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

Abstract

Flavobacterium seoulense strain EM1321T is the type strain of Flavobacterium seoulense sp. nov., a proposed novel species within the genus Flavobacterium. This strain is a Gram-reaction-negative, aerobic, rod-shaped bacterium isolated from stream water in Bukhansan National Park, Seoul. This organism is motile by gliding. Here, we describe the features of Flavobacterium seoulense EM1321T, together with its genome sequence and annotation. The genome comprised 3,792,640 bp, with 3,230 protein-coding genes and 52 RNA genes.

Introduction

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 [48]. 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 EM1321T (= KACC 18114T = JCM 30145T) was isolated from stream water in Bukhansan National Park, Seoul, Korea. Here, we present a summary classification and the features of Flavobacterium seoulense EM1321T as well as its genome sequence and annotation.

Classification and features

Based on its 16S rRNA gene phylogeny and phenotypic characteristics, strain EM1321T 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 EM1321T was most closely related to F. granuli Kw05T (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 EM1321T 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 EM1321T formed a monophyletic clade together with Flavobacterium soli [5] in both the NJ and ML trees; however, the clustering was not supported by the bootstrap analysis (Figure 1). Flavobacterium nitratireducens [8] was further recovered as a sister group of the monophyletic clade in the ML tree only. Based on these phylogenetic trees, F. soli KACC 17417T and F. nitratireducens JCM 17678T were selected as reference strains and were obtained from the corresponding culture collections for comparative study.

Table 1 Classification and general features of Flavobacterium seoulense EM1321T according to the MICG recommendations [14]
Figure 1
figure1

Phylogenetic tree highlighting the position of Flavobacterium seoulense EM 1321T relative 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 EM1321T was Gram-reaction negative. Cells of strain EM1321T were rod shaped with rounded ends and motile by gliding. The cells were 1.0–1.5 μm × 0.3–0.5 μm 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 EM1321T 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, β-galactosidase, and valine arylamidase (Table 2). No activity was detected for lipase, trypsin, α-chymotrypsin, α-galactosidase, β-glucuronidase, α-glucosidase, N-acetyl-β-glucosaminidase, cystine arylamidase, α-mannosidase, and α-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
figure2

Transmission electron micrograph of Flavobacterium seoulense EM1321T . 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 EM1321T 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 EM1321T 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 EM1321T 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
figure3

Reference mass spectrum from Flavobacterium seoulense EM1321T . Spectra from 12 individual colonies were compared and a reference spectrum was generated.

Figure 4
figure4

Gel view comparing the Flavobacterium seoulense EM1321T 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.

Genome sequencing information

Genome project history

Flavobacterium seoulense EM1321T was selected for genome sequencing based on its phylogenetic position and its 16S rRNA similarity to other members of the genus Flavobacterium. The genome sequence was deposited in GenBank under accession number JNCA00000000.1. A summary of the project and the Minimum Information about a Genome Sequence (MIGS) [14] are shown in Table 3.

Table 3 Genome sequencing project information

Growth conditions and DNA isolation

Flavobacterium seoulense EM1321T was cultured aerobically on R2A agar medium at 30°C. Genomic DNA was extracted using the QIAamp DNA mini kit (Qiagen).

Genome sequencing and assembly

The genome of strain EM1321T was sequenced at ChunLab, Inc. by using an Illumina Miseq_PE_300 system with 2 × 300 paired-end reads. The Illumina platform provided 166× coverage (for a total of 3,792,640 sequencing reads) of the genome. CLC Genomics Workbench (ver. 6.5.1) was used for sequence assembly and quality assessment. The final draft assembly contained 56 contigs.

Genome annotation

The genes in the assembled genome were predicted with Rapid Annotation using Subsystem Technology (RAST) server databases [25] and the gene-caller GLIMMER 3.02 [26]. The predicted ORFs were annotated by searching clusters of orthologous groups (COGs) [11] using the SEED database [27]. RNAmmer 1.2 [28] and tRNAscan-SE 1.23 [29] were used to identify rRNA genes and tRNA genes, respectively. CRISPR repeats were examined using CRISPR recognition tool (CRT) [30]. CLgenomics™ 1.06 (ChunLab) was used to visualize the genomic features.

