Journal of Microbiology (2017) Vol. 55, No. 8, pp. 607–611 DOI 10.1007/s12275-017-7257-y

eISSN 1976-3794 pISSN 1225-8873

Pseudaminobacter granuli sp. nov., isolated from granules used in a wastewater treatment plant Young Ki Hahn1, Minseok S. Kim2*, and Wan-Taek Im3,4* 1

Samsung Electronics, Seoul 06620, Republic of Korea Department of New Biology, Daegu Gyeongbuk Institute of Science & Technology (DGIST), Daegu 42988, Republic of Korea 3 Department of Biotechnology, Hankyong National University, Kyonggi-do 17579, Republic of Korea 4 Center for Genetic Information, Graduate School of Bio and Information Technology, Hankyong National University, Kyonggi-do 17579, Republic of Korea 2

(Received Jun 26, 2017 / Revised Jul 18, 2017 / Accepted Jul 18, 2017)

A Gram negative, aerobic, non-motile and rod-shaped bacterial strain designated as Gr-2T was isolated from granules used in a wastewater treatment plant in Korea, and its taxonomic position was investigated using a polyphasic approach. Strain Gr-2T grew at 18–37°C (optimum temperature, 30°C) and a pH of 6.0–8.0 (optimum pH, 7.0) on R2A agar medium. Based on 16S rRNA gene phylogeny, the novel strain showed a new branch within the genus Pseudaminobacter of the family Phyllobacteriaceae, and formed clusters with PseudaT minobacter defluvii THI 051 (98.9%) and Pseudaminobacter T salicylatoxidans BN12 (98.7%). The G+C content of the genomic DNA was 63.6%. The predominant respiratory quinone was ubiquinone-10 (Q-10) and the major fatty acids were cyclo-C19:0 ω8c, C18:1 ω7c, and iso-C17:0. The overall polar lipid patterns of Gr-2T were similar to those determined for the other Pseudaminobacter species. DNA-DNA relatedness values between strain Gr-2T and its closest phylogenetically neighbors were below 18%. Strain Gr-2T could be differentiated genotypically and phenotypically from the recognized species of the genus Pseudaminobacter. The isolate therefore represents a novel species, for which the name Pseudaminobacter granuli T T sp. nov. is proposed with the type strain Gr-2 (=KACC 18877 T =LMG 29567 ).

by Kämpfer et al. (1999). Members of the genus are Gramnegative, rod-shaped, oxidase and catalase-positive. It contains ubiquinone-10 (Q-10) as the predominant respiratory quinone. The major polyamines are spermidine, sys-homospermidine and putrescine. The overall polar lipid patterns are phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidyl-dimethylethanolamine (PDE), phosphatidylmono-methylethanolamine (PME), phosphatidyl-ethanolamine (PE), and diphosphatidylglycerol (DPG). At the time of writing this manuscript, the genus consisted of 2 species with validly published names including the recently described species Pseudaminobacter defluvii and Pseudaminobacter salicylatoxidans (Kämpfer et al., 1999). The aim of this study was to determine the taxonomic posiT tion of strain Gr-2 by performing phylogenetic analysis based on the 16S rRNA gene sequence, and to analyze its chemotaxonomic and phenotypic characteristics. The results of this study demonstrate that strain Gr-2T represents a new bacterial species within the genus Pseudaminobacter. Materials and Methods Strain isolation Strain Gr-2T was originally isolated from granules used in a wastewater treatment plant located in the Cheong-ju province (36°50���70���N, 127°41���71���E), South Korea. The granule samples were suspended completely in sterilized water, followed by serial dilution and spreading onto R2A agar medium (BD). The plates were incubated at 30°C for two weeks. Single colony was purified by subculture, by transferring to new R2A agar plates. Strain Gr-2T was found and was routinely cultured on R2A agar medium at 30°C and preserved as a suspension in R2A broth with glycerol (20%, w/v), at -80°C. Phenotypic and biochemical characteristics

