High quality draft genomic sequence of Flavobacterium enshiense DK69(T) and comparison among Flavobacterium genomes.

Flavobacterium enshiense DK69(T) is a Gram-negative, aerobic, rod-shaped, non-motile and non-flagellated bacterium that belongs to the family Flavobacteriaceae in the phylum Bacteroidetes. The high quality draft genome of strain DK69(T) was obtained and has a 3,375,260 bp genome size with a G + C content of 37.7 mol % and 2848 protein coding genes. In addition, we sequenced five more genomes of Flavobacterium type strains and performed a comparative genomic analysis among 12 Flavobacterium genomes. The results show some specific genes within the fish pathogenic Flavobacterium strains which provide information for further analysis the pathogenicity.

Introduction

DK69T (= n>an class="Chemical">CCTCC AB2011144T = KCTC 23775) is a type strain that belongs to the genus of the family [1]. In recent years, members of were identified and widely distributed in soil, fresh water, marine water, sediment, microbial mat, and glaciers [2-5]. Some strains are fish pathogens including ATCC 49512 causing columnaris disease [6], JIP02/86T causing cold-water disease [7] and FL-15T causing bacterial gill disease [8].

The common characters of strains are Gram-negative, non-spore-forming, yellow-pigmented, rod-shaped, aerobic and with a low DNA G + C content (30–41 mol %) [2-12]. The strains contained iso-C15:0 as the major fatty acid, phosphatidylethanolamine as the major polar lipid and menaquinone-6 as the major respiratory quinone [9-12].

In order to provide genome information of species, we sequenn>an class="Chemical">ced six strains including DK69T [1], F44-8T [13], R2A-7T [14], WB 3.3-2T [15], WB 4.1-42T [15] and GH29-5T [2]. In this study, we compared 12 genomes including the six strains that we sequenced and other six available genomes in the NCBI, GPTSA100-9T [16], PS1T [17], Flavobacterium sp. F52 [18], ATCC 49512, JIP02/86T and FL-15T. Here, we present the description of the non-contiguous finished genomic sequencing of DK69T and the comparative genome analysis of the 12 genomes.

Organism information

Classification and features

DK69T is a Gram-negative, strictly aerobic, yellow-pigmented rod shaped bacterium isolated from soil collected at a pharmaceutical company in Enshi, Hubei province, China. The total soil C, N, P, S and Fe concentrations were 39.83, 3.34, 0.68, 0.36, 33.80 g kg−1, respectively, and the pH was 6.97 [1]. A neighbor-joining phylogenetic tree based on the 16S rRNA gene sequences was built using MEGA 6 [19] and showed that strain DK69T was clustered within a branch containing other species in the genus (Fig. 1). In addition, the sequence of DK69T was compared with other sequenced strains of the family use BioLinux [20], and a total of 24 core protein sequences were obtained with 50 % identity and E-value exponent of e−10. A phylogenetic tree based on the 24 core protein sequences of the core genome (Fig. 2) is similar to the 16S rRNA gene based tree.

Fig. 1

A NJ phylogenetic tree of the strains within family Flavobacteriaceae based on 16S rRNA gene sequence comparisons. GenBank accession numbers are shown in parentheses. The sequences were aligned using CLUSTALX, and the phylogenetic tree was obtained using MEGA 6 [19] software of neighbor-joining method [39], with the bootstrap values of 500 replicates. *represents the strains sequenced by us

Fig. 2

A NJ phylogenetic tree of the strains within family Flavobacteriaceae based on core-protein sequence comparisons. GenBank accession numbers are shown in parentheses. *represents the strains sequenced by us

The colonies of DK69T are smooth with regular edges, circular, yellowish and about 1 mm in diameter after grown on R2A agar at 28 °C for 48 h. Growth occurs at 4–32 °C, pH 6.0–8.0 on R2A and TSA, but not on NA or LB media, and NaCl is not required [1]. Cells are non-flagellated, non-spore-forming, non-motile, rod-shaped (Fig. 3). Oxidase- and catalase- positive. The DNA G + C content is 34.4 mol% [1]. The general description of this strain is shown in Table 1.

