Non-contiguous finished genome sequence and description of the gliding bacterium Flavobacterium seoulense sp. nov.

Flavobacterium is the type genus of the family Flavobacteriaceae in the phylum Bacteroidetes. Flavobacterium was proposed by Bergey et al.1,2] and the description was emended by Bernardet et al. 3]. Flavobacterium species have been isolated from various environments, including seawater, freshwater,
river sediments, and soil 4-8]. Members of the genus Flavobacterium are Gram-negative, rod-shaped, yellow-pigmented, aerobic bacteria. At the time of
writing, about 118 Flavobacterium species with validly published names have been described 9]; however, the genomes of only 14 type strains in this genus have been sequenced.

Flavobacterium seoulense sp. nov. strain EM1321^T (= KACC 18114^T =?JCM 30145^T) was isolated from stream water in Bukhansan National Park, Seoul, Korea. Here, we
present a summary classification and the features of Flavobacterium seoulense EM1321^T as well as its genome sequence and annotation.

Classification and features

Based on its 16S rRNA gene phylogeny and phenotypic characteristics, strain EM1321^T was classified as a member of the genus Flavobacterium (Table 1). Preliminary sequence-based identification using the 16S RNA gene sequences in the
EzTaxon database 10] indicated that strain EM1321^T was most closely related to F. granuli Kw05^T (GenBank accession no. AB180738) with a sequence similarity of 96.54%. This value
was lower than the 98.7% 16S rRNA gene sequence similarity as a threshold recommended
by Stackebrandtia and Ebers 11] to delineate a new species without carrying out DNA-DNA hybridization. Subsequent
phylogenetic analysis was performed using the 16S rRNA gene sequences of strain EM1321^T and related species. Sequences were aligned according to the bacterial rRNA secondary
structure model using the jPHYDIT 12]. Phylogenic trees were constructed using neighbor-joining (NJ) and maximum-likelihood
(ML) methods implemented in MEGA version 5 13]. The resultant tree topologies were evaluated by bootstrap analyses with 1,000 random
samplings. Strain EM1321^T formed a monophyletic clade together with Flavobacterium soli5] in both the NJ and ML trees; however, the clustering was not supported by the bootstrap
analysis (Figure 1). Flavobacterium nitratireducens8] was further recovered as a sister group of the monophyletic clade in the ML tree
only. Based on these phylogenetic trees, F. soli KACC 17417^T and F. nitratireducens JCM 17678^T were selected as reference strains and were obtained from the corresponding culture
collections for comparative study.

Figure 1. Phylogenetic tree highlighting the position of Flavobacterium seoulense EM 1321^Trelative to the type strains of other species within the genus Flavobacterium. The strains and their corresponding GenBank accession numbers of 16S rRNA genes are
indicated in parentheses. The sequences were aligned using jPHYDIT and the phylogenetic
inferences were obtained using neighbour-joining method with MEGA version 5 13]. The numbers at nodes are the percentage of bootstrap values obtained by 1,000 replicates.
Solid circles indicate that the corresponding nodes were also recovered in maximum-likelihood
tree. Bar, 0.01 substitutions per nucleotide position.

Strain EM1321^T was Gram-reaction negative. Cells of strain EM1321^T were rod shaped with rounded ends and motile by gliding. The cells were 1.0–1.5 ?m?×?0.3–0.5 ?m
in size (Figure 2). No flagellum was observed. The colonies were yellow in color and translucent on
R2A agar medium. Growth occurred aerobically at 4–35°C, and optimal growth was observed
at 30°C. The cells grew in 0–4% (w/v) NaCl. Strain EM1321^T exhibited catalase and oxidase activities. Physiological and biochemical properties
were tested by using the API 20NE, API 50CH, and API ZYM systems (BioMérieux). In
the API ZYM system, enzyme activity was detected for alkaline phosphatase, esterase
(C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase,
?-galactosidase, and valine arylamidase (Table 2). No activity was detected for lipase, trypsin, ?-chymotrypsin, ?-galactosidase, ?-glucuronidase, ?-glucosidase, N-acetyl-?-glucosaminidase, cystine arylamidase, ?-mannosidase, and ?-fucosidase. In the API 20NE system, positive reactions were observed for nitrate
reduction and negative reactions were observed for indole production, glucose fermentation,
arginine dihydrolase, urease activity, and aesculin and gelatin hydrolysis. The strain
assimilated d-glucose and l-arabinose, but not d-mannitol, d-mannose, d-maltose, potassium
gluconate, N-acetylglucosamine, capric acid, adipic acid, malic acid, trisodium citrate, or phenylacetic
acid. Acid was produced from l-arabinose, d-xylose, d-galactose, d-glucose, d-fructose,
d-mannose, and d-lactose (API 50CH).

Figure 2. Transmission electron micrograph of Flavobacterium seoulense EM1321^T. Scale bar, 200 nm.

Table 2. Phenotypic characteristics of Flavobacterium seoulense EM1321^T and phylogenetically related Flavobacterium species

Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) MS protein
analysis was carried out as previously described 24]. Deposits were done from 12 isolated colonies for each strain (strain EM1321^T and reference strains). Measurements were made with a Microflex spectrometer (Bruker
Daltonics, Leipzig, Germany). Spectra were recorded in the positive linear mode for
the mass range of 2,000 to 20,000 Da (parameter settings: ion source 1 (IS1), 20 kV;
IS2, 18.5 kV; lens, 7 kV). The time of acquisition was between 30 seconds and 1 minute
per spot. The twelve EM1321^T spectra were imported into the MALDI BioTyper software (version 2.0; Bruker) and
analyzed by standard pattern matching (with default parameter settings) against 4,613
bacterial spectra including eight Flavobacterium species, used as reference data, in the BioTyper database. For strain EM1321^T spectrum (Figure 3), no significant score was obtained, suggesting that our isolate was not a member
of the eight known species in the database. Spectrum differences with the two closely
related Flavobacterium species are shown in Figure 4.

Figure 3. Reference mass spectrum from Flavobacterium seoulense EM1321^T. Spectra from 12 individual colonies were compared and a reference spectrum was generated.

Figure 4. Gel view comparing the Flavobacterium seoulense EM1321^T spectrum with those of other members in the genus Flavobacterium. The gel view displays the raw spectra of all loaded spectrum files arranged in a
pseudo-gel-like look. The x-axis records the m/z value. Peak intensity is shown as
a gray-scale scheme code. The color bar and the right y-axis indicate the relation
between the color of a peak and peak intensity in arbitrary units.