Generally, halophilic microorganisms (i.e., halophiles) require a salt concentration of at least 0.5% (w/v, NaCl) in order to grow. In addition, some halophiles (e.g., moderate halophiles) can grow at higher NaCl concentrations (> 5% w/v, NaCl). These extremely or moderately halophiles have specific adapted strategies including stable active site or promotion of subunit interactions for their enzymes (DasSarma and DasSarma, 2015), indicating that these halophiles are excellent sources of enzymes due to their ability to perform stable functions under extreme conditions and their applicability to diverse biotechnologies such as food processing and industrial bioconversion (Patel and Saraf, 2015). This study investigated culturable halophilic-bacterial diversity using a slightly high salt concentration of 4 or 5% (w/v, NaCl) than seawater. To isolate bacterial cells, a sample of the marine sediment soil (33°27'15" N, 126°18'26"E) was collected with a sterile spatula and promptly transported to the laboratory at 4°C with an icepack. The sample was then slurred by autoclaved-natural seawater and finally adjusted to 4 or 5% NaCl (w/v). The slurry mixture was serially diluted (ten-fold) to 10^-4. Sequentially, 100 µl of the aliquot from the final diluted sample (10^-4) was spread onto marine agar (BD Difco^TM) plates adjusted to 4 or 5% (w/v, NaCl; named adjusted-salt marine agar, ASM agar) and incubated at 30°C for one week under aerobic conditions. After the first-round cultivation, several strains were selected based on colony morphology and color under naked eyes and transferred to new ASM agar plates. To obtain a purified single colony, colonies were successively transferred to new ASM agar plates at least five times. Finally, one isolated yellowish colony designed as GJH2-4 was collected at 4% (w/v) NaCl culture condition. The isolated strain GJH2-4 was deposited at the Korean Culture Center for Microorganisms (KCCM) (deposit number: KCCM 43405).

To analyze its phylogenetic and taxonomic positions, genomic DNA of strain GJH2-4 was extracted using a Monarch^® Genomic DNA Purification Kit (New England Biolabs, Inc.). Its 16S ribosomal RNA gene sequence was then amplified and sequenced according to the previous study (Kim et al., 2020). It was found that strain GJH2-4 was most closely related to Photobacterium atrarenae M3-4^T (HM452945, 99.6% 16S rRNA gene sequence similarities) (Kim et al., 2011) using EzBioCloud server (https://www.ezbiocloud.net/). The genus Photobacterium of the order Vibrionales and the type species, Photobacterium phosphoreum, were reclassified by Beijerinck (1889) (formerly named as Micrococcus phosphoreus; Cohn, 1878). Members of the genus are Gram-stain-negative, facultative aerobic, motile, and widely distributed in various marine associated environments such as seawater and marine animals (Urbanczyk et al., 2011). In addition, some strains belonging to the genus Photobacterium are pathogens having high virulence potentials (Matanza and Osorio, 2020; Pira et al., 2022).

Whole-genome sequencing was performed at DNA Link, Inc. using a Pacific Biosciences RSII instrument (Pacific Biosciences). A single SMRT cell produced a total of 1.7 Gb (about 317 X in depth) in 312,782 polymerase reads that passed filtering. Finally, we obtained three contigs closed using RS HGAP assembly (v3.0). Genome completeness and quality for assembled chromosomes were estimated with BUSCO (Manni et al., 2021). Resulting assembled contigs were annotated with Prokka (v1.14.5) installed stand-alone and NCBI Prokaryotic Genome Annotation Pipeline (PGAP) using GeneMarkS-2+ version 5.2 with the best-placed reference protein method (Angiuoli et al., 2008). In addition, to assign functional characteristic, protein sequences were predicted using Clusters of Orthologous Groups (COGs) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database references as described previously (Kim et al., 2020). Finally, the length of the three circularized chromosomes of strain GJH2-4 was about 5.41 Mb with G + C content of 53.0%. BUCSO estimation showed 100% genome completeness.

The complete genomes of strain GJH2-4 estimated by Prokka had 4,984 predicted genes, 34 ribosomal RNAs, and 153 transfer RNA genes (Table 1). Based on the phylogenomic tree constructed with Type (strain) Genome Sever (TYGS) (Meier-Kolthoff and Göker, 2019), strain GJH2-4 belonged to genus Photobacterium (Fig. 1), supporting the result for 16S rRNA gene phylogeny analysis. However, due to no genome sequence of Photobacterium atrarenae M3-4^T, we were unable to conducted digital DNA-DNA Hybridization (DDH) estimated by Genome-to-Genome Distance Calculator (GGDC) (Meier-Kolthoff et al., 2013) in this study.

Table 1

Genome features	Chromosome 1	Chromosome 2	Chromosome 3
Genome length (Mb)	3.46	1.86	0.09
GC content (%)	53.5	52.3	50.9
No. of total predicted genes*	3,235	1,655	94
No. of total coding sequences*	3,054	1,648	92
No. of rRNA (23S, 16S, 5S)*	34 (11, 11, 12)	0	0
No. of tRNA*	146	7	0

* Estimated by Prokka (v.1.14.5).

