Complete genome sequence of the halophile bacterium Kushneria konosiri X49T, isolated from salt-fermented Konosirus punctatus

Kushneria konosiri X49T is a member of the Halomonadaceae family within the order Oceanospirillales and can be isolated from salt-fermented larval gizzard shad. The genome of K. konosiri X49T reported here provides a genetic basis for its halophilic character. Diverse genes were involved in salt-in and -out strategies enabling adaptation of X49T to hypersaline environments. Due to resistance to high salt concentrations, genome research of K. konosiri X49T will contribute to the improvement of environmental and biotechnological usage by enhancing understanding of the osmotic equilibrium in the cytoplasm. Its genome consists of 3,584,631 bp, with an average G + C content of 59.1%, and 3261 coding sequences, 12 rRNAs, 66 tRNAs, and 8 miscRNAs.


Introduction
The gizzard shad, Konosirus punctatus, is a popular marine fish used as a food source in Northeast Asia and is usually consumed as a grilled dish, sushi, or jeotgal (or jeot). Jeotgal is a traditional Korean fermented food made by adding a substantial amount of solar salt to seafood such as fish, shrimp, or shellfish. During fermentation, jeotgal gains an extra flavor that may be caused by a microorganism derived from the environment, solar salt, or sea organisms. In an analysis of the microbiota of salt-fermented seafood, the strain X49 T (= KACC 14623 T = JCM 16805 T ) was isolated from the salt-fermented larval gizzard shad, known as Daemi-jeot in Korean [1]. Blast analysis and phylogenetic analysis using the 16S rRNA sequence revealed that the strain X49 T belongs to the genus Kushneria.
The genus Kushneria was first proposed as a novel genus by Sanchez-Porro in 2009, on the basis of phylogenetic analyses of 16S and 23S rRNA gene sequences [2]. It comprises a group of related Gram-negative, aerobic, motile, and rod-or oval-shaped bacteria. Most Kushneria strains have been isolated from saline environments and possess hypersaline resistance [2][3][4][5][6][7]. Strain X49 T also has a halophilic character [1]. At present, there are four sequenced Kushneria strains, but only the genome of Kushneria marisflavi KCCM 80003 T has been reported [8]. Thus, the genomic analysis of strain X49 T should help us to understand the genetic basis of adaptation to a hypersaline environment. The present study determined the classification and features of strain X49 T , as well as its genome sequence and gene annotations.

Classification and features
Serially diluted suspensions of Daemi-jeot were plated directly on Marine agar medium and maintained under aerobic condition at 25 ± 1°C for 14 days. To obtain pure isolates, a single colony was repeatedly transferred to new agar plates. Comparison between the 16S rRNA gene sequence of strain X49 T (Accession number: GU198748) and those obtained using NCBI BLASTN [9] with the settings for highly similar sequences produced 100 hits: 47, 36, 1, and 1 from the genera Kushneria, Halomonas, Halomonadaceae, and Chromohalobacter, respectively, and the remaining 15 from uncultured bacteria. The validated species with the maximum sequence similarity was K. marisflavi SW32 T (NR_025094), which shared a sequence identity of 98.63%. Phylogenetic analysis using MEGA6 [10] based on 16S rRNA gene sequences of the Kushneria members and related taxa showed that strain X49 T was within the cluster comprising the genus Kushneria (Fig. 1). Strain X49 T is classified as Proteobacteria, Oceanospirillales, Halomonadaceae, and Kushneria, and is named Kushneria konosiri. Characteristics of K. konosiri X49 T are presented in Table 1. The cells were aerobic, Gram-negative, rod-or oval-shaped, and 1.2-3.2 μm in length and 0.5-1.0 μm in width. A flagellum was observed (Fig. 2). The colonies were orange-colored and circular with entire margins on marine agar medium. Growth was observed at 10-37°C, at pH 4.5-8.5, and in the presence of 0-26% (w/v) NaCl. The physiological characteristics, such as the growth substrates of K. konosiri X49 T , have been described in detail previously [1].

