Pseudaminobacter Granuli Sp. Nov., Isolated from Granules Used in a Wastewater Treatment Plant

Journal of Microbiology (2017) Vol. 55, No. 8, pp. 607–611 eISSN 1976-3794 DOI 10.1007/s12275-017-7257-y pISSN 1225-8873

Pseudaminobacter granuli sp. nov., isolated from granules used in a wastewater treatment plant

Young Ki Hahn1, Minseok S. Kim2*, by Kämpfer et al. (1999). Members of the genus are Gram- 3,4 negative, rod-shaped, oxidase and catalase-positive. It con- and Wan-Taek Im * tains ubiquinone-10 (Q-10) as the predominant respiratory quinone. The major polyamines are spermidine, sys-homo- 1 Samsung Electronics, Seoul 06620, Republic of Korea spermidine and putrescine. The overall polar lipid patterns 2Department of New Biology, Daegu Gyeongbuk Institute of Science & Technology (DGIST), Daegu 42988, Republic of Korea are phosphatidylcholine (PC), phosphatidylglycerol (PG), 3Department of Biotechnology, Hankyong National University, phosphatidyl-dimethylethanolamine (PDE), phosphatidyl- Kyonggi-do 17579, Republic of Korea mono-methylethanolamine (PME), phosphatidyl-ethanol- 4 Center for Genetic Information, Graduate School of Bio and amine (PE), and diphosphatidylglycerol (DPG). At the time Information Technology, Hankyong National University, Kyonggi-do 17579, Republic of Korea of writing this manuscript, the genus consisted of 2 species with validly published names including the recently described (Received Jun 26, 2017 / Revised Jul 18, 2017 / Accepted Jul 18, 2017) species Pseudaminobacter defluvii and Pseudaminobacter salicylatoxidans (Kämpfer et al., 1999). The aim of this study was to determine the taxonomic posi- A Gram negative, aerobic, non-motile and rod-shaped bac- T terial strain designated as Gr-2T was isolated from granules tion of strain Gr-2 by performing phylogenetic analysis based used in a wastewater treatment plant in Korea, and its taxo- on the 16S rRNA gene sequence, and to analyze its chemotaxonomic and phenotypic characteristics. The results of this nomic position was investigated using a polyphasic approach. T Strain Gr-2T grew at 18–37°C (optimum temperature, 30°C) study demonstrate that strain Gr-2 represents a new bacte- and a pH of 6.0–8.0 (optimum pH, 7.0) on R2A agar medium. rial species within the genus Pseudaminobacter. Based on 16S rRNA gene phylogeny, the novel strain showed a new branch within the genus Pseudaminobacter of the fa- mily Phyllobacteriaceae, and formed clusters with Pseuda- Materials and Methods minobacter defluvii THI 051T (98.9%) and Pseudaminobacter salicylatoxidans BN12T (98.7%). The G+C content of the ge- Strain isolation nomic DNA was 63.6%. The predominant respiratory quinone Strain Gr-2T was originally isolated from granules used in a was ubiquinone-10 (Q-10) and the major fatty acids were wastewater treatment plant located in the Cheong-ju prov- cyclo-C19:0 ω8c, C18:1 ω7c, and iso-C17:0. The overall polar lipid ince (36°50�70�N, 127°41�71�E), South Korea. The granule T patterns of Gr-2 were similar to those determined for the samples were suspended completely in sterilized water, fol- other Pseudaminobacter species. DNA-DNA relatedness values lowed by serial dilution and spreading onto R2A agar me- T between strain Gr-2 and its closest phylogenetically neighbors dium (BD). The plates were incubated at 30°C for two weeks. T were below 18%. Strain Gr-2 could be differentiated genoty- Single colony was purified by subculture, by transferring to pically and phenotypically from the recognized species of the new R2A agar plates. Strain Gr-2T was found and was rou- genus Pseudaminobacter. The isolate therefore represents a tinely cultured on R2A agar medium at 30°C and preserved novel species, for which the name Pseudaminobacter granuli as a suspension in R2A broth with glycerol (20%, w/v), at T T sp. nov. is proposed with the type strain Gr-2 (=KACC 18877 -80°C. =LMG 29567T). Phenotypic and biochemical characteristics Keywords: 16S rRNA gene, polyphasic taxonomy, Pseuda- minobacter granuli, granules 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 Introduction (Hitachi) and Nikon light microscope at 1000× using the hanging drop technique (Perry, 1973), with cells grown on Genus Pseudaminobacter was first defined and described 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 correspondence. (W.T. Im) E-mail: [email protected]; Tel.: +82-31- 670-5335; Fax: +82-31-670-5339 / (M.S. Kim) E-mail: [email protected]; for