DOI: 10.15468/d5ayrb
title: Flavobacterium niveum sp. nov., isolated from a freshwater creek

description: This dataset contains the digitized treatments in Plazi based on the original journal article Chen, Wen-Ming, Chen, Wei-Ting, Young, Chiu-Chung, Sheu, Shih-Yi (2019): Flavobacterium niveum sp. nov., isolated from a freshwater creek. International Journal of Systematic and Evolutionary Microbiology 69 (1): 271-277, DOI: 10.1099/ijsem.0.003150, URL: http://dx.doi.org/10.1099/ijsem.0.003150AbstractStrain TAPW14 T was isolated from a freshwater creek in Taiwan. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain TAPW14 T belonged to the genus Flavobacterium and was most closely related to Flavobacterium akiainvivens IK-1 T (96.6 % sequence identity) and Flavobacterium hauense BX 12 T (96.0 %) and less than 96 % sequence similarity to other members of the genus. Cells of strain TAPW14 T were Gram-negative, strictly aerobic, motile by gliding, rod-shaped and formed white colonies. Optimal growth occurred at 20 Ǫ C, pH 7 and in the presence of 0.5 % NaCl. Strain TAPW14 T contained summed feature 3 (C 16: 1 Ɯ 6 c and/or C 16: 1 Ɯ 7 c), iso-C 15: 0 and C 16: 0 as the predominant fatty acids. The polar lipid profile consisted of phosphatidylethanolamine, three uncharacterized aminophospholipids, one uncharacterized phospholipid and one uncharacterized lipid. The major polyamine was homospermidine. The major isoprenoid quinone was MK-6. The DNA G+C content of the genomic DNA was 46.0 mol%. On the basis of the phylogenetic inference and phenotypic data, strain TAPW14 T should be classified as a novel species, for which the name Flavobacteriumniveum sp. nov. is proposed. The type strain is TAPW14 T (=BCRC 81055 T =LMG 30057 T =KCTC 52808 T).The genus Flavobacterium, type genus of the family Flavobacteriaceae (order Flavobacteriales, class Flavobacteriia, phylum Bacteroidetes [1]), was established by Bergey et al. [2] and emended by Bernardet et al. [3], Dong et al. [4], Kang et al. [5] and Kuo et al. [6]. More than 208 species of the genus Flavobacterium have been listed on the List of Prokaryotic Names with Standing in Nomenclature (LPSN; www.bacterio.net/flavobacterium.html), which were isolated from various habitats such as animal, plant, algae, freshwater, seawater, sediment, soil, compost, lagoon, glacier, Antarctic lakes, water reservoir, activated sludge and wastewater treatment plant sources. Members of the genus Flavobacterium are typically Gram-negative rods, non-motile or motile by gliding that form yellow colonies. Menaquinone 6 (MK-6) is the major respiratory quinone, while C 15: 0, iso-C 15: 0, iso-C 15:1 G, iso-C 15: 0 3-OH, summed feature 3 (C 16: 1 Ɯ 7 c and/or C 16: 1 Ɯ 6 c), iso-C 16: 0 3-OH, iso-C 17: 1 Ɯ 9 c and iso-C 17: 0 3-OH are the predominant fatty acids. The DNA G-+C contents are between 30 and 52 mol% [3 – 9]. The present study was carried out to clarify the taxonomic position of a novel species of the genus Flavobacterium by a polyphasic taxonomic approach.During our investigations on the biodiversity of bacteria in Wanan Creek (22 Ǫ 37′ 21′′ N 120 Ǫ 38′ 48′′ E) in Pingtung County, Taiwan, a novel bacterium, designated TAPW14 T, producing white colonies, was isolated and selected for detailed taxonomy analyses. Strain TAPW14 T was isolated on Reasoner’ s 2A (R2A) agar (BD Difco) after incubation at 25 Ǫ C for 3 days, sub-cultured under the same conditions and stored at ―80 Ǫ C in R2A broth (BD Difco) with 20 % (v/ v) glycerol or by lyophilization.Genomic DNA was isolated using a bacterial genomic DNA purification kit and the 16S rRNA gene sequence