Genome sequencing information
Genome project history K. konosiri X49 T was selected for genome sequencing based on its environmental potential and this genome sequencing was part of the Agricultural Microbiome R&D Program (grant number: 914006-4) at the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry (IPET) funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA). The genome sequence was deposited in DDBJ/EMBL/ GenBank under accession number CP021323, and the genome project was deposited in the GOLD [11] under Gp0223024. The sequencing and annotation were performed by Macrogen (Seoul, Korea). The details of the project information and the associations with MIGS [12] are shown in Table 2.
Growth conditions and genomic DNA preparation K. konosiri X49 T (lab stored, = KACC 14623 T = JCM 16805 T ) was cultured aerobically in LB broth (BD, USA) containing NaCl (4% w/v) at 30 ± 1°C for 3 days. The genomic DNA of K. konosiri X49 T was extracted using a MG™ Genomic DNA Purification kit (Macrogen, Korea) according to the manufacturer's instructions.

Genome sequencing and assembly
For library preparation, gDNA was sheared with g-TUBE (Covaris Inc., USA) and then used for library preparation Fig. 1 Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences of K. konosiri X49 T and closely related taxa. Numbers at nodes indicate bootstrap values (over 70%, 1000 replicates) for neighbor-joining, maximum-likelihood, and maximum-parsimony. Closed circles indicate the nodes that were also generated by maximum-likelihood and maximum-parsimony. Scale bar, 0.005 accumulated changes per nucleotide by ligating SMRTbell adaptors (20 kb SMRTbell library).
The sequences of the generated library were sequenced using the PacBio RSII system, SMRT sequencing with DNA Sequencing Reagent Kit P6 and SMRT Cells 8Pac V3 (Pacific Biosciences). The sequencing generated 1,199,776,790 bp with 160,304 reads. After filtering of sequences that were shorter than 50 bp, 1,199,771,927 bp sequences with 160,189 subreads remained. Assembly was performed using software RS HGAP v3, which consists of pre-assembly, de novo assembly with Celera® Assembler, and assembly polishing with Quiver. Assembly resulted in one scaffold with the complete genome in circular form and 244-fold coverage.

Genome annotation
Annotation of the assembled genome was performed using the DOE-JGI Microbial Genome Annotation Pipeline v.4.15.1 [13]. The gene prediction was carried out using the IMG-ER platform. Comparisons of the predicted ORFs using the KEGG [14], NCBI COG [15], Pfam [16], TIGRfam [17], and InterPro [18] databases were conducted during gene annotation. Additional gene prediction analyses and functional assignment were carried out using the NCBI PGAP [19] and the RAST with the gene caller classicRAST [20] based on the SEED [21]. CRISPR system analysis was carried out using the web-based interface CRISPRFinder (http://crispr.i2bc.paris-saclay.fr/). The chromosome map of K. The evidence codes are as follows. TAS: traceable author statement (i.e., a direct report exists in the literature). NAS: non-traceable author statement (i.e., not observed directly in a living, isolated sample, but based on a generally accepted property of the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [13] konosiri X49 T was obtained from the output of the IMG pipeline (Fig. 3).

Genome properties
The genome of K. konosiri X49 T comprised a single circular chromosome with a length of 3,584,631 bp and a G + C content of 59.1% ( Fig. 3 and Table 3). Of the 3347 predicted genes, 3261 were protein-coding. According to tRNAscan-SE and RNAmmer 1.2, 66 tRNA, 12 rRNA (four 5S rRNA, four 16S rRNA, and four 23S rRNA genes), and 8 miscRNA genes were found in the genome. The genome contained one CRISPR structure with 25 spacers of 28 bp and two putative CRISPRs. The number of CDSs including signal peptides was 239 (7.14%). The majority of the protein-coding genes (2815 genes; 84.1%) were assigned to functional categories, while the remainder were annotated as hypothetical proteins (446 genes). The properties and statistics of the genome are summarized in Table 3, and the distributions of genes among the functional categories of COG are shown in Table 4.

