Introduction

The genus Flavobacterium was proposed by Bergey et al. (1923) for strains that were previously described as Bacillus aquatilis by Frankland and Frankland (1889) to accommodate rod-shaped, non-spore-forming, Gram staining-negative bacteria (Bergey et al. 1923; Frankland and Frankland 1889; Weeks 1955). This genus belongs to the Cytophaga–Flavobacterium–Bacteroides (CFB) group of the phylum Bacteroidetes (Ludwig and Klenk 2001; Bernardet and Bowman 2010). At the time of writing, there are more than 180 species with validly published names (http://www.bacterio.net/flavobacterium.html). However, descriptions of the genus have been extensively emended; the key characteristics are yellow coloured colonies, inability to produce indole, inability to grow in anaerobic conditions, DNA G + C content in the range of 30–41 mol% (except Flavobacterium caeni; 52 mol%), most of the members contain iso-C15:0 as a major fatty acid, menaquinone-6 as major respiratory quinone, and phosphatidylethanolamine as major polar lipid (Bernardet et al. 1996; Dong et al. 2013; Kang et al. 2013; Liu et al. 2010). Members of the genus Flavobacterium are cosmopolitan in distribution and have been frequently isolated from soil, water, sludge, sediments, plants, fish, and food products. Although most species are non-pathogenic, some species notably Flavobacterium columnare, Flavobacterium branchiophila, Flavobacterium psychrophilum, and Flavobacterium spartansii are recognized as fish pathogens (Pilarski et al. 2008; Wakabayashi et al. 1989; Duchaud et al. 2007; Starliper 2001; Louch and Faisal 2014). Some species have been isolated from wastewater polluted with heavy metal and sediment with very high arsenic content (Ao et al. 2014; Yoon et al. 2009). In addition, some urea hydrolyzing members of the genus Flavobacterium have also been reported (Pilarski et al. 2008; Nupur et al. 2013).

Strain S-42T was isolated during a study of bacterial diversity of soil near Kyonggi University. This study describes a novel member of the genus Flavobacterium, isolated from stream bank soil near Kyonggi University, Suwon, South Korea and subjected to polyphasic taxonomic characterization.

Materials and methods

Isolation, cultivation and maintenance

Strain S-42T was isolated using a modified culture technique with 6-well polycarbonate transwell plates (Corning, Inc.) from stream bank soil near Kyonggi University, Suwon, South Korea. Isolation, routine culture, and preservation were done as described previously (Dahal and Kim 2016; Dahal et al. 2017). Based on 16S rRNA gene sequence similarities and phylogenetic analyses, Flavobacterium maotaiense JCM 19927T, Flavobacterium hibernum DSM 12611T, and Flavobacterium granuli KACC 11820T were selected for comparative analyses and were used as reference strains. In addition, the type species of the genus Flavobacterium, Flavobacterium aquatile KACC 11692T was also selected for comparative physiological and chemotaxonomic analyses. All the reference strains were cultivated on R2A agar plate at 28 °C for 3–4 days.

Cell morphology

The morphologies of cells grown on R2A agar for 3–4 days at 28 °C were observed by light microscopy (BX50 microscope; Olympus, Japan), and transmission electron microscopy (Bio-TEM, Hitachi, H-7650, Japan). Colony morphology was observed on R2A agar after incubation at 28 °C for 4 days using a Zoom Stereo Microscope (SZ61; Olympus, Japan). Gram staining was performed according to the procedure described by Doetsch (1981).

