactosidase activity are negative. Positive enzymatic activity for alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), valine arylamidase, trypsin, α-chymotrypsin, acid phosphatase and naphthol-AS-BI-phosphohydrolase. The API 32GN kit for carbon source assimilation shows that the strain assimilates D-ribose, sucrose, itaconic acid, malonate, acetate, L-arabinose, valeric acid, citrate, and L-proline, but does not assimilate L-rhamnose, N-acetyl-glucosamine, inositol, D-maltose, suberic acid, lactic acid, L-alanine, potassium 5-ketogluconate, glycogen, 3-hydroxybenzoic acid, L-serine, D-mannitol, D-glucose, salicin, D-melibiose, L-fucose, D-sorbitol, capric acid, L-histidine, 2-ketogluconate, 3-hydroxybutyric acid, and 4-hydroxybenzoic acid. Its major isoprenoid quinone is Q-10. The major fatty acids (> 10%) are summed feature 8 (C_18:1 ω7c and/or C_18:1 ω6c) (51.0%) and C_18:1 ω7c 11-methyl (39.0%). The polar lipids are diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, six unidentified glycolipids, three unidentified phospholipids and an unidentified aminolipid.

The type strain NMCR1094^T (= KACC 19706^T = NBRC 113394^T = FBCC-B10017) was isolated from the freshwater of Oship stream, Republic of Korea. The G + C content of the genomic DNA of the type strain is 65.8%.

경상북도 영덕의 오십천에서 그람 음성, 막대 모양이며 극편모를 가진 세균(NMCR1094^T)가 분리되었다. 이 균주는R2A 배지에서 10–40°C (최적 25°C), pH 6.5–10.0 (최적 pH 7.0) 및 0–0.5% (w/v; 0% NaCl최적)에서 생장이 관찰되었다. 16S rRNA 유전자 서열에 기초한 계통발생학적 분석은 균주 NMCR1094^T가 Phreatobacter 속에 속하고, 염기서열 비교에서 Phreatobacter 종들과 98.4~98.6%의 유사도를 나타냈다. 균주 NMCR1094^T의 전체 게놈 크기는 4,974,952 bp이며 G + C 함량은 65.8 mol%이다. ANI및 디지털 DNA-DNA 혼성화 값은 NMCR1094^T 균주와 Phreatobacter 종 간에 각각 78.3~78.5% 및 20.1~20.5%였다. 균주 NMCR1094^T에서 발견된 주요 호흡 퀴논은 Ubiquinone-10 (Q-10)이었다. 주요 지방산은 summed feature 8 (C_18:1 ω7c/C_18:1 ω6c) 및 C_18:1 ω7c 11-methyl이었다. 극성 지질은 디포스파티딜글리세롤, 포스파티딜글리세롤, 포스파티딜콜린, 포스파티딜에탄올아민, 6개의 미확인 당지질, 3개의 미확인 인지질 및 1개의 미확인 아미노지질로 구성되었다. 다상 분류학 연구의 결합된 데이터를 기반으로 NMCR1094^T 균주가 Phreatobacter속의 신종임을 나타내며, 신규 학명 Phreatobacter aquaticus sp. nov.를 제안한다. 표준 균주는 NMCR1094^T (= KACC 19706^T = NBRC 113394^T = FBCC-B10017)이다.

This work was supported by the Nakdonggang National Institute of Biological Resources (NNIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (NNIBR202301108) and Korea Environment Industry & Technology Institute (KEITI) through project to make multi-ministerial national biological research resources more advanced program, funded by Korea Ministry of Environment (MOE) (2021003420001).

The authors declare that there are no conflicts of interest.