Comparative genomics
To determine the genomic relatedness between K. konosiri X49 T and closest relative strain K. marisflavi SW32 T , the ANI value was calculated using an online calculator (https://www.ezbiocloud.net/tools/ani). The ANI value between two whole genome sequences was 89.32%. This value was well below the threshold of 95%; this suggested that two strains represent genotypically distinct species.
To balance the osmotic pressure between the inside and outside of the cell in the hypersaline habitat, theses halophile microorganisms increase the internal osmolarity of the cytoplasm using inorganic ions (mostly potassium ions) or organic compounds (mainly ectoine, choline, glycine betaine, and proline betaine, etc.), which are known as compatible solutes for the exclusion of salt ions. Genome analysis through the IMG pipeline revealed that K. konosiri X49 T can adapt its salt tolerance using several functional genes (Fig. 4). In response to changes in the external osmolality, potassium is accumulated in the cytoplasm by transport via the Trk-type transport systems, KUP system, or Kdp two-component system [22][23][24]. With increasing potassium levels, osmoprotective compounds are accumulated by synthesis and/or uptake in the cytoplasm as a bi-phasic response [22]. Also, expression of membrane proteins such as OMPs and of MDOs is affected by environmental osmolarity [25,26].
To regulate the osmolarity between cytoplasm and environment, the genome of K. konosiri X49 T encodes potassium uptake-related loci: the Trk system (low-affinity potassium transport; two TrkA and two TrkH genes), KUP system (two potassium uptake permeases), and Kdp two-component system (one sensor histidine kinase KdpD and one operon response regulator KdpE).

Carotenoid biosynthesis
Carotenoids are naturally occurring pigments that not only act as antioxidants but also enhance salt stress tolerance [27]. The first step of carotenoid biosynthesis starts by formation of one phytoene from two molecules of GGPP, and phytoene is converted to αor β-carotene along the biosynthesis pathway. αor β-carotene is transformed into xanthophyll by obtaining an oxygen atom (Fig. 5). The colonies of K. konosiri X49 T were orange. The genome of K. konosiri X49 T encodes genes Fig. 3 Circular map of the complete K. konosiri X49 T genome. Marked characteristics are shown from the outside to the center: the number of bases, COG on forward strand, COG on reverse strand, RNA genes (tRNAs, green bars; rRNAs, red bars; other RNAs, black bars), GC content, and GC skew. Individual genes are colored according to COG categories related to carotenoid biosynthesis: two GGPP synthases, one phytoene synthase, two phytoene desaturases, one lycopene β-cyclase, one β-carotene hydroxylase, and one enhancing lycopene biosynthesis protein 2. However, there is no xanthophyll cycle-related gene, ZEP, which is key for conversion of zeaxanthin to violaxanthin, suggesting that the orange color of the colony of K. konosiri is derived from β-carotene or β-cryptoxanthin. These carotenoids may act also as the potential osmoprotectants (Fig. 5).

Conclusions
The orange pigmented K. konosiri X49 T was isolated from a salt-fermented food, Daemi-jeot, and was resistant to a hypersaline environment. Whole genome sequence analysis and physiological observations leads us to conclude that K. konosiri X49 T is a orange-coloured halophile and its capabilities of a cellular response are enabled by a variety of genes determining the 'carotenoid biosynthesis' and 'inorganic or organic transport and metabolism'. The presence of ectoine and betaine biosynthesis genes or transport system related genes  The total is based on the total number of protein coding genes (3261) in the genome demonstrates the possibility of a cellular response to high osmolarity through biosynthesis of ectoine and betaine to protect the cell from stress. As ectoine or betaine can play as a protectant under stress condition, the genome sequence of K. konosiri X49 T may provide the molecular basis for its hypersaline tolerance and may lead to new development in its diverse biotechnological applications comprising environmental, medical and biofuel industries. Authors' contributions JHY performed the experiments, analyzed the data, prepared and displayed elements, and wrote the main manuscript text. HJS and HSK performed the experiments. EJT, WRK, JYL, DWH, and PSK prepared elements. JWB is the guarantor of this work, has full access to all the data in the study, and takes responsibility for the integrity of the data and the accuracy of the data analyses. All authors read and approved the final manuscript.