Fig. 3

A transmission electron micrograph of F. enshiense DK69T cells

Table 1

Classification and general features of F. enshiense DK69T according to the MIGS recommendations [21]

MIGS ID	Property	Term	Evidence code
MIGS-6	Classification	Domain Bacteria	TAS [22]
		Phylum Bacteroidetes	TAS [23]
		Class Flavobacteriia	TAS [24]
		Order Flavobacteriales	TAS [24]
		Family Flavobacteriaceae	TAS [25]
		Genus Flavobacterium	TAS [5, 26]
		Species Flavobacterium enshiense	TAS [1]
		Type strain: DK69 T (=CCTCC AB 2011144 T = KCTC 23775 T)	TAS [1]
	Gram stain	negative	TAS [1]
	Cell shape	Rod	TAS [1]
	Motility	non-motile	TAS [1]
	Sporulation	non-sporulating	TAS [1]
	Temperature range	4-32 °C	TAS [1]
	Optimum temperature	28 °C	TAS [1]
	pH range; Optimum	6.0-8.0; 7.0	TAS [1]
	Carbon source	casein, gelatin, egg yolk, tyrosine, sucrose, D-mannitol	TAS [1]
	Habitat	soil	TAS [1]
MIGS-6.3	Salinity	0 % NaCl (w/v)	TAS [1]
MIGS-22	Oxygen requirement	aerobic	TAS [1]
MIGS-15	Biotic relationship	free-living	NAS
MIGS-14	Pathogenicity	non-pathogen	NAS
MIGS-4	Geographic location	Enshi city, Hubei Province, China	TAS [1]
MIGS-5	Sample collection	2010	TAS [1]
MIGS-4.1	Latitude	not reported
MIGS-4.2	Longitude	not reported
MIGS-4.4	Altitude	not reported

Evidence codes–IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., 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 [27]

A transmission elepan class="Chemical">ctron mipan class="Chemical">crograph of F. enshiense DK69T cells

Chemotaxonomic data

The major cellular n>an class="Chemical">fatty acids of DK69T were iso-C15:0, iso-C17:1ω9c, C15:0, iso-C17:0 3-OH and iso-C15:0 3-OH. The major polar lipids were phosphatidylethanolamine, one unidentified aminolipid and one unidentified lipid. DK69T contained menaquinone 6 as the major quinone [1].

Genome sequencing information

Genome project history

Genome of DK69T was sequenn>an class="Chemical">ced by Majorbio Bio-pharm Technology Co., Ltd, Shanghai, China. The high-quality draft genome sequence was deposited in the National Center for Biotechnology Information. Contigs less than 200 bp were not included. The GenBank accession number is JRLZ00000000. The summary of the genome sequencing project information is shown in Table 2.

Table 2

Project information of F. enshiense DK69T

MIGS ID	Property	Term
MIGS 31	Finishing quality	High-quality draft
MIGS-28	Libraries used	Illumina Paired-End library (300 bp insert size)
MIGS 29	Sequencing platforms	Illumina Hiseq2000
MIGS 31.2	Fold coverage	487.4 x
MIGS 30	Assemblers	SOAPdenovo v1.05
MIGS 32	Gene calling method	GeneMarkS+
	Locus Tag	Q767
	Genbank ID	JRLZ00000000
	Genbank Date of Release	October 28, 2014
	BIOPROJECT	PRJNA221771
	Project relevance	Genome comparison
MIGS 13	Source Material Identifier	DK69T

pan class="Chemical">Projepan class="Chemical">ct information of F. enshiense DK69T

Growth conditions and genomic DNA preparation

DK69T was grown on R2A medium at 28 °n>an class="Chemical">C for 2 d with 160 rpm shaking. Cells in late-log-phase growth were harvested and lysed by EDTA, lysozyme, and detergent treatment, followed by proteinase K and RNase digestion. The DNA was extracted and purified using the QiAamp kit according to the manufacturer’s instruction (Qiagen, Germany). The quantity of DNA was measured by the NanoDrop Spectrophotometer to ensure that the DNA concentration is greater than 20 ng/μl, then 5 μg of DNA was sent to Majorbio (Shanghai, China) for sequencing.

Genome sequencing and assembly

The Illumina Hiseq2000 with the Paired-End library strategy was used to determine the whole-genome sequenn>an class="Chemical">ce of DK69T. TruSeq DNA Sample Preparation Kits are used to prepare DNA libraries with insert sizes of 300–500 bp for single, paired-end, and multiplexed sequencing. The protocol used 1 μg of DNA sheared by either sonication or nebulization [28]. The genome raw data of DK69T generated 8,329,997 x 2 reads totaling 1,682,659,394 bp data with an average coverage of 498.4 x. Then SOAPdenovo v1.05 [29] was used to perform the following steps to assemble the sequencing data: (1) removing the adapter sequences in the reads; (2) cutting the 5’ end bases without clear A, T, C and G; (3) trimming the quality read scores lower than 20; (4) removing the reads containing more than 10 % Ns; (5) removing the reads which the length were less than 25 bp. A total of 8,217,761 x 2 high quality reads totaling 1,645,393,073 bp data with an average coverage 487.4 × was generated. The assembled sequence contained 67 scaffolds with a genome size of 3.38 Mbp.