Physiological and biochemical tests

Production of flexirubin-type pigment was investigated by flooding with 20% (w/v) KOH solution (Reichenbach 1992). Catalase activity was determined by production of bubbles with 3% (v/v) hydrogen peroxide (H2O2). Oxidase activity was determined using 1% (w/v) tetra-methyl-p-phenylenediamine dihydrochloride. Motility was assessed in SIM (Oxoid) medium and/or R2A medium containing 0.4% agar. Growth was assessed on various media, including R2A agar (MB Cell), nutrient agar (NA; Oxoid), tryptone soya agar (TSA; Oxoid), sorbitol MacConkey agar (MA; Oxoid), marine agar (Difco), brain heart infusion agar (BHI; Bacto), mueller–hinton agar (MHA; BBL), veal infusion agar (Difco), and Luria–Bertani agar (LBA; Oxoid). Growth at various temperatures, 4–37 °C (4, 10, 15, 20, 25, 28, 30, 32, 35, and 37 °C) was determined on R2A plates for 7 days. The pH range for growth was determined by cultivation at 28 °C in R2A broth adjusted to pH 4–12 (at 0.5 pH unit intervals) prior to sterilization using 5 M citrate/NaH2PO4 buffer (for pH 4.0–5.5), 5 M phosphate buffer (for pH 6–7.5), 5 M Tris buffer (for pH 8–10) (Breznak and Costilow 2007), and 5 M NaOH (for pH 10.5–12.0) each at a final concentration of 20 mM. Growth in NaCl was examined by cultivation in R2A broth containing 0–5% NaCl (w/v, at 0.5% intervals). Anaerobic growth was assessed by cultivation on R2A agar at 28 °C for 10 days in a BBL (Becton–Dickinson) anaerobic jar with a GasPak™ EZ Gas Generating Container (Becton–Dickinson). Hydrolysis of Tween 80, Tween 60, and Tween 40 were assessed according to the method of Smibert and Krieg (1994). Hydrolysis of chitin, CM-cellulose, hypoxanthine, tyrosine, starch, and casein were evaluated as described previously (Dahal and Kim 2017). Production of hydrogen sulphide and indole was assessed using sulphide indole motility medium (SIM; Oxoid). A DNase assay was performed on DNase agar (Oxoid) by flooding 6 N HCl. Endospore formation was evaluated by staining with malachite green. Other physiological and biochemical tests were performed using API 20NE, and API ID 32GN test kits (bioMérieux). Enzyme activities were tested using an API ZYM kit (bioMérieux) according to the manufacturer’s instructions.

Phylogenetic analysis

For 16S rRNA gene sequencing, genomic DNA was extracted using an InstaGene Matrix kit (Bio-Rad, Hercules, CA, USA), and the 16S rRNA gene was amplified by PCR using universal bacterial primers 27F (forward) and 1492R (reverse) (Frank et al. 2008). PCR product was purified with multiscreen-filter plate (Millipore Corp., Bedford, MA, USA), and was sequenced with an Applied Biosystems 3770XL DNA analyser using a BigDye Terminator cycle sequencing kit v.3.1 (Applied Biosystems, USA). Almost complete sequence was assembled with SeqMan software (DNASTAR Inc.). The nearly complete length of 16S rRNA gene sequence for strain S-42T was 1475 bp. The close phylogenetic neighbours were identified using the EzBioCloud (Yoon et al. 2017). All the 16S rRNA sequences of the close members were retrieved from NCBI GenBank and subjected to multiple sequence alignment using Clustal X 2.1 (Larkin et al. 2007). After multiple alignments, gaps at the 5′ and 3′ ends were deleted using the software package BioEdit (Hall 1999). Phylogenetic trees were constructed using MEGA6 (Tamura et al. 2013) by three different treeing methods: neighbour–joining method (Saitou and Nei 1987), maximum–parsimony algorithm (Fitch 1971), and maximum–likelihood algorithm (Felsenstein 1981). During phylogenetic analysis, evolutionary distances were calculated using Kimura two-parameter model (Kimura 1980), and bootstrap values were calculated based on 1000 replications (Felsenstein 1985).

Chemotaxonomic characterization

For the fatty acid analysis, cellular biomass of strain S-42T and reference strains were harvested from R2A plate incubated at 28 °C for 3 days. Fatty acids were extracted using the standard MIDI protocol (Sherlock Microbial Identification System, version 6.0B). The fatty acids were analysed with a gas chromatograph (HP 6890 Series GC System; Hewlett Packard) and identified using the TSBA6 database of the Microbial Identification System (Sasser 1990).

The respiratory quinone was extracted and analysed from freeze-dried cells as described by Minnikin et al. (1984). Isoprenoid quinone was extracted with methanol–water (10:1 v/v; water containing 0.3% sodium chloride) and petroleum ether at 80 °C, evaporated under a vacuum, re-extracted with acetone, and analysed by HPLC (Minnikin et al. 1984; Collins and Jones 1981). The polar lipids were extracted by the method described by Nguyen and Kim (2017). The polar lipids were analysed by two-dimensional TLC using chloroform/methanol/water (65:25:4; v/v/v) in the first dimension and chloroform/methanol/acetic acid/water (40:7.5:6:2, v/v/v/v) in the second. Appropriate detection reagents (Minnikin et al. 1984; Komagata and Suzuki 1987) were used to identify the spots; molybdophosphoric acid (phosphomolybdic acid reagent, 5% v/v solution in ethanol; Sigma-Aldrich, Germany) was used to detect total polar lipids, ninhydrin reagent (0.2% solution; Sigma Life Science, USA) was used to detect amino lipids, Zinzadze reagent (molybdenum blue spray reagent, 1.3%; Sigma Life Sciences) was used to detect phospholipids, and α-naphthol reagent was used to detect glycolipids.

