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Flavobacterium Di Cile Sp. Nov., Isolated from a Freshwater Waterfall

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Flavobacterium Di Cile Sp. Nov., Isolated from a Freshwater Waterfall Flavobacterium Dicile sp. nov., Isolated from a Freshwater Waterfall Wen-Ming Chen National Kaohsiung College of Marine Technology: National Kaohsiung University of Science and Technology - Nanzih Campus Ya-Xiu You National Kaohsiung College of Marine Technology: National Kaohsiung University of Science and Technology - Nanzih Campus Chiu-Chung Young National Chung Hsing University Shih-Yao Lin National Chung Hsing University Shih-Yi Sheu ( [email protected] ) National Kaohsiung Marine University https://orcid.org/0000-0002-4097-2037 Research Article Keywords: Flavobacterium dicile, Flavobacteriaceae, Flavobacteriales, Flavobacteriia, Bacteroidetes Posted Date: April 13th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-407420/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/23 Abstract A bacterial strain designated KDG-16T is isolated from a freshwater waterfall in Taiwan and characterized to determine its taxonomic aliation. Cells of strain KDG-16T are Gram-stain-negative, strictly aerobic, motile by gliding, rod-shaped and form light yellow colonies. Optimal growth occurs at 20-25 oC, pH 6-7, and with 0% NaCl. Phylogenetic analyses based on 16S rRNA gene sequences and an up-to-date bacterial core gene set reveal that strain KDG-16T is aliated with species in the genus Flavobacterium. Analysis of 16S rRNA gene sequences shows that strain KDG-16T shares the highest similarity with Flavobacterium terrigena DSM 17934T (97.7%). The average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization values between strain KDG-16T and the closely related Flavobacterium species are below the cut-off values of 95-96, 90 and 70%, respectively, used for species T demarcation. Strain KDG-16 contains iso-C15:0, iso-C15:1 G and iso-C17:0 3-OH as the predominant fatty acids. The polar lipid prole consists of phosphatidylethanolamine, one uncharacterized aminophospholipid, one uncharacterized phospholipid, two uncharacterized aminolipids and two uncharacterized lipids. The major polyamine is homospermidine. The major isoprenoid quinone is MK-6. Genomic DNA G+C content of strain KDG-16T is 31.6%. Based on the polyphasic taxonomic data obtained, strain KDG-16T is considered to represent a novel species in the genus Flavobacterium, for which the name Flavobacterium dicile sp. nov. is proposed. The type strain is KDG-16T (=BCRC 81194T =LMG 31332T). Introduction The genus Flavobacterium (type species, Flavobacterium aquatile), a member of the family Flavobacteriaceae in the order Flavobacteriales of class Flavobacteriia within the phylum Bacteroidetes (2011), was rst established by Bergey et al. (1923) and emended by Bernardet et al. (1996), Dong et al. (2013), Kang et al. (2013) and Kuo et al. (2013). The genus Flavobacterium comprises 242 species with validly published names so far stated on the List of Prokaryotic Names with Standing in Nomenclature (https://lpsn.dsmz.de/genus/avobacterium). Species of the genus Flavobacterium have been isolated from habitats worldwide such as animal, plant, algae, freshwater, seawater, sediment, soil, compost, lagoon, glacier, decaying wood, water reservoir, activated sludge, sequencing batch reactor and wastewater treatment plant sources (Bernardet et al. 1996, 2006, 2011; Dong et al. 2013; Fujii et al. 2014; Kang et al. 2013; Kuo et al. 2013; Liu et al. 2010; Yoon et al. 2007). The present study was carried out to clarify the taxonomic position of a putative novel species belonging to the genus Flavobacterium, designated KDG-16T, by a polyphasic taxonomic approach. Materials And Methods Bacterial isolation and culture conditions Page 2/23 During a survey on cultivable bacterial resources from freshwater environment, a water sample (20 oC, pH 7, 0% NaCl) was collected from the Dajin Waterfall (GPS location: 22° 51' 40'' N 120° 38' 43'' E) in the Sandimen Township of Pingdong County, Taiwan on 5 April 2015 (Supplementary Fig. S1). The water sample was plated on R2A agar medium (BD Difco) through serial dilution technique. After incubation at 25 oC for 3 days, strain KDG-16T was isolated as a single light yellow colony and subjected to detailed taxonomy analyses. Strain KDG-16T was sub-cultured on R2A agar and stored at -80 oC in R2A broth with 20% (v/v) glycerol or stored following lyophilization. Strain KDG-16T has been deposited to Bioresource Collection and Research Center, Taiwan (BCRC 81194T) and BCCM/LMG Bacteria Collection, Belgium (LMG 31332T). Flavobacterium terrigena DSM 17934T, Flavobacterium urocaniciphilum JCM 19142T and Flavobacterium aquatile DSM 1132T were obtained from corresponding culture collections. The three type strains were used as reference strains and evaluated together with strain KDG-16T under identical experimental conditions. Morphological, physiological, and biochemical characterizations Cell morphology of strain KDG-16T