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Flavobacterium Sasangense Sp. Nov., Isolated from a Wastewater Stream Polluted with Heavy Metals

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Flavobacterium Sasangense Sp. Nov., Isolated from a Wastewater Stream Polluted with Heavy Metals International Journal of Systematic and Evolutionary Microbiology (2009), 59, 1162–1166 DOI 10.1099/ijs.0.004978-0 Flavobacterium sasangense sp. nov., isolated from a wastewater stream polluted with heavy metals Hwan Sik Yoon,1 Zubair Aslam,1 Geun Cheol Song,1 Seon Won Kim,1 Che Ok Jeon,2 Tae Soo Chon3 and Young Ryun Chung1 Correspondence 1Division of Applied Life Science (BK21), PMBBRC and EB-NCRC, Gyeongsang National Young Ryun Chung University, Jinju 660-701, Republic of Korea [email protected] 2Division of Life Science, Chung-Ang University, Seoul 156-756, Republic of Korea 3Division of Biological Sciences, Pusan National University, Busan 609-735, Republic of Korea A Gram-negative, rod-shaped bacterium, designated strain YC6274T, was isolated from a stream (Sasang) carrying wastewater polluted with heavy metals in Busan, Korea. Growth was observed at 10–35 6C (optimum, 30 6C) and pH 6.0–9.5 (optimum, pH 7.5–8.0). Comparative 16S rRNA gene sequence analyses showed that the strain was most closely related to Flavobacterium cucumis R2A45-3T (96.6 % similarity), F. aquatile ATCC 11947T (93.7 %), F. croceum EMB47T (93.3 %), F. indicum GPTSA100-9T (93.3 %) and F. terrigena DS-20T (93.2 %). Sequence similarities with strains of other Flavobacterium species with validly published names were lower than 93.0 %. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain YC6274T formed a distinct phyletic lineage within the genus Flavobacterium. The predominant T fatty acids of strain YC6274 were iso-C15 : 0, iso-C16 : 0, iso-C15 : 1 G, iso-C17 : 0 3-OH, iso-C16 : 0 3-OH and iso-C15 : 0 3-OH. The G+C content of the genomic DNA was 32.5 mol% and the major quinone was MK-6. On the basis of phenotypic, chemotaxonomic and molecular data, it is clear that strain YC6274T represents a novel species within the genus Flavobacterium, for which the name Flavobacterium sasangense sp. nov. is proposed. The type strain is YC6274T (5KCTC 22246T 5DSM 21067T). Members of the genus Flavobacterium are Gram-negative, Tamaki et al., 2003; Zhang et al., 2006). Several species are aerobic, yellow-pigmented, rod-shaped bacteria that are known to cause diseases in freshwater fish (Wakabayashi motile by gliding. The genus consists of 52 recognized et al., 1989; Bernardet & Bowman, 2006). Reflecting their species, including the recently described species ecological diversity, Flavobacterium species have been Flavobacterium terrae, F. cucumis (Weon et al., 2007), F. isolated from various habitats such as fresh water, seawater, filum (Ryu et al., 2007), F. ceti (Vela et al., 2007) and F. wastewater, Antarctic lakes, soil, the gut of an earthworm anhuiense (Liu et al., 2008). Because of the complex and and sediments (McCammon & Bowman, 2000; Zhu et al., heterogeneous characteristics of the genus, its description 2003; Van Trappen et al., 2004, 2005; Aslam et al., 2005; has been emended repeatedly; the present description was Cousin et al., 2007). We report here the isolation and provided by Bernardet et al. (1996, 2002). Flavobacterium identification of a strain designated YC6274T that repre- species are physiologically diverse and found in a variety of sents a novel species of the genus Flavobacterium. environments. Many species are psychrotolerant, psychro- In order to isolate bacteria for the removal of heavy metals, philic or mesophilic and the genus includes species that are samples of wastewater were collected from the Sasang halotolerant, halophilic or sensitive to salts, and others stream, which receives wastewater from many industrial produce a variety of enzymes (Humphry et al., 2001; factories in Busan, Korea. The stream was found to be polluted with high concentrations of heavy metals such as Abbreviations: ML, maximum-likelihood; MP, maximum-parsimony; NJ, neighbour-joining. Cr, Fe and Ni (Chon, 2007). Collected samples were diluted with sterilized water and the diluted solutions were The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene u sequence of strain YC6274T is EU423319. then spread on R2A agar (Difco) and incubated at 30 C T for 10 days. Among several bacterial colonies, one of the A transmission electron micrograph of a cell of strain YC6274 and 16S dominant colonies with a yellowish colour was selected for rRNA gene sequence-based maximum-likelihood and maximum-par- simony trees are available as supplementary material with the online characterization and sequence analysis of the 16S rRNA T version of this paper. gene. Since the sequence similarity of strain YC6274 to Downloaded from www.microbiologyresearch.org by 1162 004978 G 2009 IUMS Printed in Great Britain IP: 54.70.40.11 On: Wed, 12 Dec 2018 23:30:14 Flavobacterium sasangense sp. nov. bacterial strains with validly published names was lower available in MEGA4 (Tamura et al., 2007). Bootstrap