- Short genome report
- Open Access
Genome sequence of a high agarase-producing strain Flammeovirga sp. SJP92
Standards in Genomic Sciencesvolume 12, Article number: 13 (2017)
Flammeovirga sp. SJP92 is a Gram-negative, aerobic, rod-shaped, non-motile and non-flagellated strain that belongs to the family Flammeovirgaceae of the class Cytophagia. The strain was isolated from the intestine of abalone, which produces many extracellular agarases and exhibits efficient degradation activities on various polysaccharides, especially agarose. Here we present the high-quality draft genome of Flammeovirga sp. SJP92, together with its phenotypic characteristics. The genome sequence is 8, 534, 834 bp, which comprised with one chromosome and no plasmid. It contained 6, 291 protein-coding and 99 RNA genes, including 93 tRNA, 5 rRNA and 1 ncRNA genes.
Flammeovirga is one of genera belonging to the family Flammeovirgaceae of the class Cytophagia . There are five species have been reported in this genus, including F. aprica , F. arenaria , F. yaeyamensis , F. kamogawensis  and F. pacifica . They are all marine bacterium and have a potent ability to degrade marine complex polysaccharides, such as agar, carrageenan [3,6,7,, 5–8]. Among them, only two draft genome sequences have been published , namely Flammeovirga sp. OC4 (NZ_JTAM01000001.1)  and F. pacifica WPAGA1T (=CCTCC AB 2010364T=LMG 26175T=DSM 24597T=MCCC 1A06425T) .
Flammeovirga sp. SJP92 with high-producing agarase was isolated and identified from the intestine of abalone in Xiamen, China. It is closely related with Flammeovirga sp. NBRC 100896 (AB681288.1) and shared 99% similarities of 16S rRNA. In order to provide more genome information of Flammeovirga species and realize the function of Flammeovirga sp. SJP92 when degradingmarine complex polysaccharides, the genome of Flammeovirga sp. SJP92 was sequenced. In this study, we summarized its genomic characteristics, as well as general phenotypic properties. Other species of Flammeovirga genus were also compared with Flammeovirga sp. SJP92 in both phenotypic and genomic aspects.
Classification and features
Flammeovirga sp. SJP92 was isolated from the digestion guts of abalone with high agar-degrading ability, and deposited in China General Microbiological Culture Collection Center (CGMCC 10071). Based on the phylogenetic tree constructed with 16S rRNA, Flammeovirga sp. SJP92 is closely related with Flammeovirga sp. NBRC 100896 (AB681288.1) (Fig. 1). It is Gram-negative, curved-rods (0.75 μm wide and 11–13 μm long) after growth on 2216E plate for 3 days at 30 °C. It is aerobic and not motile without any flagella (Fig. 2). Also it is able to utilize a relatively wide spectrum of carbon substrates for growth, including agar, starch, carrageenan, L-fructose, Tween40, Tween80, galactose, lactose and so on, but it cannot utilize cellulose. Its growth temperature ranges from 15 to 40 °C with optimum between 25 and 30 °C. In addition, the optimum salinities for the growth of Flammeovirga sp. SJP92 were 2 ~ 4% (Table 1). When compared with other Flammeovirga species, this strain is different from F. pacifica WPAGA1T  and F. aprica NBRC 15941 T  in catalase, urease and esterase lipase and in the utilization of starch, D-Mannitol, L-fructose, Tween40&80 and D-xylose, differences were also observed in growth temperature range (Table 2).
Genome sequencing information
Genome project history
This organism was initially selected for sequencing on the basis of its high agar-degrading ability. Sequencing of the Flammeovirga sp. SJP92 genome was performed at the Beijing Novogene Bioinformatics Technology Co., Ltd. The Whole Genome Shotgun project has been deposited at the DDBJ/EMBL/GenBank database under the accession number LQAQ00000000. The project information and its association with MIGS version 2.0 compliance were presented in Table 3 .
Growth conditions and genomic DNA preparation
Flammeovirga sp. SJP92 was incubated aerobically in the modified 2216E medium (2.2% NaCl, 0.365% MgCl2·6H2O, 0.729% MgSO4 · 7H2O, 0.03% CaCl2 · 2H2O, 0.05% KCl, 0.042% KH2PO4, 0.005% NaBr, 0.002% SrCl · 6H2O, 0.002% Fe (NH4) Citrate, 1.326% tryptone) supplied with 0.2% agar. After incubation at 32 °C, 200 rpm for 24 h, the bacteria was collected at 13000 rpm for 30–60 min at 4 °C. The CTAB/NaCl method  was used for the extraction of chromosomal DNA of Flammeovirga sp. SJP92.