Keywords: 16S rRNA gene, polyphasic taxonomy, Pseudaminobacter granuli, granules Introduction Genus Pseudaminobacter was first defined and described *For correspondence. (W.T. Im) E-mail: [email protected]; Tel.: +82-31670-5335; Fax: +82-31-670-5339 / (M.S. Kim) E-mail: [email protected]; Tel.: +82-53-785-1740; Fax: +82-53-785-1819 The GenBank accession number for the 16S rRNA gene sequence of strain T Gr-2 is KX066102. Copyright G2017, The Microbiological Society of Korea

The Gram reaction was carried out using the non-staining method, as described by Buck (1982). Cell morphology and motility was observed under scanning electron microscopy (Hitachi) and Nikon light microscope at 1000× using the hanging drop technique (Perry, 1973), with cells grown on R2A agar for 2 days at 30°C. Catalase and oxidase tests were performed as outlined by Cappuccino and Sherman (2002). Cells grown on R2A agar for 1 day were used as an inoculum for the physiological and biochemical tests. In addition, biochemical and phenotypic tests were carried out using API 20NE, API ID 32GN, and API ZYM test kits according to the manufacturer’s instructions (bioMérieux). Tests for hy-

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drolysis of DNA (using DNase agar from Scharlau, with DNase activity determined by flooding the plates with 1M HCl), casein, starch, Tween 80 (Atlas, 1993), and carboxymethyl-cellulose (Ten et al., 2004) were performed. The results were evaluated after 7 days of incubation at 30°C. Growth at different temperatures (4, 10, 15, 20, 25, 30, 37, 42, and 45°C) and various pH values (pH 4.5–10.0 at intervals of 0.5 pH units) was assessed after 7 days of incubation. Three different buffers (final concentration, 50 mM) were used to adjust the pH of R2A broth. Acetate buffer was used for pH 5.0–5.5, phosphate buffer was used for pH 6.0–8.0 and Tris buffer was used for pH 8.5–10.0. Salt tolerance was tested on R2A medium supplemented with 1–10% (w/v at intervals of 1% unit) NaCl after 7 days of incubation. Growth on nutrient agar (NA, Difco), trypticase soy agar (TSA, Difco) and MacConkey agar (Difco) was also evaluated at 30°C. 16S rRNA gene sequencing and phylogenetic analysis The genomic DNA of the novel isolate was extracted and purified using a genomic DNA extraction kit (Macrogen) and PCR-mediated amplification of the 16S rRNA gene and sequencing of the purified PCR product were carried out according to the protocol of Kim et al. (2015). A near full length sequence of the 16S rRNA gene was compiled using the SeqMan software (DNASTAR). The 16S rRNA gene sequences of related taxa were obtained from GenBank and EzBiocloud [http://www.ezbiocloud.net] server (Yoon et al., 2017). Multiple alignments were performed by Clustal_X program (Thompson et al., 1997) and gaps were edited in the BioEdit program (Hall, 1999). Evolutionary distances were calculated using the Kimura two-parameter model (Kimura, 1983). The phylogenetic trees were constructed using neighbor-joining (Saitou and Nei, 1987), maximum-parsimony (Fitch, 1971), and maximum likelihood methods with the MEGA 6 Program (Tamura et al., 2013) with bootstrap values based on 1,000 replications (Felsenstein, 1985).

Determination of DNA G+C content For measurement of the G+C content of chromosomal DNA, T the genomic DNA of strain Gr-2 was extracted and purified as described by Moore and Dowhan (1995) and degraded enzymatically into nucleosides. The G+C content was then determined as described by Mesbah et al. (1989), using reverse-phase HPLC. DNA-DNA hybridization DNA-DNA hybridization experiments were performed beT tween strain Gr-2 and the two reference strains Pseudaminobacter defluvii KACC 11352T, Pseudaminobacter salicylatoxidans KACC 11264T, using the method described by Ezaki et al. (1989) using photobiotin-labeled DNA probes and micro-dilution wells. Hybridizations were performed at 50.4°C with five replications for each sample. The highest and lowest values obtained for each sample were excluded and the means of the remaining three values were converted to percentage DNA-DNA relatedness values. Results and Discussion Morphological and phenotypic characteristics T