Genome properties

The genome comprised a circular chromosome with a length of 3,792,640 bp and 33.25% G + C content (Figure 5 and Table 4). It is composed of 56 contigs. Of the 3,282 predicted genes, 3,230 were protein-coding genes and 52 were RNA genes (2 rRNA genes and 50 tRNA genes). The sequencing coverage of rRNA operon (673×) indicated that 4 copies of rRNA operons are exist in this genome. The majority of the protein-coding genes (2,054 genes, 62.58%) were assigned putative functions, while the remaining genes were annotated as hypothetical proteins (1,176 genes, 35.83%). The properties of and statistics for the genome are summarized in Table 4. The distribution of genes into COG functional categories is presented in Table 5 and Figure 5.

Figure 5
figure5

Graphical circular map of the genome. Starting from the outmost circle and moving inwards, each ring of the circle contains information on a genome: rRNA/tRNA, genes on the reverse strand (colored according to the COG categories), genes on the forward strand (colored according to the COG categories), GC skew, and GC ratio.

Table 4 Genome statistics
Table 5 Number of genes associated with the 25 general COG functional categories

Conclusions

Based on the results from phylogenetic and phenotypic analyses, we formally propose the creation of the new species Flavobacterium seoulense sp. nov. for strain EM1321T. The non-contiguous genome sequence of the type strain was determined and described here.

Description of Flavobacterium seoulense sp. nov

Flavobacterium seoulense (seo.ul.en’se. N.L. neut. adj., named after Seoul, Korea, the geographical origin of the type strain).

Aerobic, Gram-reaction negative. Cells are rod shaped and motile by gliding. Does not have a flagellum. The colonies are yellow in color and translucent on R2A agar medium. Grows at 4–35°C, with optimum growth at 30°C and in 0–4% (w/v) NaCl. Catalase- and oxidase-positive. Positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, β-galactosidase, and valine arylamidase. Positive for nitrate reduction, but negative for indole production, glucose fermentation, arginine dihydrolase, urease activity, and aesculin and gelatin hydrolysis. Negative for lipase, trypsin, α-chymotrypsin, α-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, or cystine arylamidase activity. This strain assimilated d-glucose and l-arabinose, but not d-mannitol, d-mannose, d-maltose, N-acetylglucosamine, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, or phenylacetic acid. Produces acid from l-arabinose, d-xylose, d-galactose, d-glucose, d-fructose, d-mannose, and d-lactose.

The G + C content of the genome is 33.25%. The 16S rRNA and genome sequences are deposited in GenBank under accession numbers KJ461685 and JNCA00000000.1, respectively. The type strain EM1321T (= KACC 18114T = JCM 30145T) was isolated from stream water in Bukhansan National Park, Seoul, Korea.

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Acknowledgements

This work was supported by the Basic Science Research Programs through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, and Future Planning (NRF-2013R1A1A3010041) and supported by a Korea University Grant.

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Correspondence to Hana Yi.

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The authors declare that they have no competing interests.

Authors’ contributions

SS drafted the manuscript, performed laboratory experiments, and analyzed the data. HG cultured samples and performed the electron micrograph and phylogenetic analysis. YC, SK and DY sequenced, assembled, and annotated the genome. HY organized the study and drafted the manuscript. All authors read and approved the final manuscript.

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Shin, SK., Goo, H., Cho, YJ. et al. Non-contiguous finished genome sequence and description of the gliding bacterium Flavobacterium seoulense sp. nov.. Stand in Genomic Sci 9, 34 (2014). https://doi.org/10.1186/1944-3277-9-34

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Keywords

  • Flavobacterium
  • Gliding motility
  • Aerobic
  • Flavobacteriaceae