the physiological and biochemical tests. In addition, bio- Tel.: +82-53-785-1740; Fax: +82-53-785-1819 chemical and phenotypic tests were carried out using API The GenBank accession number for the 16S rRNA gene sequence of strain 20NE, API ID 32GN, and API ZYM test kits according to T Gr-2 is KX066102. the manufacturer’s instructions (bioMérieux). Tests for hy- Copyright G2017, The Microbiological Society of Korea 608 Hahn et al. drolysis of DNA (using DNase agar from Scharlau, with Determination of DNA G+C content DNase activity determined by flooding the plates with 1M For measurement of the G+C content of chromosomal DNA, HCl), casein, starch, Tween 80 (Atlas, 1993), and carboxyme- T the genomic DNA of strain Gr-2 was extracted and purified thyl-cellulose (Ten et al., 2004) were performed. The results as described by Moore and Dowhan (1995) and degraded were evaluated after 7 days of incubation at 30°C. Growth enzymatically into nucleosides. The G+C content was then at different temperatures (4, 10, 15, 20, 25, 30, 37, 42, and determined as described by Mesbah et al. (1989), using re- 45°C) and various pH values (pH 4.5–10.0 at intervals of verse-phase HPLC. 0.5 pH units) was assessed after 7 days of incubation. Three different buffers (final concentration, 50 mM) were used to DNA-DNA hybridization 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 DNA-DNA hybridization experiments were performed be- T buffer was used for pH 8.5–10.0. Salt tolerance was tested tween strain Gr-2 and the two reference strains Pseudami- T on R2A medium supplemented with 1–10% (w/v at intervals nobacter defluvii KACC 11352 , Pseudaminobacter salicy- T of 1% unit) NaCl after 7 days of incubation. Growth on nu- latoxidans KACC 11264 , using the method described by trient agar (NA, Difco), trypticase soy agar (TSA, Difco) and Ezaki et al. (1989) using photobiotin-labeled DNA probes MacConkey agar (Difco) was also evaluated at 30°C. and micro-dilution wells. Hybridizations were performed at 50.4°C with five replications for each sample. The highest 16S rRNA gene sequencing and phylogenetic analysis and lowest values obtained for each sample were excluded and the means of the remaining three values were converted The genomic DNA of the novel isolate was extracted and to percentage DNA-DNA relatedness values. 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 Results and Discussion according to the protocol of Kim et al. (2015). A near full length sequence of the 16S rRNA gene was compiled using Morphological and phenotypic characteristics the SeqMan software (DNASTAR). The 16S rRNA gene se- T quences of related taxa were obtained from GenBank and Cells of strain Gr-2 were Gram-reaction-negative, aerobic EzBiocloud [http://www.ezbiocloud.net] server (Yoon et al., and rod shaped (Fig. 1). Tests for oxidase and catalase were 2017). Multiple alignments were performed by Clustal_X positive. The colonies obtained after growth on R2A agar program (Thompson et al., 1997) and gaps were edited in the plates for 3 days were smooth, circular, creamy in color, and T BioEdit program (Hall, 1999). Evolutionary distances were 2–3 mm in diameter. On R2A agar, Gr-2 was able to grow at calculated using the Kimura two-parameter model (Kimura, 18–37°C, but not at 10 and 42°C. The isolate showed growth 1983). The phylogenetic trees were constructed using neigh- on nutrient agar and TSA, but not on MacConkey agar. The bor-joining (Saitou and Nei, 1987), maximum-parsimony phenotypic and chemotaxonomic characteristics that differ- T (Fitch, 1971), and maximum likelihood methods with the entiate the strain Gr-2 from other closely related Pseuda- MEGA 6 Program (Tamura et al., 2013) with bootstrap val- minobacter species are listed in Table 1. ues based on 1,000 replications (Felsenstein, 1985). Phylogenetic analysis The 16S rRNA gene sequence of strain Gr-2T determined in Chemotaxonomic analysis this study is a continuous stretch of 1,405 bp, which has been Isoprenoid quinone, cellular fatty acids, polar lipids, and poly- deposited in the GenBank database (accession number KX- amine analyses: Cell biomass of strain Gr-2T 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 Gr-2T was determined using the method of Minnikin et al. (1984). Polyamines were extracted and analyzed according to Schenkel et al. (1995). Fig. 1. Scanning electron micrograph of strain Gr-2T. Bar represents 2 μm. Pseudaminobacter granuli sp. nov. 609

