Genome sequence of a high agarase-producing strain Flammeovirga sp. SJP92

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 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 WPAGA1 T [8] and F. aprica NBRC 15941 T [2] in catalase, urease and esterase lipase and in the utilization of starch, D-Mannitol, Lfructose, Tween40&80 and D-xylose, differences were also observed in growth temperature range ( Table 2). Sequences were aligned using ClustalX [14] and a neighbor-joining tree obtained using the maximum-likelihood method within the MEGA version4.0 [20]. Numbers adjacent to the branches represent percentage bootstrap values based on 1000 replicates

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 [9].

Growth conditions and genomic DNA preparation
Flammeovirga sp. SJP92 was incubated aerobically in the modified 2216E medium (2.2% NaCl, 0.365% MgCl 2· 6H 2 O, 0.729% MgSO 4 · 7H 2 O, 0.03% CaCl 2 · 2H 2 O, 0.05% KCl, 0.042% KH 2 PO 4 , 0.005% NaBr, 0.002% SrCl · 6H 2 O, 0.002% Fe (NH 4) 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. , not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [28]. If the evidence code is IDA, then the property should have been directly observed for a live isolate by one of the authors, or an expert or reputable institution mentioned in the acknowledgement The CTAB/NaCl method [10] 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 pairedend library with an insert size of 500 bp and two matepair 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 215folds 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.

Genome annotation
The genes of Flammeovirga sp. SJP92 was identified by NCBI Prokaryotic Genome Annotation Pipeline server online [13]. 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 [17], and transmembrane helice was analyzed by TMHMM program [18]. CRISPRFinder was used for CRISPR identification [19].

Genome properties
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 WPAGA1 T 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 WPAGA1 T , but almost the same as Flammeovirga sp. OC4. The G + C content of   The total is based on either the size of the genome in base pairs or on the total number of protein coding genes in the annotated genome NA not available 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.

Conclusions
Flammeovirga sp. SJP92 is another strain with the genome sequence of the genus Flammeovirga together with F. pacifica WPAGA1 T 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.  Genes of agarase 13 10 5