DOI: 10.15468/yqrnwr
title: Flavobacterium tibetense sp. nov., isolated from a wetland

description: This dataset contains the digitized treatments in Plazi based on the original journal article Phurbu, Dorji, Lu, Hubin, Xue, Xiaofeng, Tian, Yanling, Pema, Yangzom, Ma, Chao, Xing, Peng (2019): Flavobacterium tibetense sp. nov., isolated from a wetland. International Journal of Systematic and Evolutionary Microbiology 69 (1): 165-170, DOI: 10.1099/ijsem.0.003124, URL: http://dx.doi.org/10.1099/ijsem.0.003124AbstractA novel, Gram-stain-negative, non-motile and rod-shaped bacterium, designated YH5 T, was isolated from the YonghuCo wetland on the Tibetan Plateau. The strain was able to grow optimally with 1 % (w/v) NaCl and tolerated up to 3 % NaCl. Growth occurred at pH 6–9 (optimum pH 7) and 10–37 Ǫ C (optimum 30 Ǫ C). Vitamins were not required for growth. The major polar lipid of strain YH5 T was phosphatidylethanolamine. The predominant respiratory quinone was menaquinone 6 (MK-6). The major fatty acids were iso-C 15: 0, C 16: 0 10-methyl and/or iso-C 17: 1 Ɯ 9 c, iso-C 17: 0 3-OH, iso-C 15: 1 G and iso-C 15: 0 3-OH. Genome sequencing revealed a genome size of 2.74 Mbp and a G+C content of 33.3 mol%. Analysis of 16S rRNA gene sequences showed that strain YH5 T belonged to the genus Flavobacterium, with the closest neighbours Flavobacterium luticocti xz20 T (96.7 % similarity), Flavobacterium jejuense EC 11 T (96.4 %), Flavobacterium jumunjinense HME 7102 T (95.9 %) and Flavobacterium dongtanense LW 30 T (95.6 %). DNA–DNA relatedness between strain YH5 T and the closest phylogenetically related strain F. luticocti xz20 T was 27.0 %. Strain YH5 T was clearly distinguished from the reference type strains based on phylogenetic analysis, DNA–DNA hybridization, fatty acid composition and a range of physiological and biochemical characteristics. Based on its phenotypic and chemotaxonomic characteristics, strain YH5 T is classified as a representative of a novel species of the genus Flavobacterium, for which the name Flavobacterium tibetense sp. nov. is proposed. The type strain is YH5 T (= CICC 24247 T = KCTC 62174 T).The genus Flavobacterium belongs to the large family Flavobacteriaceae in the phylum Bacteroidetes, which currently contains 141 genera (http://www.bacterio.net/ -classifphyla. html#flavobacteriaceae). Flavobacterium was initially proposed by Bergey et al. [1] and the description was later emended by Bernardet et al. [2], Dong et al. [3] and Kang et al. [4]. At the time of writing, 208 species, including synonyms, have been identified in the genus Flavobacterium (http://www.bacterio.net/flavobacterium.html), isolated from a wide range of habitats such as soil [5], freshwater [6], seawater [7], wastewater [8] and sediment [9], and displaying a variety of physiological characteristics. Based on current knowledge, all species in the genus Flavobacterium are Gram-stain-negative, aerobic, rod-shaped, non-sporeforming and yellow-pigmented, and contain menaquinone-6 (MK-6) as the major respiratory quinone [10, 11].During a study of microbial diversity on the Tibetan Plateau, water samples were collected from different sites in the wetland YonghuCo (29 Ǫ 46′ 08.40″ N 85 Ǫ 42′ 30.84″ E, 5151 m a.s.l.) on 10 August 2016. Water temperature was 10.96 Ǫ C at the time of sampling. pH and conductivity were measured in the field, and the corresponding values were 9.42 and 3356 µS/cm. The water sample used for microbial isolation was kept at 4 Ǫ C until transfer to the lab. Lake water (100 µl) was sprayed on tryptic soy agar (TSA; BD) plates and strains were purified by serial dilutions and spreading. A Gram-stain-negative, aerobic, non-motile and rod-shaped strain, designated as YH5 T, was isolated. The isolate was routinely cultured on TSA plates at 30 Ǫ C and preserved as a suspension in 30 % (v/v) glycerol at ―70 Ǫ C. 16S rRNA gene sequence analysis indicated that strain YH5 T was most closely related to Flavobacterium luticocti xz20 T (96.7 % similarity). The reference strains F. luticocti xz20 T (CCTCC AB2015421 T) [12] ordered from China Centre for Type Culture Collection (CCTCC), Flavobacterium jejuense EC 11 T (KCTC 42149 T) [13], Flavobacterium jumunjinense HME 7102 T (KCTC 23618 T) [14] and Flavobacterium dongtanense LW 30 T (KCTC 22671 T) [15] ordered from Korean Collection for Type Culture (KCTC), were used for comparisons in this study. Based on a taxonomic investigation using a polyphasic approach, we proposed to assign YH5 T as the type strain of a novel species in the genus Flavobacterium.Author affiliations: 1 Tibet Plateau Institute of Biology, Lhasa 850000, PR China; 2 State Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, Nanjing 210008, PR China; 3 University of Chinese Academy of Sciences, Beijing 100049, PR China; 4 College of Life Sciences, Nanjing Normal University, Nanjing 210046, PR China.