Known as extremophiles, extremely halophilic microorganisms, mostly reported haloarchaea, inhabit and recognize in high-salinity environments such as solar saltern, salt marshes, salt lakes, and high salt-fermented foods (Oren, 2002; Roh et al., 2007; Cui and Dyall-Smith, 2021). They have been well-reported for distinctive metabolic characteristic (i.e., nutrient-available limitation) to adapt to their high concentrations for sodium ions. In addition, recent investigations have reported valuable insights into their potential industrial or therapeutic applications (Koller and Rittmann, 2022; Moopantakath et al., 2023). We successfully isolated the strain halo-7 from brine sediment of solar saltern and validly named species, Halolamina sediminis as an extremely halophilic archaeon, as described in our previous study (Koh et al., 2015). The genus Halolamina, a member of the family Halorubraceae (class Halobacteria of the phylum Euryarchaeota) (Gupta et al., 2016), was firstly proposed with the description of Halolamina pelagica isolated from brine sample of Taibei marine solar saltern as a type species by Cui et al. (2011). At the time of writing (accessed on April 2024), only six species have been validly named in this genus and isolated from various extremely saline environments (https://lpsn.dsmz.de/genus/halolamina). Cells of the recognized species in the genus Halolamina are pleomorphic shape and Gram-stain-negative. Some species (Halolamina rubrua and Halolamina sediminis) require magnesium ion for growth (Cha et al., 2014; Koh et al., 2015). Indole production from tryptophan and anaerobic growth are only reported from Halolamina litorea isolated from marine solar saltern (Xu et al., 2016). The strain halo-7 was unable to grow under anaerobic condition with nitrate, L-arginine, DMSO or TMAO. In addition, the cells of the strain halo-7 were also pleomorphic shape, Gram-stain-negative, and positive activity for catalase and oxidase (Koh et al., 2015). It was capable of growing within a range of 15–30% NaCl (w/v, optimum 20–25%), with the optimal pH (7.0–8.0). The cells were lysed in distilled water. Here, we interpreted the genomic traits of strain halo-7, which includes its potential-capabilities for bioremediation.

Data description

Strain halo-7 was isolated from a sediment sample of a crystallizing pond in the Gomso solar saltern, South Korea (35°35' N 126°36' E). For routine cultivation, the isolated strain was grown at 30 °C on DBCM2 agar under aerobic conditions (Koh et al., 2015). The polyphasic taxonomic results were described in our previous study (Koh et al., 2015). Briefly, the colonies exhibited a smooth, round shape and were red-pigmented. The cells were Gram-stain-negative, pleomorphic (short rod or oval), and motile. The strain tested positive for oxidase and catalase activity. The growth conditions were a temperature range of 25–45°C (optimal 37–40°C), pH range of 6.5–9.5 (optimal 7.0–8.0), and NaCl concentration of 15–30% (w/v; optimal 20–25%).

For genome sequencing, genomic DNA from strain halo-7 was extracted using a commercial Genomic DNA extraction Kit (Exgene^TM Cell SV, GeneAll Biotechnology Co., Ltd.). PacBio RSII sequencing (performed at Macrogen) utilized the SMRTbell^TM Template prep kit following standard protocols. De novo assembly of the strain halo-7 genome using the hierarchical genome assembly process (HGAP) from raw reads at 215X coverage resulted in a single closed contig approximately 2.84 Mb in size (Table 1). Gene prediction and coding sequences (CDSs) annotation were performed by Prokka (ver. 1.14.5) (Seemann, 2014). Digital DNA-DNA relatedness (species boundary; ANI and AAI) was estimated as previously described (Kim and Park, 2023). CDSs were compared to the COG and KEGG databases by EggNOG-mapper (ver. 2.1.12) (Cantalapiedra et al., 2021). Additional microbial metabolism was predicted using ‘anvi-estimate-metabolism’ (Veseli et al., 2023) within Anvi’o (Eren et al., 2021). Putative CRISPR-Cas (CRPISR-associated) elements and prophage were predicted using CRISPRCasFinder (Couvin et al., 2018) and PHASTEST (Wishart et al., 2023), respectively.