Fig. 1. Tree inferred with FastME 2.1.6.1 from GBDP distances calculated from genome sequences. Branch lengths are scaled in terms of GBDP distance formula d₅. Numbers above branches are GBDP pseudo-bootstrap support values > 50% from 100 replications. The tree was rooted at the midpoint by Vibrio fluvialis NBRC 103150 (GCA_001598835).

Resultant COGs revealed that the category of amino acid transport and metabolism (E, 9.94%) was the most dominant one in the genome, followed by transcription category (K, 8.17%) and the category of translation, ribosomal structure, and biogenesis (J, 7.85%) (Fig. 2).

Fig. 2. Distribution of clusters of orthologous groups (COGs) for strain GJH2-4 genome. The x-axis represents single letter for COG class and the y-axis represents gene frequency. Each COG functional class with single letter is presented in the right panel.

KEGG analysis (n = 4,794) showed that complete chromosomes of strain GJH2-4 harbored genes for central metabolisms such as glycolysis, gluconeogenesis, glyoxylate cycle as an anabolic tricarboxylic acid cycle, and biosynthesis and degradation for glycogen. In addition, the genome contained genes for assimilatory sulfate reduction (cysNDCHJI), dissimilatory nitrate reduction (nitrate to ammonia, napAB, nirBD, and nrfAH), and denitrification (nitrate to nitrogen, napAB, nirK, norBC, and nosZ). Strain GJH2-4 genome harbored a complete gene cluster for benzene degradation, which could degrade benzene to acetyl-CoA via catechol. Lots of the ABC transporters (n = 107) were identified in the genome, including phosphate, phosphonate, amino acids, phospholipid, oligopeptide, glycine betaine, and osmoprotectant. Interestingly, vitamin B₁₂ (also called cobalamin) transporter (BtuFCD) was found in the genome with complete genes for cobalamin biosynthesis. These results imply that strain GJH2-4 contributes to nitrogen and carbon cycles in marine sediment (Herbert, 1999) with a possibility for growth factors supply to other organisms (Sañudo-Wilhelmy et al., 2014). However, contributions of the genus Photobacterium in the family Vibrionaceae are largely unknown. In the same class of Gammaproteobacteria, the cobalamin-mediated metabolic process of the family Psychromonadaceae has been analyzed based on ecogenomics (Jin et al., 2022).

In addition, the genome of strain GJH2-4 harbored many genes for two-component system (n = 115), quorum sensing (n = 47), biofilm formation (n = 131), pilus system (twitching motility and type IV pilus, n = 18), and nearly the entire flagellar assembly (n = 41). The genome harbored cbb₃-type (cytochrome c oxidase) as a high oxygen affinity under low-oxygen conditions (Pitcher and Watmough, 2004). Taken together, these genetic potentials indicate that strain GJH2-4 might have ecophysiologically dominant position against other competitors in its habitats.

Nucleotide sequence accession number

The complete genome sequence of strain GJH2-4 (= KCCM 43405) has been deposited at DDBJ/ENA/GenBank under accession numbers CP101508 to CP101510.

Pseudomonadota 문, Vibrionales 목에 속하는 Photobacterium 속은 그람 음성, 통성 호기성 및 운동성을 지닌 것으로 알려져 있다. 또한, 병원균을 포함한 Photobacterium 속 세균들은 다양한 생활사를 가지고 있다. 본 연구에서는 제주의 해양 퇴적물에서 Photobacterium 속의 GJH2-4 균주를 분리하여 완전한 게놈 서열을 분석하였다. 균주의 유전체 염기서열은 새로이 (de novo) 조립되어, 총 3개의 완전 전장유전체가 확인되었으며, 전체 게놈 크기와 G + C 함량은 각각 약 5.41 Mb와 53.0%였다. 3개의 완전 전장유전체는, 4,794개의 단백질을 암호화하는 서열을 비롯하여, 34개의 리보솜 RNA, 153개의 운반 RNA 유전자가 확인되었다. 유전체 분석 결과, GJH2-4 균주의 게놈은 벤젠 분해, 완전 탈질화, 이화적 질산염 환원, 동화적 황산염 환원, 편모 조립과 다양한 비타민 생합성과 관련된 유전자를 지니고 있다. 또한, 저산소 조건에서 높은 산소 친화력을 가지는 cbb₃ 타입의 시토크롬 산화효소를 암호화하고 있다. 본 연구를 통하여, GJH2-4균주가 물질 순환에 기여하는 것을 발견하였으며, 이는 Photobacterium 속에 속하는 세균의 보다 다양한 능력에 대한 이해에 도움이 될 것이다.

This research was supported by the 2022 scientific promotion program funded by Jeju National University.

The authors have no conflict of interest to report.