was analysed as described by Chen et al. [10]. Primers 27F (5′- AGAGTTTGATCCTGGCTCAG-3′) and 1541R (5′-AAGGAGGTGATCCAGCC-3′) were used for amplification of bacterial 16S rRNA genes by PCR [11, 12]. The PCR product was purified, and direct sequencing was performed by using sequencing primers 27F, 1541R, 520F and 800R [11, 12] with an ABI Prism 3730 DNA sequencer (Applied Biosystems). The sequenced length of the 16S rRNA gene was 1482 bp for strain TAPW14 T and this gene sequence was compared to those available from EzBioCloud [13]. Multiple sequence alignments were performed with CLUSTAL_W [14] (BioEdit software [15]). The neighbour-joining, maximum-likelihood and maximum-parsimony phylogenetic trees were generated by using the tree-making algorithms as described previously [16].Author affiliations: 1 Laboratory of Microbiology, Department of Seafood Science, National Kaohsiung University of Science and Technology, No. 142, Hai-Chuan Rd. Nan-Tzu, Kaohsiung City 811, Taiwan, ROC; 2 College of Agriculture and Natural Resources, Department of Soil and Environmental Sciences, National Chung Hsing University, Taichung 402, Taiwan, ROC; 3 Department of Marine Biotechnology, National Kaohsiung University of Science and Technology, No. 142, Hai-Chuan Rd. Nan-Tzu, Kaohsiung City 811, Taiwan, ROC.*Correspondence: Shih-Yi Sheu, sys816@nkust.edu.twKeywords: Flavobacterium niveum sp. nov.; freshwater creek; Flavobacteriaceae; Flavobacteriales; Flavobacteriia; Bacteroidetes.Abbreviations: APL, uncharacterized aminophospholipid; L, uncharacterized lipid; PE, phosphatidylethanolamine; PL, uncharacterized phospholipid. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Flavobacterium niveum strain TAPW14 T is LT703450.Two supplementary figures are available with the online version of this article.Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain TAPW14 T formed a separate phylogenetic branch cluster with Flavobacterium akiainvivens IK-1 T within the genus Flavobacterium in the neighbour-joining tree (Fig. 1). The overall topologies of the maximum-likelihood and maximum-parsimony trees were similar (data not shown). Sequence identity calculations (over 1400 bp) indicated that strain TAPW14 T was closely related to the species of the genus Flavobacterium, and had the highest sequence identity with F. akiainvivens IK-1 T (96.6 %) and Flavobacterium hauense BX 12 T (96.0 %). Sequence identities &lt;96.0 % were observed with the type strains of all other species listed in Fig. 1.Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the position of Flavobacterium niveum TAPW 14 T and other Flavobacterium species. Numbers at nodes are bootstrap percentages (&gt;70 %) based on the neighbour-joining (above nodes) and maximum-parsimony (below nodes) tree-making algorithms. Filled circles indicate branches of the tree that were also recovered using the maximum-likelihood and maximum-parsimony tree-making algorithms. Open circles indicate that the corresponding nodes were also recovered in the tree generated with the maximum-parsimony algorithm. Myroides odoratus ATCC 4651 T was used as an outgroup. Bar, 0.01 substitutions per nucleotide position.F, akiainvivens ATCC BAA-2412 T, F. hauense KCTC 32147 T, Flavobacterium subsaxonicum DSM 21790 T and Flavobacterium rivuli DSM 21788 T were obtained from culture collections. The four type strains were used as reference strains and evaluated together under identical experimental conditions to those for strain TAPW14 T.Cell morphology of strain TAPW14 T was observed by phase-contrast microscopy (DM 2000, Leica) and transmission electron microscopy (H-7500, Hitachi; Fig. S1, available in the online version of this article) using cells grown on R2A agar for 48 h at 25 Ǫ C. Flagellar motility was tested using the hanging drop method, and Spot Test flagella stain (BD Difco) was used for flagellum staining. Gliding motility was studied using phase-contrast microscopy as described by Bernardet et al. [17]. The Gram reaction, the presence of flexirubin and carotenoid types of pigments and Congo red adsorption were investigated as described previously [16]. Colony morphology was observed on R2A agar under a stereoscopic microscope (SMZ 800, Nikon).The physiological characteristics of strain TAPW14 T and the four reference strains were examined by growing each at various pH values, temperatures and NaCl concentrations as described previously [16]. The pH range for growth was estimated at pH 4.0–9.0 (at intervals of 0.5 pH unit). The temperature range for growth was determined in R2A agar at 4–50 Ǫ C. To investigate the tolerance to NaCl, R2A broth was prepared with sodium chloride concentrations of 0, 0.5 and 1.0–5.0 %, w/v (at intervals of 1.0 %). Growth under anaerobic conditions was determined after incubating strain TAPW14 T on R2A and on R2A supplemented with nitrate (0.1 % KNO 3) in the Oxoid AnaeroGen system. Growth was studied on R2A, nutrient, Luria–Bertani and trypticase soy agars (all from Difco) under aerobic condition at 25 Ǫ C.Strain TAPW14 T and the four reference strains were examined for activities of catalase, oxidase, DNase, urease and lipase (corn oil), and hydrolysis of starch, casein, gelatin, lecithin and Tweens 20, 40, 60 and 80 using standard approaches [18]. Chitin hydrolysis was assessed on chitinase-detection agar [19] and visualized by the formation of clear zones around the colonies. Hydrolysis of carboxymethyl cellulose (CM-cellulose) was tested as described by Bowman using R2A agar as the basal medium [20]. Additional biochemical tests were performed using API ZYM and API 20NE kits (both from bioḾerieux) and carbon source utilization was evaluated using the GN2 MicroPlate (Biolog). All commercial phenotypic tests were performed according to the manufacturers’ instructions.Sensitivity of strain TAPW14 T to antibiotics was tested by the disc diffusion method after spreading cell suspensions (0.5 McFarland standard) on R2A agar plates as described by Nokhal and Schlegel [21]. The discs (Oxoid) contained the following antibiotics: ampicillin (10 µg), chloramphenicol (30 µg), gentamicin (10 µg), kanamycin (30 µg), nalidixic acid (30 µg), novobiocin (30 µg), penicillin G (10 U), rifampicin (5 µg), streptomycin (10 µg), sulfamethoxazole (23.75 µg) plus trimethoprim (1.25 µg) and tetracycline (30 µg). The effect of antibiotics on cell growth was assessed after 3 days at 25 Ǫ C. Strain TAPW14 T was sensitive to chloramphenicol, gentamicin, kanamycin, nalidixic acid, novobiocin, rifampicin, streptomycin, tetracycline, ampicillin, penicillin G and sulfamethoxazole plus trimethoprim. Detailed results from the phenotypic and biochemical analyses of strain TAPW14 T are provided in Table 1 and in the species description.The fatty acid profiles of strain TAPW14 T and the four reference strains were determined in cells grown on R2A agar at 25 Ǫ C for 2 days. Fatty acid methyl esters were prepared and separated according to the standard protocol of MIDI (Sherlock Microbial Identification System, version 6.0), analysed by GC (Hewlett-Packard 5890 Series II) and identified by using the RTSBA6.00 database of the Microbial Identification System [22].The predominant cellular fatty acids of strain TAPW14 T were summed feature 