*Correspondence: Peng Xing, pxing@niglas.ac.cnKeywords: Flavobacterium tibetense; Gram-negative; wetland YonghuCo; genotypic and phenotypic analysis; Tibetan Plateau.Abbreviations: MK-6, menaquinone 6; PE, phosphatidylethanolamine.†These authors contributed equally to this work.The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and draft genome sequences of strain YH5 T are MF511809 and QLST00000000.1, respectively.Five supplementary figures are available with the online version of this article.Cell morphology was observed under a light microscope (BH-2; Olympus) and a scanning electron microscope with cells grown on TSA plates for 96 h at 30 Ǫ C according to the method described by Liu et al. [16]. Gram-staining was performed as described by Gerhardt et al. [17]. KOH reagent was used in Gram-staining [18]. Flagellar motility was investigated using motility test medium (Difco) and gliding motility was assessed by direct microscopic examination according to the protocol of Bernardet et al. [19]. The presence of flexirubin-type pigments was investigated by using the bathochromatic shift test with a 20 % (w/v) KOH solution [19].Growth in the absence of NaCl was investigated using trypticase soy broth prepared according to the formula of the Difco medium except that NaCl was excluded. Growth at various NaCl concentrations [0.5–3.0 % (w/v) at increments of 0.5 %] was investigated in trypticase soy broth [20]. Based on the optimum NaCl concentration determined, cell growth was investigated at 10, 15, 20, 25, 30, 37 and 40 Ǫ C in trypticase soy broth with 1 % NaCl. To determine the optimal growth temperature, the spectrophotometric absorbance of the samples was measured at 600 nm (OD 600) after 48 h of growth. The pH response was determined in trypticase soy broth with 1 % NaCl at pH values ranging from 4 to 12 (using increments of 1 pH unit). Different pH values were achieved using the buffer system as described by Tang et al [21]. The cells were also incubated in an anaerobic chamber for 7 days in order to determine the extent of growth under anaerobic conditions. Oxidase and catalase activities were determined according to Park et al. [22]. Biochemical tests, including determination of gelatin liquefaction, the ability to hydrolyse starch, Tweens 20, 40, 80 and casein, the production of indole and H 2 S, motility and nitrate reduction, were carried out according to Dong and Cai [23]. Rapid identification systems, including API 20E, API 20NE and API 50 CH (bioḾerieux), were used to detect carbon substrate utilization and acid production in three replicates at 30 Ǫ C for 72 h in accordance with the manufacturer’ s instructions. API ZYM strips were used to detect enzyme activity.Genomic DNA of strain YH5 T was prepared from an overnight cell suspension culture by using a Wizard Genomic DNA Purification Kit (Promega). The genome of YH5 T was sequenced on an Illumina Hiseq X Ten platform (Shanghai Biozeron Biotechnology). The draft genome was 2 744 270 bp with a G+C content of 33.3 mol%. The obtained genome sequence was subsequently deposited at DDBJ/ EMBL/GenBank with accession number QLST00000000.1. 