Genotypic characterization

For DNA–DNA hybridization and DNA G + C mol%, genomic DNAs of strains were extracted according to the method presented by Cheng and Jiang (2006). DNA G + C content was determined according to the procedure described by Mesbah et al. (1989). DNA–DNA hybridization was measured fluorometrically according to the method developed by Ezaki using photobiotin-labelled DNA probes and micro-dilution plates (Ezaki et al. 1989). All the assays were carried out in triplicate.

Results and discussion

Morphological and physiological characteristics

Cells (Fig. S1) appear as rod-shaped and are Gram stain-negative, non-motile, non-spore-forming, and aerobic. Colonies on R2A are yellow-pigmented, circular, entire, and convex. Cells are 1.8–2.2 µm long and 0.5–0.8 µm wide. After incubation on R2A agar for 4 days at 28 °C, the size of the colonies was 2–3 mm in diameter. The strain can grow at 15–35 °C, pH 6.0–10.0, and at 0.5% (w/v) NaCl concentration. Urea is hydrolysed. Flexirubin-type pigments are absent. The differential phenotypic features of strain S-42T are presented in Table 1 with other closely related members of the genus Flavobacterium.

Table 1 Phenotypic characteristics that differentiate strain S-42T from phylogenetically closely related type strains of the genus Flavobacterium
Full size table

Phylogenetic analysis

The nucleotide sequence of the 16S rRNA gene of strain S-42T has been deposited in GenBank/EMBL/DDBJ under the accession number KY117464. Preliminary comparisons with the 16S rRNA gene sequences in GenBank indicated that strain S-42T belongs to the genus Flavobacterium and is closely related to F. maotaiense T9T (97.4% sequence similarity), F. hibernum ATCC 51468T (97.4%), and F. granuli Kw05T (97.1%). The 16S rRNA gene sequence identity between strain S-42T and other members of the genus Flavobacterium were <97.0%. A phylogenetic tree based on these sequences and ones with lower similarities is presented in Fig. 1.

Fig. 1
figure 1

Maximum–likelihood tree based on 16S rRNA gene sequences showing the phylogenetic position of strain S-42T among closely related members of the genus Flavobacterium. Filled circles indicate nodes recovered by all three treeing methods (neighbour–joining, maximum–likelihood, and maximum–parsimony). The numbers at the nodes indicate the percentage of 1000 bootstrap replicates yielding this topology; only values > 50% are shown. Chryseobacterium balustinum LMG 8329T was used as an out-group. GenBank accession numbers are given in parentheses. Bar, 0.05 substitutions per nucleotide position

Full size image

Based on 16S rRNA gene sequence similarities and phylogenetic analysis, F. maotaiense JCM 19927T, F. hibernum DSM 12611T, F. granuli KACC 11820T, and F. aquatile KACC 11692T were selected for comparative analysis and were used as reference strains for biochemical tests, fatty acid analysis, menaquinone, and DNA–DNA hybridization.

Chemotaxonomic characteristics

The major cellular fatty acids were identified as iso-C15:0, summed feature 3 (C16:1ω7c and/or C16: 1ω6c), C16:0, anteiso-C15:0, iso-C17:0 3-OH, iso-C15:0 3-OH, and iso-C15:1 G (Table 2). Menaquinone-6 (MK-6) was detected as sole respiratory quinone. The major polar lipids of strain S-42T were identified as phosphatidylethanolamine (PE) and an unidentified aminolipid (AL4). In addition, three unidentified aminolipids (AL1–AL3), and four unidentified polar lipids (L1–L4) were also detected in moderate amounts (Fig. S2).

Table 2 Cellular fatty acid profiles (% of totals) of strain S-42T and phylogenetically closely related species of the genus Flavobacterium
Full size table

DNA G + C content and DNA–DNA hybridization

The G + C content of type strain S-42T was 35.8 mol%, which is in line with that (30–52 mol%) of members of the genus Flavobacterium. DNA–DNA relatedness of the strain S-42T with reference strains showed DNA similarities of 45.6 ± 3.2% (reciprocal, 51.1 ± 2.7%)with F. maotaiense JCM 19927T, 43.7 ± 2.9% (reciprocal, 45.3 ± 2.6%) with F. hibernum DSM 12611T, and 29.7 ± 2.3% (reciprocal, 28.1 ± 3.3%) with F. granuli KACC 11820T. DNA–DNA relatedness between the species demonstrated that, strain S-42T differs genetically from closely related type strains of the genus Flavobacterium at the species level as the relatedness is below the recommended 70% cut off value (Wayne et al. 1987).

