- Short genome report
- Open Access
Non-contiguous finished genome sequence and description of the gliding bacterium Flavobacterium seoulense sp. nov.
Standards in Genomic Sciences volume 9, Article number: 34 (2014)
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.
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. . 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 ; 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  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  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 . Phylogenic trees were constructed using neighbor-joining (NJ) and maximum-likelihood (ML) methods implemented in MEGA version 5 . The resultant tree topologies were evaluated by bootstrap analyses with 1,000 random samplings. Strain EM1321T formed a monophyletic clade together with Flavobacterium soli  in both the NJ and ML trees; however, the clustering was not supported by the bootstrap analysis (Figure 1). Flavobacterium nitratireducens  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.
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).
Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) MS protein analysis was carried out as previously described . 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.
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)  are shown in Table 3.
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.
The genes in the assembled genome were predicted with Rapid Annotation using Subsystem Technology (RAST) server databases  and the gene-caller GLIMMER 3.02 . The predicted ORFs were annotated by searching clusters of orthologous groups (COGs)  using the SEED database . RNAmmer 1.2  and tRNAscan-SE 1.23  were used to identify rRNA genes and tRNA genes, respectively. CRISPR repeats were examined using CRISPR recognition tool (CRT) . CLgenomics™ 1.06 (ChunLab) was used to visualize the genomic features.
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.
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.
Bergey DH, Harrison FC, Breed RS WHB, Huntoon FM: Bergey’s Manual of Determinative Bacteriology, Volume 4. 1st edition. Baltimore: The Williams and Wilkins Co; 1923:1–442.
Skerman VBD, Mcgowan V, Sneath PHA: Approved lists of bacterial names. Int J Syst Bacteriol 1980, 30: 225–420. 10.1099/00207713-30-1-225
Bernardet JF, Segers P, Vancanneyt M, Berthe F, Kersters K, Vandamme P: Cutting a gordian knot: Emended classification and description of the genus Flavobacterium , emended description of the family Flavobacteriaceae , and proposal of Flavobacterium hydatis nom nov (basonym, Cytophaga aquatilis Strohl and Tait 1978). Int J Syst Bacteriol 1996, 46: 128–148. 10.1099/00207713-46-1-128
Kim BY, Weon HY, Cousin S, Yoo SH, Kwon SW, Go SJ, Stackebrandt E: Flavobacterium daejeonense sp. nov. and Flavobacterium suncheonense sp. nov., isolated from greenhouse soils in Korea. Int J Syst Evol Microbiol 2006, 56: 1645–1649. 10.1099/ijs.0.64243-0
Yoon JH, Kang SJ, Oh TK: Flavobacterium soli sp nov., isolated from soil. Int J Syst Evol Microbiol 2006, 56: 997–1000. 10.1099/ijs.0.64119-0
Tamaki H, Hanada S, Kamagata Y, Nakamura K, Nomura N, Nakano K, Matsumura M: Flavobacterium limicola sp. nov., a psychrophilic, organic-polymer-degrading bacterium isolated from freshwater sediments. Int J Syst Evol Microbiol 2003, 53: 519–526. 10.1099/ijs.0.02369-0
Kim JH, Kim KY, Cha CJ: Flavobacterium chungangense sp. nov., isolated from a freshwater lake. Int J Syst Evol Microbiol 2009, 59: 1754–1758. 10.1099/ijs.0.007955-0
Nupur Bhumika V, Srinivas TN, Kumar PA: Flavobacterium nitratireducen s sp. nov., an amylolytic bacterium of the family Flavobacteriaceae isolated from coastal surface seawater. Int J Syst Evol Microbiol 2013, 63: 2490–2496.
NamesforLife, LLC: http://doi.namesforlife.com/10.1601/tx.8071
Kim OS, Cho YJ, Lee K, Yoon SH, Kim M, Na H, Park SC, Jeon YS, Lee JH, Yi H, Won S, Chun J: Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 2012, 62: 716–721. 10.1099/ijs.0.038075-0
Stackebrandt E, Ebers J: Taxonomic parameters revisited: tarnished gold standards. Microbiol Today 2006, 33: 152–155.