16S rRNA gene amplification was performed according to Lane [24] using the universal primers 27F (5′-AGAG TTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACC TTGTTACGACTT-3′). The PCR product was then cloned into pGEM-T Easy Vector and sequenced by the automated DNA sequencer (model ABI 3730; Applied Biosystems). The 16S rRNA gene sequence of strain YH5 T was aligned with closely related species within the genus Flavobacterium and one species outside the genus by using the program CLUSTAL X v1.8 [25]. Phylogenetic trees based on 16S rRNA gene sequences were reconstructed via the neighbour-joining [26], maximum-likelihood [27] and maximum-parsimony methods [28] according to Kimura’ s two-parameter model [29] in the MEGA 5.0 program [30]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) was shown next to the branches [31]. Since no genome data were available for the reference strains, DNA–DNA hybridization experiments were carried out between strain YH5 T and the reference type strains using the method described by De Ley et al. [32] with a Beckman DU 800 spectrophotometer.Cells of strain YH5 T and the other reference type strains of the genus Flavobacterium grown on TSA plates at 30 Ǫ C for 72 h were used in the chemotaxonomic comparisons. Fatty acid methyl esters were extracted, separated and identified according to the instructions of the Microbial Identification System (MIDI; Microbial ID) from a culture mass of approximately 50 mg. Polar lipids were extracted with chloroform/methanol (1: 2, v/v) and identified by twodimensional TLC followed by spraying with the appropriate detection reagent [33]. Preparation of the cell wall and determination of peptidoglycan composition were performed as described by Hasegawa et al. [34] with the modification that TLC on cellulose sheets was used instead of paper chromatography. Respiratory quinones were analysed according to a previously described method [21].Colonies of strain YH5 T on TSA plates were yellow and circular with entire margins. Cells of strain YH5 T were Gramstain-negative, non-spore-forming rods, 0.4–0.5 µm in width and 1.0–1.6 µm in length (Fig. S2, available in the online version of this article). Growth of strain YH5 T was observed at 10 and 37 Ǫ C (optimum growth at 30 Ǫ C), at pH 6–9 (optimum pH 7) and in the presence of 0–3 % (w/v) NaCl (optimum 1 %). Phototrophic growth was not observed. Based on the ability to grow at low concentrations of NaCl and relatively moderate temperatures, strain YH5 T was regarded as a halotolerant and mesophilic bacterium. Differences in physiological characteristics among strain YH5 T and the type strains of closely related species of the genus Flavobacterium are shown in Table 1. Strain YH5 T was positive for catalase and oxidase activity. Casein was hydrolysed. Nitrate reduction was not detected for strain YH5 T. The Voges–Proskauer test was positive, which was distinct from all the other reference strains. Both strain YH5 T and its closest phylogenetic neighbour F. luticocti CCTCC AB 2015421 T were positive for arginine dihydrolase.The partial 16S rRNA gene sequence of strain YH5 T was a continuous stretch of 1482 nt. Phylogenetic analysis of 16S rRNA gene sequences showed that strain YH5 T was most closely related to F. luticocti xz20 T (96.7 % similarity), a higher value than for other species within the genus Flavobacterium, such as F. jejuense EC 11 T (96.4 %), F. jumunjinense HME 7102 T (95.9 %) and F. dongtanense LW 30 T (95.6 %). The five strains formed a subclade within the genus Flavobacterium (Fig. 1). The affiliation of strain YH5 T and its closest neighbours was also confirmed by the maximum-likelihood and maximum-parsimony methods (Fig. S1). The G+C content of the genomic strain YH5 T was 33.3 mol%, which was similar to values for the type strain of the other species within the genus Flavobacterium (Table 1).Table 1. Comparative characteristics between strain YH5T and four reference strains Strain: 1, YH5T; 2, Flavobacterium luticocti CCTCC AB2015421T; 3, Flavobacterium jejuense KCTC 42149T; 4, Flavobacterium jumunjinense KCTC 23618T; 5, Flavobacterium dongtanense KCTC 22671T. All data were generated in this study. The five strains were able to grow at pH 6.0–9.0, with optimum pH of 7.0. Activity of b- galactosidase, lysine decarboxylase, tryptophan decarboxylase, ornithine decarboxylase and urease was negative. All strains were negative for the following characteristics: citrate utilization, H2S production, indole production and glucose fermentation. All strains were negative for hydrolysis of starch and Tweens (20/40/60/80). The five strains were positive for DNA hydrolysis and gelatin liquefaction. All were able to assimilate glucose, but not mannitol, capric acid or phenylacetic acid. The strains were unable to ferment the following substrates: glycerol, erythritol, D- arabinose, L- arabinose, D- ribose, D- xylose, L- xylose, adonitol, methyl