Table 1. Differentiating characteristics of Pseudaminobacter granuli Gr-2T 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 pro- duction, glucose acidification, arginine dihydrolase, gelatin hydrolysis, β-galactosidase, lipase, valine arylamidase, α-chymotrypsin, naphthol-AS-BI-phos- phohydrolase, α-galactosidase, β-galactosidase, β-glucuronidase, β-glucosidase, N-acetyl-β-glucosaminidase, a-mannosidase, α-fucosidase, salicin, D-me- libiose, 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 EzBio- Table 2. Cellular fatty acid profiles of strain Gr-2T and recognized species cloud (http://www.ezbiocloud.net/identify) indicated that of genus Pseudaminobacter T Strain: 1, GR-2T; 2, Pseudaminobacter defluvii KACC 11352T; 3, Pseuda- strain Gr-2 shared less than 98.9% 16S rRNA gene sequence T similarity with all taxa with validly published names of the minobacter salicylatoxidans KACC 11264 . All cells were cultured on R2A agar for 48 h at 30°C. tr, trace amount (<0.5%); Major fatty acids (>15%) genus Pseudaminobacter. The strain showed the highest se- T are shown in bold type. Some fatty acids that account for less than 0.5% of quence similarity with Pseudaminobacter defluvii THI 051 the total fatty acids in all strains are not listed. –, not detected; tr, traces (98.9%) and Pseudaminobacter salicylatoxidans BN12T (98.7%). amount. In the phylogenetic trees analysis, strain Gr-2T clearly indi- Fatty acids 1 2 3 cated a separate branch within the genus Pseudaminobacter Saturated T and formed a group with P. defluvii THI 051 and P. salicyla- C16:0 4.4 9.2 4.5 T toxidans BN12 , as shown in Fig. 2. Therefore, based on the C17:0 tr 1.5 1.1 phylogenetic tree analysis these two reference strains P. de- C18:0 2.6 2.0 4.7 T T fluvii THI 051 and P. salicylatoxidans BN12 were selected Unsaturated    T as the closest recognized neighbors of strain Gr-2 . C18:1 ω7c 20.5 26.7 19.6

C18:1 ω9c tr 0.6 - Cellular fatty acid, quinone, polar lipid composition, and poly- Branched-chain fatty acid    amine analyses C13:0 iso 3OH 0.8 0.9 - T The predominant respiratory quinone of strain Gr-2 was C15:0 iso 1.7 3.8 - Q-10, similar to that in all the other members of the genus C15:0 iso 3OH - - 1.1 T Pseudaminobacter. The cellular fatty acids of strain Gr-2 C17:0 iso 12.2 5.2 4.5 and related type strains are listed in the Table 2. The major C17:0 anteiso 0.6 0.5 tr cellular fatty acids in all 3 strains were cyclo-C19:0 ω8c, C18:1 Hydroxy fatty acids    ω7c and iso-C17:0. According to the Table 2, some qualitative C18:0 3OH - - 0.6 and quantitative differences in fatty acid content were ob- Cyclo fatty acid    T served between strain Gr-2 and its phylogenetically closest C17:0 cyclo - 1.6 1.3 relatives. The main polar lipids were phosphatidylcholine C19:0 cyclo ω8c 52.1 43.4 58.2 (PC), phosphatidylglycerol (PG), phosphatidyl-dimethyle- Methyl ester    thanolamine (PDE), phosphatidylmono-methylethanolamine C18:1 ω7c 11 methyl 1.2 1.2 2.5 (PME), phosphatidyl-ethanolamine (PE) and diphosphati- Summed features* dylglycerol (DPG); while, minor polar lipid was an uniden- C18:2 ω6,9c/C18:0 ante 3.3 2.4 0.9 tified phospholipid (PL) and two unidentified polar lipids *Summed features represent groups of two or three fatty acids that could not be se- (L1 and L2) as shown in Fig. 3. The major polyamines were parated by GLC with the MIDI system. 610 Hahn et al.