Cells of strain Gr-2 were Gram-reaction-negative, aerobic and rod shaped (Fig. 1). Tests for oxidase and catalase were positive. The colonies obtained after growth on R2A agar plates for 3 days were smooth, circular, creamy in color, and 2–3 mm in diameter. On R2A agar, Gr-2T was able to grow at 18–37°C, but not at 10 and 42°C. The isolate showed growth on nutrient agar and TSA, but not on MacConkey agar. The phenotypic and chemotaxonomic characteristics that differentiate the strain Gr-2T from other closely related Pseudaminobacter species are listed in Table 1. Phylogenetic analysis

Chemotaxonomic analysis Isoprenoid quinone, cellular fatty acids, polar lipids, and polyT amine analyses: Cell biomass of strain Gr-2 for analyzing isoprenoid quinones was obtained from cultures grown on R2A agar for 2 days at 30°C. Isoprenoid quinones were extracted with chloroform/methanol (2:1, v/v), evaporated under a vacuum and re-extracted in n-hexane-water (1:1, v/v). The crude quinone in n-hexane solution was purified using Sep-Pak Vac Cartridges Silica (Waters) and subsequently analyzed by reverse- phase HPLC system (Younglin), as described by Hiraishi et al. (1996). Cellular fatty acid profiles were determined for strains grown on R2A agar for 48 h at 30°C. The cellular fatty acids were saponified, methylated and extracted according to the protocol of the Sherlock Microbial Identification System (MIDI). The fatty acid methyl esters were then analyzed by gas chromatography (model 6890; Hewlett Packard) using the TSBA library (version 6.1), and the MIDI system (Sasser, 1990). Polar lipid profile of the strain T Gr-2 was determined using the method of Minnikin et al. (1984). Polyamines were extracted and analyzed according to Schenkel et al. (1995).

The 16S rRNA gene sequence of strain Gr-2T determined in this study is a continuous stretch of 1,405 bp, which has been deposited in the GenBank database (accession number KX-

T

Fig. 1. Scanning electron micrograph of strain Gr-2 . Bar represents 2 μm.

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Table 1. Differentiating characteristics of Pseudaminobacter granuli Gr-2 and the type strains of related Pseudaminobacter species. Strain: 1, Gr-2T; 2, Pseudaminobacter defluvii KACC 11352T; 3, Pseudaminobacter salicylatoxidans KACC 11264T. In API kit system (ZYM, 20NE and 32GN), all strains were positive for urease, alkaline phosphatase, esterase, esterase lipase, leucine arylamidase, trypsin, acid phosphatase, D-glucose, propionate, L-histidine, 3-hydroxy-butyrate, L-proline, lactate, and L-alanine. Negative for nitrate reduction, indole production, glucose acidification, arginine dihydrolase, gelatin hydrolysis, β-galactosidase, lipase, valine arylamidase, α-chymotrypsin, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, β-glucuronidase, β-glucosidase, N-acetyl-β-glucosaminidase, a-mannosidase, α-fucosidase, salicin, D-melibiose, caprate, L-rhamnose, inositol, D-sucrose, D-maltose, itaconate, suberate, malonate, 5-ketogluconate, Glycogen, 3-hydroxy-benzoate. +, positive; w, weak positive; -, negative. Characteristics 1 2 3 Cell size (μm) 0.4-0.6×1.2-2.5 0.5-0.8×0.8-1.2† 0.5-0.8×1.0-1.5† Temperature range (°C) 18-37 10-40† 20-40† API 20 NE & ID32 GN tests    Esculin hydrolysis w L-Arabinose + w N-Acetyl-glucosamine + Citrate + + L-Fucose + D-Mannitol + D-Ribose + L-Serine + + D-Sorbitol + Valerate w + API ZYM test    Cystine arylamidase w α-Glucosidase + DNA G+C content (mol %) 63.6 62.9† 63.9† †

data taken from Kämpfer et al. (1999).