b- D- xyloside, D- galactose, D- glucose, D- fructose, D- mannose, L- sorbose, L-rhamnose, dulcitol, inositol, D- mannitol, D- sorbitol, methyl a- D- mannoside, methyl a- D- glucoside, N -acetylglucosamine, amygdalin, arbutin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, inulin, melezitose, raffinose, starch, glycogen, xylitol, D- gentiobiose, turanose, D- lyxose, D-tagatose, D- fucose, L- fucose, D- arabinitol, L- arabinitol, gluconate, 2-keto-gluconate and 5-keto-gluconate. The following enzyme activities were detected for all the strains: alkaline phosphatase, leucine arylamidase, valine arylamidase, trypsin, acid phosphatase and naphthol-AS-BI-phosphohydrolase. All the strains were negative for lipase C14, a- galactosidase, b- galactosidase, b- glucuronidase, b- glucosidase, N -acetyl-b- glucosaminidase, a- mannosidase and b- fucosidase. +, Positive; ―, negative; W, weakly positive.Characteristic 1 2 3 4 5 Growth temperature (Ǫ C) 10–37 15–37 10–37 15–30 10–37 Optimum temperature (Ǫ C) 30 25–30 25 25 30 Growth NaCl concentration (%, w/v) 0–3 0–3 0–4 0–3 0–3 Optimum NaCl (%, w/v) 1 1 2 0–1 1 Oxidase + + + + W Catalase + + + + W Arginine dihydrolase + + ― ― ― Voges–Proskauer test + ― ― ― ― Nitrate reduction ― ― + ― ― Assimilation of: Arabinose ― + ― + ― Mannose ― ― + + ― N -Acetylglucosamine ― + ― + ― Maltose ― ― + + ― Gluconate ― ― ― ― + Adipic acid ― ― ― W ― Malic acid ― ― ― W ― Acid production from: Aesculin + + + ― ― Enzyme activity of: Esterase C4 W W W ― + Esterase lipase C8 W + W W W Cystine arylamidase + ― W W W Chymotrypsin W ― ― ― ― a- Glucosidase ― ― W + ― DNA G+C content (mol%) 33.3 32.9 28.1 36.5 30.0DNA–DNA hybridization experiments were performed on strain YH5 T with F. luticocti CCTCC AB 2015421 T, F. jejuense KCTC 42149 T, F. jumunjinense KCTC 23618 T and F. dongtanense KCTC 22671 T, respectively. The results indicated low levels of relatedness between strain YH5 T and its closest phylogenetic neighbour F. luticocti CCTCC AB 2015421 T (27.0 %). Furthermore, the relatedness values between strain YH5 T and F. jejuense KCTC 42149 T, F. jumunjinense KCTC 23618 T and F. dongtanense KCTC 22671 T were 20.0, 33.4 and 46.5 % respectively. These values were clearly below the 70 % cut-off point recommended for the assignment of strains to the same genomic species [35], which indicated that strain YH5 T represented a novel species of the genus Flavobacterium.The fatty acid and polar lipid profilesof strain YH5 T were found to be similar to other members of the genus Flavobacterium. In particular, the major fatty acids (when the strain was grown on TSA plates) comprised iso-C 15: 1 (49.7 %), C 16: 0 10-methyl and/or iso-C 17: 1 Ɯ 9 c (11.7 %), iso-C 17: 0 3-OH (10.9 %), iso-C 15: 1 G (8.8 %) and iso-C 15: 0 3- OH (5.9 %). A full comparison of the fatty acid profiles of YH5 T and other reference strains of the genus Flavobacterium is presented in Table 2. iso-C 15: 1 was the predominant fatty acid in all the reference strains. The predominant polar lipid of strain YH5 T was phosphatidylethanolamine (PE). Trace amounts of several unidentified aminolipids (UAL1– 4) and unidentified lipids (UL1–3) were also detected in the cells (Fig. S3). PE was present as a main component in many species of the genus Flavobacterium [36], including F. luticocti xz20 T, F. jejuense EC 11 T and F. jumunjinense HME 7102 T. The predominant respiratory quinone was MK-6 (Fig. S4), which was also true for all other members of the genus Flavobacterium. The diamino acid in the cellwall peptidoglycan was identified as meso -diaminopimelic acid (Fig. S5).Fig. 1. Phylogenetic tree of strain YH5 T and related type strains within the genus Flavobacterium based on almost-complete 16S rRNA gene sequences. The tree was calculated using the neighbour-joining algorithm in MEGA 5 software. Numbers at nodes indicate the percentages of bootstrap support based on 1000 resampled datasets; only values&gt;50 % are shown. Zobellia galactanivorans Dsij T (FP476056) was used as outgroup. Bar, 0.01 substitutions per nucleotide position.Based on phenotypic, genotypic, phylogenetic and chemotaxonomic characterization, strain YH5 T represents a novel species in the genus Flavobacterium, for which the name Flavobacterium tibetense sp. nov. is proposed.