Genome annotation

The annotation of the genomic sequenn>an class="Chemical">ces was completed using the NCBI Prokaryotic Genome Annotation Pipeline which was combined using Best-placed reference protein set and the gene caller GeneMarkS+. SignalP [30] and SOSUI [31] were used to predict signal peptides and transmembrane helices. The predicted CDSs were also used to search against the Pfam protein family database [32]. The GenBank database [33] and the COG databases [34] BLASTP search were used to predict protein sequences.

Genome properties

The genome statistics are provided in Table 3 and Fig. 4. After genome annotation, the genome of n>an class="Species">DK69T was found to have a total length of 3,375,260 bp, a G + C content of 1,273,385 bp (37.7 mol %) and 74 contigs. From a total of 3,054 genes predicted, 2,848 genes are protein-coding genes, 50 are RNA genes, 57.9 % are assigned with putative functions and the remaining are annotated as hypothetical proteins or proteins of unknown functions. The distribution of genes into COGs functional categories is shown in Table 4.

Table 3

Genome statistics of F. enshiense DK69T

Attribute	Value	% of Totala
Genome size (bp)	3,375,260	100.00
DNA coding (bp)	2,808,588	83.21
DNA G + C (bp)	1,273,385	37.73
DNA scaffolds	67	-
Total genes	3054	100.00
Protein coding genes	2848	93.25
RNA genes	50	1.64
Pseudo genes	156	44.67
Genes in internal clusters	1113	3908
Genes with function prediction	1649	57.90
Genes assigned to COGs	1718	60.32
Genes with Pfam domains	2495	87.61
Genes with signal peptides	735	25.81
Genes with transmembrane helices	651	22.86
CRISPR repeats	0	-

aThe total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome

Fig. 4

A graphical circular map of F. enshiense DK69T. From outside to inside, 1, 4 circles show forward strand or reverse strand protein-coding genes according to COG categories; 2, 3 circles show forward strand or reverse strand genes; ring 5 shows G + C% content, ring 6 shows GC skew

Table 4

Number of genes in F. enshiense DK69T associated with general COG functional categories

Code	Value	% agea	Description
J	142	4.99	Translation, ribosomal structure and biogenesis
A	0	0.00	RNA processing and modification
K	76	2.67	Transcription
L	93	3.27	Replication, recombination and repair
B	1	0.04	Chromatin structure and dynamics
D	20	0.70	Cell cycle control, Cell division, chromosome partitioning
V	56	1.97	Defense mechanisms
T	67	2.35	Signal transduction mechanisms
M	176	6.18	Cell wall/membrane biogenesis
N	4	0.14	Cell motility
U	29	1.02	Intracellular trafficking and secretion
O	75	2.63	Posttranslational modification, protein turnover, chaperones
C	100	3.51	Energy production and conversion
G	54	1.90	Carbohydrate transport and metabolism
E	158	5.55	Amino acid transport and metabolism
F	60	2.11	Nucleotide transport and metabolism
H	108	3.79	Coenzyme transport and metabolism
I	69	2.42	Lipid transport and metabolism
P	81	2.84	Inorganic ion transport and metabolism
Q	39	1.37	Secondary metabolites biosynthesis, transport and catabolism
R	192	6.74	General function prediction only
S	118	4.14	Function unknown
-	1130	39.68	Not in COGs

aThe total is based on the total number of protein coding genes in the annotated genome

Genome statistipan class="Chemical">cs of n>an class="Species">F. enshiense DK69T

aThe total is based on either the size of the genome in base pairs or the total number of protein pan class="Chemical">coding genes in the annotated genome

Number of genes in pan class="Species">F. enshiense DK69T assopan class="Chemical">ciated with general COG functional categories

aThe total is based on the total number of protein pan class="Chemical">coding genes in the annotated genome

Insights from the genome sequences

Profiles of metabolic network and pathway

The metabolic network and pathways of n>an class="Species">DK69T (Fig. 5) were predicted using the Kyoto Encyclopedia of Genes and Genomes [35]. The metabolic network showed that DK69T possesses glycolysis, TCA cycle and pentose phosphate pathways and could utilize casein, tyrosine, sucrose and D-mannitol. The genome analysis results are in agreement with the phenotypes [1].

Fig. 5

Metabolic network and pathways of Flavobacterium enshiense DK69T as predicted using KEGG [35]. Green lines indicate pathways that are possessed by this strain

Comparison of the 12 genomes

The genomic information of the 12 genomes are summarized in Table 5. OrthoMCL [36] analysis was performed to identify the set of orthologs among the 12 genomes. DK69T shared 1,190 genes with the other 11 strains, and had 437 strain-specific genes which may contribute to the species-specific features (Fig. 6).