066102). The sequence similarity calculated using the EzBiocloud (http://www.ezbiocloud.net/identify) indicated that strain Gr-2T shared less than 98.9% 16S rRNA gene sequence similarity with all taxa with validly published names of the genus Pseudaminobacter. The strain showed the highest sequence similarity with Pseudaminobacter defluvii THI 051T (98.9%) and Pseudaminobacter salicylatoxidans BN12T (98.7%). T In the phylogenetic trees analysis, strain Gr-2 clearly indicated a separate branch within the genus Pseudaminobacter and formed a group with P. defluvii THI 051T and P. salicylaT toxidans BN12 , as shown in Fig. 2. Therefore, based on the phylogenetic tree analysis these two reference strains P. defluvii THI 051T and P. salicylatoxidans BN12T were selected T as the closest recognized neighbors of strain Gr-2 . Cellular fatty acid, quinone, polar lipid composition, and polyamine analyses The predominant respiratory quinone of strain Gr-2T was Q-10, similar to that in all the other members of the genus Pseudaminobacter. The cellular fatty acids of strain Gr-2T and related type strains are listed in the Table 2. The major cellular fatty acids in all 3 strains were cyclo-C19:0 ω8c, C18:1 ω7c and iso-C17:0. According to the Table 2, some qualitative and quantitative differences in fatty acid content were obT served between strain Gr-2 and its phylogenetically closest relatives. The main polar lipids were phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidyl-dimethylethanolamine (PDE), phosphatidylmono-methylethanolamine (PME), phosphatidyl-ethanolamine (PE) and diphosphatidylglycerol (DPG); while, minor polar lipid was an unidentified phospholipid (PL) and two unidentified polar lipids (L1 and L2) as shown in Fig. 3. The major polyamines were

Table 2. Cellular fatty acid profiles of strain Gr-2T and recognized species of genus Pseudaminobacter T T Strain: 1, GR-2 ; 2, Pseudaminobacter defluvii KACC 11352 ; 3, Pseudaminobacter salicylatoxidans KACC 11264T. All cells were cultured on R2A agar for 48 h at 30°C. tr, trace amount (<0.5%); Major fatty acids (>15%) are shown in bold type. Some fatty acids that account for less than 0.5% of the total fatty acids in all strains are not listed. –, not detected; tr, traces amount. Fatty acids 1 2 3 Saturated C16:0 4.4 9.2 4.5 C17:0 tr 1.5 1.1 C18:0 2.6 2.0 4.7 Unsaturated    C18:1 ω7c 20.5 26.7 19.6 C18:1 ω9c tr 0.6 Branched-chain fatty acid    C13:0 iso 3OH 0.8 0.9 C15:0 iso 1.7 3.8 C15:0 iso 3OH 1.1 C17:0 iso 12.2 5.2 4.5 C17:0 anteiso 0.6 0.5 tr Hydroxy fatty acids    C18:0 3OH 0.6 Cyclo fatty acid    C17:0 cyclo 1.6 1.3 C19:0 cyclo ω8c 52.1 43.4 58.2 Methyl ester    C18:1 ω7c 11 methyl 1.2 1.2 2.5 * Summed features C18:2 ω6,9c/C18:0 ante 3.3 2.4 0.9 *Summed features represent groups of two or three fatty acids that could not be separated by GLC with the MIDI system.