3 (C 16: 1 Ɯ 6 c and/or C 16: 1 Ɯ 7 c; 25.9 %), iso-C 15: 0 (21.3 %) and C 16: 0 (10.1 %), and the major hydroxyl fatty acids were iso-C 17: 0 3-OH (6.8 %). The complete fatty acid composition is shown in Table 2. Strain TAPW14 T and the four reference strains had summed feature 3, iso-C 15: 0 and C 16: 0 as the predominant cellular fatty acids. Furthermore, the fatty acid profile of strain TAPW14 T differed markedly from those of the four reference strains by the presence of minor amounts of C 18: 0, iso-C 18: 1 H and anteiso-C 14: 0.The DNA G+C content of strain TAPW14 T, as determined by HPLC according to Mesbah et al. [23], was 46.0 mol%, a value within the range reported for Flavobacterium strains [7 – 9]. The isoprenoid quinones of strain TAPW14 T were extracted and purified according to the method of Collins and analysed by HPLC [24]. The only respiratory quinone was menaquinone (MK-6), which is consistent with other members of the family Flavobacteriaceae [17].Polyamines were extracted from strain TAPW14 T and analysis was carried out as described by Busse and Auling [25] and Busse et al. [26]. Cells of strain TAPW14 T were cultivated and prepared as described previously [16]. Strain TAPW14 T contained homospermidine (HSPD, 88.1 %) as the major polyamine and putrescine (PUT, 6.4 %) and spermidine (SPD, 5.5 %) as the minor components. HSPD was the major polyamine of strain TAPY14 T in line with all other Flavobacterium species which polyamine composition was analysed [3, 8].The polar lipids of strain TAPW14 T were extracted and analysed by two-dimensional TLC according to Embley and Wait [27]. Ethanolic molybdophosphoric acid was used for the detection of the total polar lipids, ninhydrin for amino lipids, the Zinzadze reagent for phospholipids and the a-naphthol reagent for glycolipids. Strain TAPW14 T exhibited a complex polar lipid profile consisting of phosphatidylethanolamine (PE), three uncharacterized aminophospholipids (APL 1– APL 3), one uncharacterized phospholipid (PL 1) and one uncharacterized lipid (L1; Fig. S2). Strain TAPW14 T contained PE as the predominant polar lipid; the possession of PE as the common major polar lipid is consistent with previous descriptions of species of Flavobacterium [4, 5, 16, 28 – 33]. Furthermore, strain TAPW14 T differed from the other type strains of the genus Flavobacterium in the presence and proportions of some minor uncharacterized polar lipids.Table 1. Differential characteristics of Flavobacterium niveum TAPW14T and phylogenetically closely related Flavobacterium species Strains: 1, TAPW14T; 2, Flavobacterium akiainvivens ATCC BAA-2412T; 3, Flavobacterium hauense KCTC 32147T; 4, Flavobacterium subsaxonicum DSM 21790T; 5, Flavobacterium rivuli DSM 21788T. All data from this study except the G+C content of F. akiainvivens ATCC BAA-2412T [6], F. hauense KCTC 32147T [28], F. subsaxonicum DSM 21790T and F. rivuli DSM 21788T [34]. +, Positive reaction;, negative reaction. All strains are positive for activi- ― ties of alkaline phosphatase, C8 esterase lipase, leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and a- glucosidase, assimilation of glucose, arabinose, mannose and maltose, and hydrolysis of aesculin and Tween 80. All strains are negative for Gram staining; Congo red absorption; nitrate reduction; indole production; D- glucose acidification; arginine dihydrolase, urease, b- glucuronidase, N -acetyl-b- glucosaminidase, a- mannosidase and a- fucosidase activities; assimilation of mannitol, N -acetyl-glucosamine, gluconate, caprate, adipate, malate, citrate and phenyl-acetate; and hydrolysis of chitin and