was observed by phase-contrast microscopy (DM 2000; Leica) and transmission electron microscopy (Model H-7500; Hitachi) using cells grown on R2A agar at 25 oC for 3 days. The gliding motility was studied using phase-contrast microscopy as described by Bernardet et al. (2002). Gram-staining was performed with Stain Set S kit (BD Difco). Colony morphology was investigated on R2A agar using a stereoscopic microscope (SMZ 800; Nikon). The presence of exirubin and carotenoid types of pigments was examined as described by Reichenbach (1992) and Schmidt et al. (1994). The physiological characteristics of strain KDG-16T and the three reference strains were examined by growing bacteria at various temperatures, pH values and NaCl concentrations. The temperature range for bacterial growth was determined on R2A agar at 4, 10, 15, 20, 25, 30, 35, 37, 40, 45 and 50 oC. The pH range for bacterial growth was estimated at pH 4–9 (at intervals of 0.5 pH unit) using R2A broth at 25 oC. The pH of the medium was adjusted prior to sterilization to pH 4–9 using the biological buffers such as citrate/Na2HPO4, phosphate and Tris (Breznak and Costilow 2007). To measure the tolerance to NaCl, R2A broth was prepared and contained NaCl concentration, 0, 0.5% and 1–5%, w/v (at intervals of 1%). Growth under anaerobic conditions was determined after streaking strain KDG-16T on R2A agar and on R2A agar supplemented with nitrate (0.1% KNO3) and then incubated in anaerobic jars with AnaeroGen anaerobic system envelopes (Oxoid) at 25 oC for 21 days. Bacterial growth was studied on R2A agar, nutrient agar, Luria-Bertani agar and trypticase soy agar (all from Difco) under aerobic condition at 25 oC for 21 days. Activities of catalase, oxidase, DNase, urease and lipase (corn oil), and hydrolysis of starch, casein, lecithin and Tweens 20, 40, 60 and 80 were analyzed according to the methods described by Tindall et al. (2007). Chitin hydrolysis was evaluated on chitinase-detection agar as described by Wen et al. (2002) and carboxymethyl cellulose (CM-cellulose) hydrolysis was assessed using R2A agar as the basal medium as Page 3/23 the method described by Bowman (2000). Utilization of carbon sources was investigated in a basal medium as described by Chang et al. (2004). Substrates were added at a concentration of 0.1% (w/v or v/v). Incubation was prolonged for 21 days at 25 oC under aerobic condition and bacterial growth was examined every two days. Additional biochemical tests were carried out using API ZYM, API 20NE and API 50CH kits (all from bioMérieux) according to the manufacturers’ instructions. Sensitivity of strain KDG-16T to antibiotics was tested by the disc diffusion method on R2A agar at 25 oC for 3 days using the approach described by Nokhal and Schlegel (1983). The discs (Oxoid) contained the following antibiotics: rifampicin (5 µg), ampicillin (10 µg), gentamicin (10 µg), streptomycin (10 µg), kanamycin (30 µg), chloramphenicol (30 µg), nalidixic acid (30 µg), novobiocin (30 µg), tetracycline (30 µg), penicillin G (10 U) and sulfamethoxazole (23.75 µg) plus trimethoprim (1.25 µg). Chemotaxonomy The fatty acid proles of strain KDG-16T and the three reference strains were analyzed from cells that grown on R2A agar at 25 oC for 3 days. Fatty acid methyl esters were prepared and separated using a standard MIDI protocol (Sherlock Microbial Identication System, version 6.0), analyzed by GC (Hewlett- Packard 5890 Series II) and identied by using the RTSBA6.00 database (Sasser 1990). Polar lipids were extracted and analyzed by two-dimensional TLC as described by Embley and Wait (1994). Ethanolic molybdophosphoric acid was used for the detection of the total polar lipids, ninhydrin for amino lipids, the α-naphthol reagent for glycolipids and the Zinzadze reagent for phospholipids. Polyamines were extracted and identied according to the methods described by Busse and Auling (1988) and Busse et al. (1997). Cells of strain KDG-16T were cultivated in R2-PYE medium as described by Kämpfer et al. (2007) o at 25 C for 3 days, and homogenized in 0.2 M perchloric acid (HClO4) and centrifuged. Polyamines in the resultant supernatant were treated with dansyl chloride solution (7.5 µg ml− 1 in acetone), and analyzed by HPLC on a D-7000 high-speed liquid chromatograph and UV-VIS detector L-7420 (Hitachi). Isoprenoid quinones were extracted and puried according to the method of Collins (1994) and analyzed by HPLC with a Spherisorb ODS column. Determination 16S rRNA gene sequence and phylogenetic analysis Genomic DNA was extracted using a bacterial genomic DNA purication kit (DP02-150, GeneMark) for 16S rRNA gene analysis. The 16S rRNA gene was amplied using the universal primer set (27F and 1541R) and then sequenced using four primers (27F, 520F, 800R and 1541R) (Weisburg et al. 1991; Anzai et al. 1997). The sequence obtained was compared with those available from EzBioCloud (Yoon et al. 2017). Multiple sequence alignment was carried out using the clustal W (Larkin et al. 2007) and BioEdit software (Hall 1999).
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