Table 1

Genome information for Halolamina sediminis halo-7

Items	Description
Project information
Sequencing quality	Finished (single contig)
Fold coverage	215X
Sequencing method	PacBio RSII
Assembly method	HGAP
Genomic features*
Size (bp)	2,835,858
G + C content (%)	67.94
Number of predicted CDSs	2,846
Genes assigned to COG	2,176
Genes assigned to KEGG	1,556
Number of rRNA genes	2,2,2 (5S, 16S, 23S)
Number of tRNAs	46

* genomic features estimated by Prokka (v1.14.5) and emapper (v2.1.12)

The circular genomic size of strain halo-7 was 2,835,858 base pairs (bp), and its GC content was 67.94%. A total of 2,900 genes were predicted in the genome of strain halo-7, including 2,846 protein-coding genes, 46 tRNAs, and 6 rRNAs, as presented in Table 1. Coding density was estimated with 89.06%. Strain halo-7 has been validated under polyphasic taxonomic approach, including wet-based DDH (i.e., traditional DDH) (Koh et al., 2015). To further verify the wet-based DDH, the digital genomic relatedness (measured by ANI and AAI) was assessed between strain halo-7 and the publicly available genomes belonging to the genus Halolamina (H. rubura, H. pelagica, H. saliffodinae, and Halolamina sp. CBA1230). The ANI and AAI values ranged from 83–86% and 79–82%, respectively, when comparing halo-7 with other genomes of four other strains. This result supports the identification of strain halo-7 as a novel species within the genus Halomonas (Thompson et al., 2013; Meier-Kolthoff and Goker, 2019).

A total of 2,176 and 1,556 genes from the genome of strain halo-7 were categorized in the arCOG and KEGG databases, respectively. The majority of arCOG categories was observed in Function unknown (S, 21.6% of the total assigned arCOG CDSs), succeeded by Amino acid transport and metabolism (11.8%), Transcription (7.9%), Transloation, ribosomal structure and biogenesis (7.7%), and Energy production and conversion (C, 7.4%) (Fig. 1). Interestingly, Carbohydrate transport and metabolism (G, 2.5%) was observed. A few metabolic pathways were predicted as a complete pathway such as beta-oxidation (acyl-CoA synthesis) and de novo synthesis for purine and pyrimidine. The findings suggest that the functional CDSs present in strain halo-7 might be focused on energy production, despite most CDSs were assigned to functional unknwon. Additionally, this metabolic feature deduces that extremely halophilic archaea can survive and adapt to their oligotrophic (i.e, nutrient poor) environment (Stan-Lotter and Fendrihan, 2015). Across various nutrient concentrations, ATP-binding cassette (ABC) transporters facilitate the uptake and efflux of diverse nutrients into the cytoplasm by utilizing ATP hydrolysis. Each ABC transporter has a specific responsibility for transporting a target nutrient, such as carbohydrates, amino acids, peptides, lipoproteins, or metal ions. Therefore, it is evident that microorganisms possessing numerous ABC transporters can better adapt and survive in their habitats by expanding their metabolic lifestyles (Schneider and Hunke, 1998; Davidson and Chen, 2004). Few genes (n = 35) involved in ABC transporters were identified in the strain halo-7 genome. In particular, for complete nutrient transporter, there were only thiamin, glucose/mannose, phosphate, branched-chain amino acids, and biotin. Taken together the identification of only 35 genes involved in ABC transporters in the halo-7 genome suggests a relatively limited metabolic capacity and potentially (i.e., already) adapted to nutrient-poor environments. The genome of strain halo-7 completely harbored genes for glycolysis, gluconeogenesis, and TCA cycle, which involves the central carbohydrate metabolism. Moreover, these genomic traits are in accordance with the physiochemical taxonomic findings from our previous study (Koh et al., 2015).

Fig. 1. Distribution of archaeal clusters of orthologous groups (arCOGs) for the strain halo-7 genome. The x- and y-axes represent the number of genes in each arCOG category and a single letter for arCOG class. Full name of arCOG single letter is presented in the right panel.

