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

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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 chemo- taxonomic 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 ex- tracted with chloroform/methanol (2:1, v/v), evaporated un- der 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 an- alyzed by reverse- phase HPLC system (Younglin), as des- cribed 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).
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