Genome sequence of the haloarchaeon Haloterrigena jeotgali type strain A29T isolated from salt-fermented food

Haloterrigena jeotgali is a halophilic archaeon within the family Natrialbaceae that was isolated from shrimp jeotgal, a traditional Korean salt-fermented food. A29T is the type strain of H. jeotgali, and is a Gram-negative staining, non-motile, rod-shaped archaeon that grows in 10 %–30 % (w/v) NaCl. We present the annotated H. jeotgali A29T genome sequence along with a summary of its features. The 4,131,621 bp genome with a GC content of 64.9 % comprises 4,215 protein-coding genes and 127 RNA genes. The sequence can provide useful information on genetic mechanisms that enable haloarchaea to endure a hypersaline environment.


Introduction
An extremely halophilic archaeon, called a haloarchaeon, that is a member of the family Natrialbaceae [1] was isolated from various hypersaline environments such as soda and salt lakes, solar salterns, salt mines, salted soils, deepsea brine, and various salt-fermented foods. Although high salinity is toxic to most cells, extreme halophiles are adapted to their hypersaline environments [2]. Most halophilic archaea require at least 1.5 M NaCl for growth and optimum growth occurs in the range of 3.1 to 3.4 M NaCl [3]. Since halophilic enzymes from the haloarchaea are generally considered to be active and stable at high salt concentrations, they have potential for biotechnological applications such as engineering for salt-resistant plants in agriculture, environmental bioremediation of organic pollutants and production of fermented foods. The genus Haloterrigena was first proposed by Ventosa et al. [4] with the reclassification of Halococcus turkmenicus as Haloterrigena turkmenica [4], and presently includes nine species: H. turkmenica [4], H. thermotolerans [5], H. longa, H. limicola [6], H. saccharevitans [7], H. hispanica [8], H. jeotgali [9], H. salina [10], and H. daqingensis [11], all of which are pleomorphic, Gramnegative staining, and red-or light pink-pigmented. However, the genus Haloterrigena is poorly characterized at the genome level.
A29 T (= KCTC 4020 T = DSM 18794 T = JCM 14585 T = CECT 7218 T ) is the type strain of H. jeotgali and was isolated from shrimp jeotgal, a traditional Korean saltfermented food [9]. Although little is known about the roles of the haloarchaea during the fermentation process, the increasing genome information is expected to contribute to expansion of the understanding of their roles and halotolerant features. Here, we present a summary of the classification and features of H. jeotgali A29 T along with the annotated genome sequence.

Classification and features
A taxonomic analysis was conducted by comparing the H. jeotgali A29 T 16S rRNA gene sequence with the most recent release of the EzTaxon-e database [12]. Phylogenetic relationships between strain A29 T and closely related species were evaluated using MEGA6 program [13], and dendrograms were generated by the neighborjoining [14], minimum evolution [15], and maximum likelihood [16] 22.0 %, and 17.9 %, respectively. The 16S rRNA gene sequence similarity data and DNA-DNA relatedness value of less than 70 % [17] suggested that strain A29 T represents a distinct genospecies [9] ( Table 1).
The consensus phylogenetic tree based on the 16S rRNA gene sequences indicated that strain A29 T was clustered in a branch with other species of the genus Haloterrigena (Fig. 1).
H. jeotgali A29 T is Gram-negative staining, nonmotile, rod-shaped (0.4 μm wide and 1.0 μm long) (Fig. 2), and grows in irregular clusters. Colonies cultured on complex agar medium were light red, circular, and measured 0.5 mm in diameter after 7 days at 37°C. Growth occurred in the presence of 10-30 % (w/v) NaCl at temperatures ranging from 17-50°C and in the pH range of 6.5-8.5. Optimal conditions for growth were; a NaCl concentration of 15-20 % (w/v), a temperature ranging from 37-45°C, and a pH of 7.0-7.5. The isolate was catalase-positive and oxidase-negative and did not reduce nitrate to nitrite. Mg 2+ was not required for growth. Cell lysis occurred in distilled water. This strain was able to hydrolyze casein and Tween 80 but not Evidence codes -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 [29] starch, gelatin, urea, or DNA. Anaerobic growth occurred in the presence of nitrate but not of sulfate, thiosulfate, dimethyl sulfoxide, or trimethylamine N-oxide. Fructose, lactose, and acetate-but not sucrose, glucose, citrate, or formate-were utilized as carbon and energy  sources. Acid was not produced from fructose, lactose, acetate, sucrose, glucose, citrate, or formate. Strain A29 T was resistant to bacitracin, penicillin, ampicillin, chloramphenicol, and erythromycin, but was sensitive to novobiocin, anisomycin, and aphidicolin. The major polar lipids were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, and mannose-2,6-disulfate(1-2)glucose glycerol diether [9].