analysis than 97.0 %, it was studied further. The novel strain was was performed according to Kimura’s two-parameter grown routinely on R2A medium at 30 uC and was model (Kimura, 1980). Phylogenetic analysis with different preserved as a 20 % glycerol stock at 270 uC. methods based on 16S rRNA gene sequences indicated that strain YC6274T formed a distinct phyletic lineage within Gram staining was performed using the bioMe´rieux Gram the genus Flavobacterium (Fig. 1 and Supplementary Fig staining kit according to the instructions of the manufac- turer. Cell morphology, flagella and gliding motility were S1, available in IJSEM Online). Comparative 16S rRNA gene sequence analyses showed that strain YC6274T was studied using phase-contrast microscopy and transmission T electron microscopy (JEM-1010; JEOL) as described most closely related to F. cucumis R2A45-3 , Flavobacterium aquatile ATCC 11947T, F. croceum previously (Bernardet et al., 2002; Jeon et al., 2005). T T T EMB47 , F. indicum GPTSA100-9 and F. terrigena DS- Physiological characteristics of strain YC6274 were T examined by growing the isolate on R2A agar at different 20 (similarity of 96.6, 93.7, 93.3, 93.3 and 93.2 %, temperatures (5–50 uCat5uC intervals) and in R2A broth respectively). Sequence similarities with strains of other adjusted to different pH values (pH 5.0–10.0 at 0.5 pH unit Flavobacterium species with validly published names were intervals) (Gomori, 1955). Salt tolerance was tested on R2A lower than 93.0 %. agar supplemented with 0–3 % NaCl (w/v, at 0.5 % Analysis of fatty acid methyl esters was performed intervals) for 5 days at 30 uC. Antibiotic susceptibility tests according to the instructions of the Microbial were performed in duplicate using filter-paper discs Identification System (MIDI; Microbial ID, Inc.). (diameter 8 mm) containing the following antibiotics: Isoprenoid quinones were identified using the methods ampicillin (10 mg), polymyxin B (100 U), streptomycin described by Komagata & Suzuki (1987). The DNA G+C (50 mg), penicillin G (10 IU), chloramphenicol (100 mg), content of strain YC6274T was determined using an HPLC gentamicin (30 mg), tetracycline (30 mg), kanamycin fitted with a reversed-phase column (GROM-SIL 100 (30 mg), lincomycin (15 mg), oleandomycin (15 mg), neo- ODS-2FE; GROM) according to the method of Tamaoka & mycin (30 mg), carbenicillin (100 mg) and novobiocin Komagata (1984). (50 mg). Oxidase activity was tested by the oxidation of 1.0 % (w/v) tetramethyl-p-phenylenediamine (Merck) and The novel strain was Gram-negative and motile by gliding. catalase activity was evaluated by the production of oxygen Cells were short rods (0.3–0.561.4–1.9 mm) (Supple- bubbles in 3.0 % (v/v) aqueous hydrogen peroxide mentary Fig. S2). Colonies on R2A agar were yellowish solution. Production of flexirubin-type pigments and of orange and circular with entire margins. Growth occurred extracellular glycans was investigated using the KOH and at 10–35 uC (optimum, 30 uC) and at pH 6.0–9.5 Congo red tests, respectively, according to the minimal (optimum, pH 7.5–8.0). The range of NaCl for growth standards for the description of new taxa in the family was 0–1.0 % (w/v); optimum growth occurred without Flavobacteriaceae (Bernardet et al., 2002). Hydrolysis of NaCl. Other physiological, biochemical and molecular casein, gelatin, Tweens 80 and 20, aesculin, urea, tyrosine, features of the strain YC6274T are presented in Table 1 and starch and CM-cellulose was investigated on R2A agar after 7 days of incubation at 30 uC according to previously described methods (Smibert & Krieg, 1994). Nitrate reduction was examined according to the method of La´nyı´ (1987). Acid production from carbohydrates, additional enzyme activities and biochemical features were determined using the API 50CH, API ZYM and API 20E kits as recommended by the manufacturer (bioMe´rieux) except that kits were incubated for 2 days at 30 uC. Sequencing of the 16S rRNA gene of strain YC6274T was carried out as described previously (Lane, 1991; Chung et al., 1999). The resulting 16S rRNA gene sequence (1343 nt) was compared with available 16S rRNA gene sequences from GenBank using the BLAST program (http:// www.ncbi.nlm.nih.gov/BLAST/) to determine an approx- imate phylogenetic affiliation, and gene sequences were aligned with those of closely related species by using the Fig. 1. Neighbour-joining tree based on 16S rRNA gene CLUSTAL W software (Thompson et al., 1994). Phylogenetic sequences showing the phylogenetic relationships of strain trees were constructed using three different methods, the YC6274T and related taxa of the genus Flavobacterium. neighbour-joining (NJ), maximum-likelihood (ML) and Bootstrap values (.50 %) are shown as percentages of 1000 maximum-parsimony (MP) algorithms. ML and MP are replicates. The sequence of Capnocytophaga granulosa LMG available in the PHYLIP software, version 3.6 (Felsenstein, 12119T was used as an outgroup. Bar, 0.01 changes per 2002), while NJ (Kimura, 1980; Saitou & Nei, 1987) is nucleotide position.
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