Table 5

General features of the twelve Flavobacterium genomes

Strains	Size (Mp)	G + C %	Total genes	CDSs	Contigs	References
F. enshiense DK69T	3.4	37.7 %	3,054	2,848	74	This study
F. beibuense F44-8T	3.8	37.7 %	3,460	3,264	61	This study
F. cauense R2A-7T	3.1	38.2 %	2,910	2,723	61	This study
F. rivuli WB 3.3-2T	4.5	39.6 %	3,975	3,691	63	This study
F. subsaxonicum WB 4.1-42T	4.6	41.6 %	4,052	3,785	80	This study
F. suncheonense GH29-5T	2.9	40.5 %	2,769	2,594	105	This study
F. frigoris PS1T	3.9	34.4 %	3,640	3,590	52	[17]
Flavobacterium sp. F52	5.3	34.4 %	4,601	4,549	54	[18]
F. indicum GPTSA100-9T	3.0	31.4 %	2,787	2,671	1	[16]
F. columnare ATCC 49512T	3.2	31.5 %	2,731	2,642	1	[6]
F. psychrophilum JIP02/86T	2.9	32.5 %	2,556	2,446	1	[7]
F. branchiophilum FL-15T	3.6	32.9 %	3,087	2,872	1	[8]

Fig. 6

A venn diagram indicates the twelve genomes of Flavobacterium analyzed by OrthoMCL [36] illustrate the number of the unique proteins and the common proteins among them

General pan class="Chemical">features of the twelve n>an class="Species">Flavobacterium genomes

A venn diagram indipan class="Chemical">cates the twelve genomes of pan class="Species">Flavobacterium analyzed by OrthoMCL [36] illustrate the number of the unique proteins and the common proteins among them

Three of the 12 strains are fish pathogenic bacteria [6-8]. Using OrthoMCL [36] analysis, a total of ten proteins we found to be unique in the three fish-pathogenic species. Three of the putative proteins were reported to be related to the pathogenicity of pathogenic bacteria including polysaccharide deacetylase [37], ABC transporter ATPase and ABC transporter permease [38] (Table 6).

Table 6

Specific proteins of three pathogenic bacteria, F. branchiophilum FL-15T, F. columnare ATCC 49512T and F. psychrophilum JIP02/86T

Strains	Accession	Putative protein
F. branchiophilum FL-15 T	WP_014083310.1	SNF2_N, HepA, PLN03142
F. columnare ATCC 49512 T	WP_014165166.1
F. psychrophilum JIP02/86 T	WP_011962958.1
F. branchiophilum FL-15 T	WP_014083635.1	hypothetical protein
F. columnare ATCC 49512 T	WP_014164281.1
F. psychrophilum JIP02/86 T	WP_011962863.1
F. branchiophilum FL-15 T	WP_014082960.1	Hexameric tyrosine-coordinated heme protein
F. columnare ATCC 49512 T	WP_014165359.1
F. psychrophilum JIP02/86 T	WP_011963152.1
F. branchiophilum FL-15 T	WP_014084059.1	polysaccharide deacetylase
F. columnare ATCC 49512 T	WP_014165336.1
F. psychrophilum JIP02/86 T	WP_011963745.1
F. branchiophilum FL-15 T	WP_014084057.1	membrane protein
F. columnare ATCC 49512 T	WP_014165338.1
F. psychrophilum JIP02/86 T	WP_011963747.1
F. branchiophilum FL-15 T	WP_014084692.1	PepSY-associated TM helix
F. columnare ATCC 49512 T	WP_014166184.1
F. psychrophilum JIP02/86 T	WP_011963892.1
F. branchiophilum FL-15 T	WP_014082991.1	S-adenosylmethionine protein
F. columnare ATCC 49512 T	WP_014164416.1
F. psychrophilum JIP02/86 T	WP_011963983.1
F. branchiophilum FL-15 T	WP_014082768.1	ABC transporter permease
F. columnare ATCC 49512 T	WP_014165791.1
F. psychrophilum JIP02/86 T	WP_011964188.1
F. branchiophilum FL-15 T	WP_014082767.1	ABC transporter ATPase
F. columnare ATCC 49512 T	WP_014165790.1
F. psychrophilum JIP02/86 T	WP_011964189.1
F. branchiophilum FL-15 T	WP_014083276.1	Transposase
F. columnare ATCC 49512 T	WP_014165862.1
F. psychrophilum JIP02/86 T	WP_011964284.1

Specifin>an class="Chemical">c proteins of three pathogenic bacteria, F. branchiophilum FL-15T, F. columnare ATCC 49512T and F. psychrophilum JIP02/86T

Conclusions

The genomic results of n>an class="Species">DK69T and related strains reveled useful information. (1) The genome based phylogenetic analysis results is in agreement with the 16S rRNA gene based one; (2) The genomic data are correlated with some phenotypes of strain DK69T; (3) Compared to the three fish pathogenic strains, no pathogenic related genes was detected in the environmental strain DK69T which indicated its non-pathogenicity; and (4) Some specific genes were found within the three fish pathogenic strains which provides information for further analysis the pathogenicity.