Genome sequencing and annotation
Genome project history H. jeotgali strain A29 T genome was sequenced to obtain information regarding mechanism(s) or molecule(s) that confer adaption to a hypersaline environment and to identify the primary structure of potentially novel halophilic enzymes with relatively low similarity to those in the sequence database. The genome project and sequence were Fig. 3 Graphical circular map of the H. jeotgali A29 T genome. RNA genes (red, tRNA and blue, rRNA) and genes on the reverse and forward strands (colored according to COG categories) are shown from the outside to the center. The inner circle shows the GC skew; yellow and blue indicate positive and negative values, respectively. GC content is indicated in red and green deposited in the Genomes OnLine Database [18] and GenBank (JDTG00000000), respectively. Sequencing and annotation were performed by ChunLab Inc. (Seoul, Korea). Project information and associated MIGS version 2.0 compliance levels [19] are shown in Table 2.
Growth conditions and genomic DNA preparation H. jeotgali A29 T was grown aerobically in DSM Medium 954 at 37°C. Genomic DNA was extracted and purified using a G-spin™ DNA extraction kit (iNtRON Biotechnology, Sungnam, Korea) according to the manufacturer's instructions.

Genome sequencing and assembly
The genome of H. jeotgali A29 T was sequenced from a total of 9,473,809 quality-trimmed sequencing reads (700.

Genome annotation
Open reading frames of the assembled genome were predicted using the Integrated Microbial Genomes-Expert Review platform as part of the Joint Genome Institute genome annotation pipeline [20]. Additional gene prediction and functional annotation were achieved using the Rapid Annotation using Subsystem Technology pipeline. Predicted ORFs were compared during gene annotation using NCBI Clusters of Orthologous Groups [21], Pfam [22], and EzTaxon-e [12] databases. rRNA and tRNA genes were identified using RNAmmer 1.2 [23] and tRNAscan-SE 1.23 [24] tools, respectively. Genomic features were visualized with CLgenomics 1.06 (ChunLab Inc.).  The total is based on the total number of protein coding genes in the genome

Genome properties
The draft genome sequence of H. jeotgali A29 T was 4,131,621 bp and comprised three scaffolds including 20 contigs, and had a GC content of 64.9 % (Fig. 3 and Table 3). Of the 4,342 predicted genes, 4,215 were protein-coding and 2,636 ORFs (60.7 %) were assigned putative functions, whereas the remaining genes were annotated as hypothetical proteins. The genome contained 127 ORFs assigned to RNA genes, including 47 predicted for tRNA, 14 for rRNA (five 5S, two 16S, and seven 23S), and 66 for miscellaneous RNA (one archaeal signal recognition particle; five for the HgcC family; one archaeal RNA P; and 59 clustered regularly interspaced short palindromic direct repeat elements). The distribution of genes across COG functional categories is presented in Table 4.

Conclusions
H. jeotgali A29 T encoded the genes associated with the mechanisms of salinity tolerance, biosynthesis and transport of compatible solutes such as glycine betaine (N,N,Ntrimethylglycine) (choline sulfatase, choline dehydrogenase, betaine reductase, and glycine betaine transporter OpuD), ion exclusion using cation (Mg 2+ and Cu 2+ ) transport and K + transport and Na + /H + antiporter systems. The sequences may contribute to expansion of our knowledge of complex osmoregulation mechanism of the haloarchaea that should facilitate biotechnological applications of the haloarchaea and provide useful information on genetic mechanisms that enable haloarchaea to endure hypersaline environments.
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