Maximum growth temperature (35° C); highest salt tolerance (0.5%); inability to assimilate l-proline and N-acetyl-glucosamine; and weak assimilation of d-mannose phenotypically differentiate strain S-42T from its phylogenetically closely related type strains. Moreover, F. maotaiense JCM 19927T, F. hibernum DSM 12611T, F. granuli KACC 11820T, and F. aquatile KACC 11692T are catalase positive but strain S-42T showed a negative result for catalase activity (Table 1). Absence of fatty acids C15:0, iso-C15:1ω10c; presence of minor fatty acids C17: 0, C13:1, C17:1ω5c, C18:1ω5c, anteiso-C17:0, iso-C14:1 E, anteiso-C15:1 A, anteiso-C17:1ω9c, C17:0 2-OH, and C17:0 3-OH and the differences in percentages of major and other minor fatty acids showed characteristic differences for strain S-42T from the most closely related type strains (Table 2). The sole respiratory quinone was MK-6, as reported as the major respiratory quinone of all members of the genus Flavobacterium. In addition, the presence of four unidentified aminolipids and four unidentified polar lipids, their spot positions and amounts also differentiates strain S-42T as a different species (Fig. S2) (Feng et al. 2015). Furthermore, strain S-42T was genotypically distinguished by DNA–DNA relatedness values (29–46% of relatedness lower than 70% cut off value for species delineation). Based on this polyphasic characterization, strain S-42T represents a novel member in the genus Flavobacterium, for which the name Flavobacterium ureilyticum sp. nov. is proposed.

Description of Flavobacterium ureilyticum sp. nov.

Flavobacterium ureilyticum (ur.e.i.ly′ti.cum. N.L. n. urea urea; N.L. neut. adj. lyticum from Gr. adj. lytikos dissolving; N.L. neut. adj. ureilyticum urea-dissolving).

Cells appear as rod-shaped and are Gram staining-negative, non-motile, non-spore-forming, and aerobic. Grows well on R2A but no growth is observed on NA, TSA, LBA, BHI, PDA, veal infusion agar, marine agar, and MacConkey agar. Colonies on R2A are yellow-pigmented, circular, entire, and convex. Cells are 1.8–2.2 µm long and 0.5–0.8 µm wide. After incubation on R2A agar for 4 days at 28 °C, the size of the colonies is 2–3 mm in diameter. Cells grow at 15–35 °C (optimum, 20–30 °C) and pH 6.0–10.0 (optimum pH, 7.0–9.0). No growth is observed in 1% NaCl. Oxidase test and urease activity are positive. Flexirubin pigments are not produced. Hydrogen sulphide is not produced from culture in SIM medium. Glucose is not fermented. Indole and catalase tests are negative. Gelatin is not liquefied. Casein, starch, and CM-cellulose are hydrolysed but chitin, tyrosine, DNA, hypoxanthine, Tween 40, Tween 60, and Tween 80 are not. Esculin ferric citrate is positive. Nitrate is not reduced to nitrite. The type strain shows the following enzyme activities: positive for, alkaline phosphatase, leucine arylamidase, valine arylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, and α-glucosidase; weakly positive for esterase (C4), esterase lipase (C8), and cystine arylamidase; and negative for, lipase (C14), α-chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, β-glucosidase, n-acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase. The following substrates are assimilated: d-glucose, d-maltose, sucrose, glycogen, and d-mannose. The major fatty acids are iso-C15:0, summed feature 3 (C16:1ω7c and/or C16: 1ω6c), C16:0, anteiso-C15:0, iso-C17:0 3-OH, iso-C15:0 3-OH, and iso-C15:1 G. The sole respiratory quinone is MK-6. The major polar lipids are phosphatidylethanolamine and an unidentified aminolipid. The DNA G + C content of the type strain is 35.8 mol%.

The type strain, S-42T (= KEMB 9005-537T = KACC 19115T = NBRC 112683T), was isolated from stream bank soil near Kyonggi University, Suwon, Gyeonggi-Do, South Korea. The Digital Protologue database TaxonNumber for strain S-42T is TA00529.