Genome sequencing and assembly
The genome of Flammeovirga sp. SJP92 was sequenced with MPS (massively parallel sequencing) Illumina technology. Three DNA libraries were constructed: a paired-end library with an insert size of 500 bp and two mate-pair libraries with an insert size of 5 kb. The 500 bp library and the 5 kb libraries were sequenced using an Illumina HiSeq2500 by PE125 strategy. Library construction and sequencing was performed at the Beijing Novogene Bioinformatics Technology Co., Ltd. Quality control of both paired-end and mate-pair reads were performed using in-house program. The final coverage reached 215-folds of the genome. SOAPdenovo [11, 12] was used for sequence assembly, and the final assembly yielded 123 contigs which generated a genome of 8.53 Mb.
The genes of Flammeovirga sp. SJP92 was identified by NCBI Prokaryotic Genome Annotation Pipeline server online . Functional predicted was performed by comparing them with sequences in RPS-BLAST against Clusters of Orthologous Groups database and pfam database [14,15,16]. SignalP was used to predict signal peptide , and transmembrane helice was analyzed by TMHMM program . CRISPRFinder was used for CRISPR identification .
The Flammeovirga sp. SJP92 genome has only one circular chromosome of a total size of about 8, 534, 834 bp with a 34.80% GC content (containing 123 contigs, 44 scaffolds).6519 genes were predicted, of which 6291 genes were protein-coding genes. 2660 genes (40.8%) were assigned to putative function and annotated as hypothetical proteins. And 99 RNAs (including 93 tRNAs, 5 rRNAs and 1 ncRNA), 127 pseudo genes were also identified. The properties and the statistics of the genome were summarized in Table 4, and Table 5 presented the distribution of genes into COGs functional categories. 3752 genes (57.55%) were assigned to COG functional categories, the most abundant COG category was “General function prediction only” (561 proteins) followed by “Signal transduction mechanisms” (401 proteins), “Transcription” (382 proteins), “Function unknown” (350 proteins), “Cell wall/membrane/envelope biogenesis” (347 proteins), “Inorganic ion transport and metabolism” (318 proteins), and “Carbohydrate transport and metabolism” (306 proteins).
Insights from the genome sequence
Until now, only two genome sequences of the strain F. pacifica WPAGA1T and Flammeovirga sp. OC4 were available within the genus Flammeovirga . Here, a whole genome comparison with these three strains have been done (Table 6). The genome of Flammeovirga sp. SJP92 is nearly 2 Mb bigger in size than F. pacifica WPAGA1T, but almost the same as Flammeovirga sp. OC4. The G + C content of Flammeovirga sp. SJP92 (34.8%) is slightly different with F. pacifica WPAGA1T (33.8%) and Flammeovirga sp. OC4 (34.9%). The gene number of Flammeovirga sp. SJP92 is different from these two strains (6, 519 & 4, 857 & 5, 898).
Annotation of the genome indicated that this strain possessed many agarase (14 agarases at least), which was coincident with its high agar-degrading ability. Many sulfatases were also predicted and sequence alignment of proteins indicated that these sulfatases were novel. It is an aerobic strain and the existence of genes encoding superoxide dismutase and catalase were consistent with this phenotype. Flammeovirga sp. SJP92 contained many genes related to the metabolism and transport of amino acids. Also, metabolic pathway analysis and Biolog GN2 experiments illustrated that this strain could utilize many amino acids. These evidences may reflect its ability to grow by using proteinaceous media as the carbon and energy source.
Flammeovirga sp. SJP92 is another strain with the genome sequence of the genus Flammeovirga together with F. pacifica WPAGA1T and Flammeovirga sp. OC4. It is an agar-degrading bacterium with efficient agarose liquefying ability and had an extracellular agarase system containing 14 agarases at least. These genomic data will provide insights into the mechanisms of how these agarases cooperation to degrade agar or other polysaccharide.
Nakagawa Y, Hamana K, Sakane T, Yamasato K. Reclassification of Cytophaga aprica (Lewin 1969) Reichenbach 1989 in Flammeovirga gen. nov. as Flammeovirga aprica comb. nov. and of Cytophaga diffluens (ex Stanier 1940; emend. Lewin 1969) Reichenbach 1989 in Persicobacter gen. nov. as Persicobacter diffluens comb. nov. Int J Syst Bacteriol. 1997;47:220–3.
Takahashi M, Suzuki K-i, Nakagawa Y. Emendation of the genus Flammeovirga and Flammeovirga aprica with the proposal of Flammeovirga arenaria nom. rev., comb. nov. and Flammeovirga yaeyamensis sp. nov. Int J Syst Evol Microbiol. 2006;56:2095–100.
Hosoya S, Yokota A. Flammeovirga kamogawensis sp. nov., isolated from coastal seawater in Japa. Int J Syst Evol Microbiol. 2007;57:1327–30.
Han W, Gu J, Yan Q, Li J, Wu Z, Gu Q, et al. A polysaccharide-degrading marine bacterium Flammeovirga sp. MY04 and its extracellular agarase system. J Ocean Univ China. 2012;11:375–82.
Liu Y, Yi Z, Cai Y, Zeng R. Draft genome sequence of algal polysaccharides degradation bacterium, Flammeovirga sp. OC4. Mar Genomics. 2015;21:21–2.