(A) (B) (C)

Fig. 3. Two-dimensional thin-layer chromatogram of polar lipids of strain Gr-2T. 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). Abbrevia- tions: 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 strain was isolated). strain Gr-2T from P. defluvii THI 051T at the species level. 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 DNA G+C content and DNA-DNA hybridization culture on R2A agar for 2 days at 30°C. Colonies grown on The DNA G+C content of strain Gr-2T was 63.6 mol%, similar R2A agar medium are smooth, circular, and cream in color to that of the previously described species of genus Pseuda- 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, minobacter as shown in Table 1. The highest and lowest values obtained for each sample were excluded and the means but not on MacConkey. Grows on R2A agar at 18–37°C of the remaining three values were converted to percentage 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. DNA-DNA relatedness values. DNA-DNA relatedness values between strain Gr-2T and P. defluvii KACC 11352T, and Positive for catalase and oxidase. Does not hydrolyze starch, P. salicylatoxidans KACC 11264T were 17.3 ± 1.0% and 7.4 DNA, casein and carboxymethyl-cellulose. Carbon assimila- ± 0.6%, respectively. According to Wayne et al. (1987), DNA- tion tests as a sole carbon sources (API ID 32 GN, API 20 NE) DNA relatedness values lower than 70% are considered to and the enzyme activities (API ZYM) are listed in Table 1. be the threshold value for the delineation of genospecies. Predominant respiratory quinone is Q-10, and cyclo-C19:0 ω8c, C ω7c, and iso-C are the major cellular fatty acids Therefore, the value obtained is low enough to assign strain 18:1 17:0 Gr-2T as a novel species of the genus Pseudaminobacter. (>15%). The major polar lipids were phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidyl-dimethylethanolamine (PDE), phosphatidylmono-methylethanolamine Taxonomic conclusion (PME), phosphatidyl-ethanolamine (PE), and diphosphati- On the basis of phylogenetic and phenotypic comparisons, dylglycerol (DPG). The major polyamines are putrescine, and T strain Gr-2 shares several common features with the mem- sys-homospermidine. The G+C content of the genomic DNA bers of the genus Pseudaminobacter, e.g. the major respira- is 63.6 mol%. tory quinone was Q-10; major polyamines were sys-homo- The type strain, Gr-2T (=KACC 18877T =LMG 29567T) was spermidine and putrescine; major polar lipid patterns were isolated from granules used in a wastewater treatment plant PC, PG, PDE, PME, PE, and DPG; and major fatty acids located in Cheong-ju province, South Korea. 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 Acknowledgements characteristics differentiate the novel isolate from previously recognized species of the genus Pseudaminobacter (Table 1 This work was carried out with the support of “Cooperative T and Table 2). Therefore, strain Gr-2 should be assigned to Research Program for Agriculture Science & Technology the genus Pseudaminobacter as the type strain, for which Development (Project No. PJ012283)” Rural Development the name Pseudaminobacter granuli sp. nov. is proposed. Administration and by the project on survey and excavation of Korean indigenous species of the National Institute of Description of Pseudaminobacter granuli sp. nov. Biological Resources (NIBR) under the Ministry of Envi- Pseudaminobacter granuli (gra.nu'li. L. gen. n. granuli of a ronment and by Basic Science Research Program through small grain, pertaining to a granule, from which the type the National Research Foundation of Korea (NRF) funded Pseudaminobacter granuli sp. nov. 611 by the Ministry of Science, ICT & Future Planning (NRF- 39, 159–167. 2015R1C1A1A01054292), Republic of Korea. Minnikin, D.E., O’Donnell, A.G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A., and Parlett, J.H. 1984. 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