Based on the KEGG analysis, the genome of the strain halo-7 contained genes for NirK and NosZ involved in denitrification, as well as NirBD for dissimilatory nitrite reduction (nitrite to ammonia). This result correlated with the finding from our previous study (Koh et al., 2015) that strain halo-7 was unable to grow under anaerobic conditions with nitrate as an electron acceptor. It suggests that strain halo-7 may have the potential to grow under anaerobic condition via an alternative respiratory pathway (e.g., NirK and NosZ) (Miralles-Robledillo et al., 2021). The genes for two distinct types of terminal oxidase: cytochrome c oxidase and cytochrome bd oxidase (CydAB, missing CydX) were identified in the genome of strain halo-7. This result indicates that strain halo-7 might exhibit a response to varying levels of oxygen (van der Oost et al., 1994; Matarredona et al., 2020). Genes involved in archaeal flagellar assembly proteins (FlaBFGHIJK) were identified, supporting cell motility as previously reported (Koh et al., 2015). The aforementioned genomic traits imply that the strain halo-7 could potentially endure various environmental stresses in hypersaline environments, including deficient in nutrients.

Based on the CRISPRCasFinder and PHASTEST analysis, only three CRISPR arrays (with three spacers) and no prophage sequence were identified in the genome of strain halo-7. However, two genes (halo7_02114 and halo7_02743) were predicted to encode Csa3 and exonuclease Cas4, respectively, in the strain halo-7 genome based on KEGG analysis. Additionally, only six phage integrase-encoded genes were found. Taken together, these results deduce that strain halo-7 might have no environmental stress against to phage infection, despite the presence of many haloarchaeal phage thriving in their habitats (Luk et al., 2014; Liu et al., 2021).

Moreover, many specific stress proteins [i.e., chaperones; GrpE, DnaJ (Hsp60), Hsp70, small Hsp20, TCP-1/cpn60 chaperonin family, and Zn-dependent protease], universal stress protein (Usp), CSP, and page-shock protein (PspA) were identified. The HrcA protein, which is an heat-inducible transcriptional regulator (i.e., repressor), contains C-terminal CBS domains (Winged helix-like DNA-binding domain) was found (Liu et al., 2005). Along with, genes involved in DNA recombination and repair proteins, and proteasome subunit were identified in the strain halo-7 genome; mutS (for DNA mismatch), uvrABCD (Nucleotide excision repair), radA (for DNA repair), ligA (DNA ligase), psmAB (for proteasome), and pan (for archaeal 20S proteasome).

In brief, the genomic traits of strain halo-7 indicate its capability to tolerate various stressful conditions in hypersaline environments, such as nutrient deficiency, UV radiation, oxidative stress, and temperature fluctuations. This tolerance is facilitated by the presence of stress-related proteins, variant oxidases, and DNA repair mechanisms evident from the genomic analysis. Moreover, despite having a relatively limited number of ABC transporters, the strain halo-7 genome suggests potential adaptation to nutrient-poor environments. Intriguingly, the identification of genes involved in bioremediation, like long-chain alkane monooxygenase and haloalkane dehalogenase, implies the possible application of strain halo-7 in bioremediation processes. Overall, this genomic study enhances our understanding of the adaptive strategies and metabolic potential of extremely halophilic archaea thriving in hypersaline habitats, paving the way for exploring their biotechnological applications in bioremediation and other relevant fields.

Genome sequence accession numbers

The complete genome sequence of Halolamina sediminis halo-7 was deposited in the European Nucleotide Archive (ENA) under the accession number CVUA01000000 (ERP010727). The strain was submitted to the Japan Collection of Microorganisms (JCM) and Spanish Type Culture Collection (CECT) with accession numbers JCM 30187 and CECT 8739, respectively.

극한환경 미생물으로 알려진 극호염성 고균은 천일염전, 소금 발효 식품 등 다양한 고염분 환경에서 서식한다. 본 연구는 국내 천일염전의 염수 퇴적물에서 분리된 극호염성 고균 halo-7 균주의 유전체 분석 결과를 제시한다. halo-7 균주의 완전 전장 유전체는 약 2.84 Mb이며, GC 함량은 67.94%이다. 흥미롭게도, 긴사슬 알칸 모노옥시게나아제 및 할로알칸 디할로게나아제와 같은 유전자가 확인되어, 생물정화에 halo-7 균주가 잠재적 적용가능성을 가지고 있음을 나타낸다. 본 유전체 연구는 고염분 환경에서 극호염성 고균의 적응전략과 대사 잠재력에 대한 이해를 높일 수 있다.

This research was supported by the 2024 scientific promotion program funded by Jeju National University.

Soo-Je Park is Editor of KJM. He was not involved in the review process of this article. Also, authors have no conflicts of interest to report.