CM-cellulose.Characteristic 1 2 3 4 5 Isolation source Freshwater creek Decaying wood Soil Hard water rivulet Hard water rivulet Colony pigmentation White Off-white Bright yellow Light yellow Pearl white Temperature range for growth (Ǫ C) (optimum) 15–30 (20) 15–35 (30) 4–30 (25) 4–40 (25–30) 4–25 (15–20) pH range for growth (optimum) 6–8 (7) 5–9 (6–8) 6–8 (7) 7–7.5 (7) 6.5–8 (7) NaCl range for growth (%, w/v) (optimum) 0–2 (0.5) 0–3 (0.5) 0–2 (0) 0–2 (0) 0–2 (1) Gliding motility + ― ― ― ― Flexirubin-type pigments ― ― ― + ― Hydrolysis of: DNA + ― ― ― ― Gelatin + ― ― + ― Starch + + ― + + Casein ― + ― ― ― Lecithin ― ― ― ― + Tween 40 ― + + + + Enzymatic activities: Oxidase + ― + + + Catalase + + ― + + C4 esterase + + + ― ― C14 lipase ― + ― ― ― Cystine arylamidase + + ― + ― Trypsin + + ― + + b- Galactosidase + + ― + + a- Glucosidase + + ― + ― b- Glucosidase + + ― ― ― Utilization of: Sucrose + ― ― ― ― L- Rhamnose ― + ― + ― Lactose + + ― ― + Melibiose + + ― + + L- Fucose + + ― + ― Acetic acid + + ― ― + a- Ketobutyric acid ― + ― + ― DL- Lactic acid + + ― + ― L- Alanine + + ― + ― L- Ornithine + + + + ― L- Proline + + ― + + L- Serine + + ― + ― DNA G+C content (mol%) 46.0 44.2 43.9 43.3 40.4This polar lipid profile, the major polyamine and the predominant isoprenoid quinone all support the affiliation of strain TAPW14 T to the genus Flavobacterium. Phenotypic examination revealed that strain TAPW14 T shared several traits in common with its four closest relatives. However, strain TAPW14 T could be clearly differentiated from these four strains by colony pigmentation; narrow temperature range for growth (15–30 Ǫ C); gliding motility; ability to hydrolyze DNA; inability to hydrolyse Tween 40; and utilization of sucrose (Table 1). In addition, strain TAPW14 T could also be distinguished from the two phylogenetically closest relatives by a number of features: for example, from F. akiainvivens ATCC BAA-2412 T by narrow pH range for growth (6-8), inability to grow at higher NaCl concentration (&gt;2 %), ability to hydrolyse gelatin, inability to hydrolyse casein, the presence of oxidase activity, the absence of C14 lipase activity, and inability to utilize L- rhamnose and a-ketobutyric acid; from F. hauense KCTC 32147 T by ability to hydrolyse gelatin and starch, the presence of catalase, cystine arylamidase, trypsin, b- galactosidase, a- glucosidase and b- glucosidase activities, and ability to utilize lactose, melibiose, L- fucose, acetic acid, DL- lactic acid, L- alanine, L-proline and L- serine (Table 1).Table 2. Cellular fatty acid composition of Flavobacterium niveum TAPW 14 T and phylogenetically closely related Flavobacterium species Strains: 1, TAPW14 T; 2, Flavobacterium akiainvivens ATCC BAA-2412 T; 3, Flavobacterium hauense KCTC 32147 T; 4, Flavobacterium subsaxonicum DSM 21790 T; 5, Flavobacterium rivuli DSM 21788 T. All data are from this study. Strains were grown on R2A agar at 25 Ǫ C for 2 days. Data are expressed as percentages of the total fatty acids. Only fatty acids representing more than 1.0 % of the total fatty acids of at least one of the strains are shown. TR, Traces (less than 1.0 % of total); ―, not detected.On the basis of the data obtained from 16S rRNA gene sequence comparison, strain TAPW14 T occupies a distinct position within the genus Flavobacterium. The phylogenetic data are supported by the chemotaxonomic and biochemical characteristics of strain TAPW14 T. It is clear from the phylogenetic and phenotypic data that the strain TAPW14 T constitutes a novel member of the genus Flavobacterium. The name Flavobacterium niveum sp. nov., is proposed for this taxon.