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(A)

(B)

(C)

T

Fig. 3. Two-dimensional thin-layer chromatogram of polar lipids of strain Gr-2 . Chloroform/methanol/water (65:25:4, by vol.) was used in the first direction, followed by chloroform/acetic acid/methanol/water (80:15:12:4, by vol.) in the second direction. The following spray reagents were used for detection: (A) 5% ethanolic molybdophosphoric acid (for total lipids); (B) ninhydrin (for aminolipids); (C) molybdenum blue (Sigma) (for phospholipids). Abbreviations: PC, phosphatidylcholine; PG, phosphatidylglycerol; PDE, phosphatidyl-dimethylethanolamine; PME, phosphatidylmono-methylethanolamine; PE, phosphatidyl-ethanolamine; DPG, diphosphatidylglycerol; PL, unidentified phospholipids; L, unidentified polar lipids (L1, L2).

sys-homospermidine and putrescine, which could differentiate T T strain Gr-2 from P. defluvii THI 051 at the species level. DNA G+C content and DNA-DNA hybridization The DNA G+C content of strain Gr-2T was 63.6 mol%, similar to that of the previously described species of genus Pseudaminobacter as shown in Table 1. The highest and lowest values obtained for each sample were excluded and the means of the remaining three values were converted to percentage DNA-DNA relatedness values. DNA-DNA relatedness values between strain Gr-2T and P. defluvii KACC 11352T, and P. salicylatoxidans KACC 11264T were 17.3 ± 1.0% and 7.4 ± 0.6%, respectively. According to Wayne et al. (1987), DNADNA relatedness values lower than 70% are considered to be the threshold value for the delineation of genospecies. Therefore, the value obtained is low enough to assign strain Gr-2T as a novel species of the genus Pseudaminobacter. Taxonomic conclusion On the basis of phylogenetic and phenotypic comparisons, T strain Gr-2 shares several common features with the members of the genus Pseudaminobacter, e.g. the major respiratory quinone was Q-10; major polyamines were sys-homospermidine and putrescine; major polar lipid patterns were PC, PG, PDE, PME, PE, and DPG; and major fatty acids were cyclo-C19:0 ω8c, C18:1 ω7c, and iso-C17:0. Thus, not only the phylogenetic distance of the 16S rRNA gene sequence but also the subsequent phenotypic and chemotaxonomic characteristics differentiate the novel isolate from previously recognized species of the genus Pseudaminobacter (Table 1 and Table 2). Therefore, strain Gr-2T should be assigned to the genus Pseudaminobacter as the type strain, for which the name Pseudaminobacter granuli sp. nov. is proposed. Description of Pseudaminobacter granuli sp. nov. Pseudaminobacter granuli (gra.nu'li. L. gen. n. granuli of a small grain, pertaining to a granule, from which the type

strain was isolated). Cells are Gram-reaction-negative, aerobic, non-motile and rods (0.4–0.6 μm in diameter and 1.2–2.5 μm in length) after culture on R2A agar for 2 days at 30°C. Colonies grown on R2A agar medium are smooth, circular, and cream in color and 2–3 mm in diameter after 3 days of incubation at 30°C on R2A agar. Growth also occurs on nutrient agar and TSA, but not on MacConkey. Grows on R2A agar at 18–37°C with pH 6.0–8.0 and 0–4% NaCl (w/v). Optimum growth occurs at 30°C and at pH 7.0 without NaCl (w/v) supplement. Positive for catalase and oxidase. Does not hydrolyze starch, DNA, casein and carboxymethyl-cellulose. Carbon assimilation tests as a sole carbon sources (API ID 32 GN, API 20 NE) and the enzyme activities (API ZYM) are listed in Table 1. Predominant respiratory quinone is Q-10, and cyclo-C19:0 ω8c, C18:1 ω7c, and iso-C17:0 are the major cellular fatty acids (>15%). The major polar lipids were phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidyl-dimethylethanolamine (PDE), phosphatidylmono-methylethanolamine (PME), phosphatidyl-ethanolamine (PE), and diphosphatidylglycerol (DPG). The major polyamines are putrescine, and sys-homospermidine. The G+C content of the genomic DNA is 63.6 mol%. The type strain, Gr-2T (=KACC 18877T =LMG 29567T) was isolated from granules used in a wastewater treatment plant located in Cheong-ju province, South Korea. Acknowledgements This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ012283)” Rural Development Administration and by the project on survey and excavation of Korean indigenous species of the National Institute of Biological Resources (NIBR) under the Ministry of Environment and by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded

Pseudaminobacter granuli sp. nov.

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