Jeon YS, Chung H, Park S, Hur I, Lee JH, Chun J: jPHYDIT: a JAVA-based integrated environment for molecular phylogeny of ribosomal RNA sequences. Bioinformatics 2005, 21: 3171–3713. 10.1093/bioinformatics/bti463
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S: MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2011, 28: 2731–2739. 10.1093/molbev/msr121
Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen MJ, Angiuoli SV, Ashburner M, Axelrod N, Baldauf S, Ballard S, Boore J, Cochrane G, Cole J, Dawyndt P, De Vos P, dePamphilis C, Edwards R, Faruque N, Feldman R, Gilbert J, Gilna P, Glockner FO, Goldstein P, Guralnick R, Haft D, Hancock D, et al.: The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol 2008, 26: 541–547. 10.1038/nbt1360
Woese CR, Kandler O, Wheelis ML: Towards a natural system of organisms: proposal for the domains Archaea , Bacteria , and Eucarya . Proc Natl Acad Sci USA 1990, 87: 4576–4579. 10.1073/pnas.87.12.4576
Krieg NR, Ludwig W, Euzéby J, Whitman WB, Phylum XIV: Bacteroidetes phyl. nov. In Bergey’s Manual of Systematic Bacteriology, Volume 4. 2nd edition. Edited by: Krieg NR, Staley JT, Brown DR, Hedlund BP, Paster BJ, Ward NL, Ludwig W, Whitman WB. New York: Springer; 2011:25.
Euzéby JP: Validation List No. 143. List of new names and new combinations previously effectively, but not validly, published. Int J Syst Evol Microbiol 2012, 62: 1–4.
Bernardet JF, Order I: Flavobacteriales ord. nov. In Bergey’s Manual of Systematic Bacteriology, Volume 4. 2nd edition. Edited by: Krieg NR, Staley JT, Brown DR, Hedlund BP, Paste RBJ, Ward NL, Ludwig W, Whitman WB. New York: Springer; 2011:105.
Bernardet JF, Family I: Flavobacteriaceae . In Bergey’s Manual of Systematic Bacteriology, Volume 4. 2nd edition. Edited by: Krieg NR, Staley JT, Brown DR, Hedlund BP, Paste RBJ, Ward NL, Ludwig W, Whitman WB. New York: Springer; 2011:106–111.
Bernardet JF, Nakagawa Y, Holmes B: Proposed minimal standards for describing new taxa of the family Flavobacteriaceae and emended description of the family. Int J Syst Evol Microbiol 2002, 52: 1049–1070. 10.1099/ijs.0.02136-0
List Editor: Validation List No. 41. Validation of publication of new names and new combinations previously effectively published outside the IJSEM. Int J Syst Bacteriol 1992, 42: 327–328.
Holmes B, Owen RJ: Proposal that Flavobacterium breve be substituted as the type species of the genus in place of Flavobacterium aquatile and emended description of the genus Flavobacterium : status of the named species of Flavobacterium. Request for an Opinion. Int J Syst Bacteriol 1979, 29: 416–426. 10.1099/00207713-29-4-416
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G: Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 2000, 25: 25–29. 10.1038/75556
Seng P, Drancourt M, Gouriet F, La Scola B, Fournier PE, Rolain JM, Raoult D: Ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Clin Infect Dis 2009, 49: 543–551. 10.1086/600885
Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O: The RAST Server: rapid annotations using subsystems technology. BMC Genomics 2008, 9: 75. 10.1186/1471-2164-9-75
Delcher AL, Bratke KA, Powers EC, Salzberg SL: Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 2007, 23: 673–679. 10.1093/bioinformatics/btm009
Overbeek R, Begley T, Butler RM, Choudhuri JV, Chuang HY, Cohoon M, de Crecy-Lagard V, Diaz N, Disz T, Edwards R, Fonstein M, Frank ED, Gerdes S, Glass EM, Goesmann A, Hanson A, Iwata-Reuyl D, Jensen R, Jamshidi N, Krause L, Kubal M, Larsen N, Linke B, McHardy AC, Meyer F, Neuweger H, Olsen G, Olson R, Osterman A, Portnoy V, et al.: The subsystems approach to genome annotation and its use in the project to annotate 1000 genomes. Nucleic Acids Res 2005, 33: 5691–5702. 10.1093/nar/gki866
Lagesen K, Hallin P, Rodland EA, Staerfeldt HH, Rognes T, Ussery DW: RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res 2007, 35: 3100–3108. 10.1093/nar/gkm160
Lowe TM, Eddy SR: tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 1997, 25: 955–964. 10.1093/nar/25.5.0955
Bland C, Ramsey TL, Sabree F, Lowe M, Brown K, Kyrpides NC, Hugenholtz P: CRISPR recognition tool (CRT): a tool for automatic detection of clustered regularly interspaced palindromic repeats. BMC Bioinformatics 2007, 8: 209. 10.1186/1471-2105-8-209
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.
The authors declare that they have no competing interests.
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.
Authors’ original submitted files for images
About this article
Cite this article
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
- Gliding motility