Han W, Gu J, Cheng Y, Liu H, Li Y, Li F. A Novel Alginate Lyase (Aly5) from a Polysaccharide-Degrading Marine Bacterium Flammeovirga sp. MY04: Effects of Module Truncation to the Biochemical Characteristics, Alginate-Degradation Patterns, and Oligosaccharide-Yielding Properties. Appl Environ Microbiol. 2015;82(1):364–74.
Chan Z, Wang R, Liu S, Zhao C, Yang S, Zeng R. Draft genome sequence of an agar-degrading marine bacterium Flammeovirga pacifica WPAGA1. Mar Genomics. 2015;20:23–4.
Xu H, Fu Y, Yang N, Ding Z, Lai Q, Zeng R. Flammeovirga pacifica sp. nov., isolated from deep-sea sediment. Int J Syst Evol Microbiol. 2012;62:937–41.
Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, et al. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol. 2008;26:541–7.
Wilson K. Preparation of genomic DNA from bacteria. Curr Protoc Mol Biol. 2001 Nov;Chapter 2:Unit 2.4. doi: 10.1002/0471142727.mb0204s56.
Li R, Li Y, Kristiansen K, Wang J. SOAP: short oligonucleotide alignment program. Bioinformatics. 2008;24:713–4.
Li R, Zhu H, Ruan J, Qian W, Fang X, Shi Z, et al. De novo assembly of human genomes with massively parallel short read sequencing. Genome Res. 2010;20:265–72.
Angiuoli SV, Gussman A, Klimke W, Cochrane G, Field D, Garrity GM, et al. Toward an online repository of Standard Operating Procedures (SOPs) for (meta) genomic annotation. OMICS. 2008;12:137–41.
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 1997;25:4876–82.
Tatusov RL, Fedorova ND, Jackson JD, Jacobs AR, Kiryutin B, Koonin EV, et al. The COG database: an updated version includes eukaryotes. BMC Bioinformatics. 2003;4:41.
Finn RD, Miller BL, Clements J, Bateman A. iPfam: a database of protein family and domain interactions found in the Protein Data Bank. Nucleic Acids Res. 2014;42:D364–D73.
Bendtsen JD, Nielsen H, von Heijne G, Brunak S. Improved prediction of signal peptides: SignalP 3. J Mol Biol. 2004;340:783–95.
Krogh A, Larsson B, Von Heijne G, Sonnhammer EL. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol. 2001;305:567–80.
Grissa I, Vergnaud G, Pourcel C. CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res. 2007;35:W52–W7.
Tamura K, Dudley J, Nei M, Kumar S. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol. 2007;24:1596–9.
Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci U S A. 1990;87:4576–9.
Krieg NR, Ludwig W, Euzéby J, Whitman WB. Bergey’s Manual of Systematic Bacteriology. In: Krieg NR, Staley JT, Brown DR, Hedlund BP, Paster BJ, Ward NL, Ludwig W, Whitman WB, editors. Phylum XIV: Bacteroidetes phyl. nov, vol. 4. 2nd ed. New York: Springer; 2011. p. 25.
Nakagawa Y, Class IV. Cytophagia class. nov. In: Krieg NR, Staley JT, Brown DR, Hedlund BP, Paster BJ, Ward NL, Ludwig W, Whitman WB, editors. Bergey’s Manual of Systematic Bacteriology, vol. 4. 2nd ed. The Bacteroidetes, Spirochaetes, Tenericutes (Mollicutes), Acidobacteria, Fibrobacteres, Fusobacteria, Dictyoglomi, Gemmatimonadetes, Lentisphaerae, Verrucomicrobia, Chlamydiae, and Planctomycetes. New York: Springer; 2010. p. 370.
List of new names and new combinations previously effectively, but not validly, published. Int J Syst Evol Microbiol. 2012;62:1–4.
Leadbetter ER, Order II. Cytophagales nomen novum. In: Buchanan RE, Gibbons NE, editors. Bergey’s Manual of Determinative Bacteriology. 8th ed. Baltimore: The Williams and Wilkins Co.; 1974. p. 99.
Skerman VBD, McGowan V, Sneath PHA, Moore WEC, Moore LVH. Approved Lists. Int J Syst Bacteriol. 1980; 30:225–420.
Yoon J, Adachi K, Park S, Kasai H, Yokota A. Aureibacter tunicatorum gen. nov., sp. nov., a marine bacterium isolated from a coral reef sea squirt, and description of Flammeovirgaceae fam. nov. Int J Syst Evol Microbiol. 2011;61:2342–7.
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Gene Ontology: tool for the unification of biology. Nat Genet. 2000;25:25–9.
This work was supported by the Marine Scientific Research Foundation for Public Sector Program (No. 201105027).
LR conceived and supervised the study. QD performed the laboratory work and performed all the bioinformatics analysis with the help of HS. QD and HS drafted the manuscript and Lingwei Ruan revised the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.