Coastal tidal flats are a unique ecosystem that is alternately covered and exposed by the tides, and it includes various habitats such as tidal flats and salt marshes (Chen et al., 2016). The intertidal zones, where seawater and freshwater meet, create unique environments due to the presence of salinity, which limits biodiversity. When salt concentrations increases, the biological diversity decreases, however, halotolerant microorganisms often require high-salt environments and can even be found at the highest salt concentrations (Gerday and Glansdorff, 2007). In particularly, coastal solar salterns are noteworthy as they utilize these environmental characteristics of tidal flat for salt production. They are man-made, thalassohaline hypersaline environments (Oren, 2007) created by evaporating seawater and are located along the coastline (Paul et al., 2020). These artificial solar salterns create an extremely hypersaline environment that results in biodiversity loss, providing evidence that the region has undergone “environmental degradation” (Rodriguez-Valera et al., 1985).

Hypersaline environments, such as solar salterns and salt lakes, are characterized by high salt levels (> 3.5%, higher than seawater), and are inhabited by halophiles (Ventosa, 2006; Javor, 2012), a group of microorganisms adapted to these extreme conditions. Halophilic bacteria can be classified based on their salt requirement and growth patterns. Slight halophiles grow optimally at 2 – 5% NaCl, moderate halophiles at 5 – 20% NaCl, and extreme halophiles at 20 – 30% NaCl (Irshad et al., 2014). To cope with high extracellular salinity, halophilic microorganisms have adopted various strategies. Bacteria and archaea accumulate K⁺ and Cl^– ions while maintaining a low Na⁺ concentration in response to external osmotic pressure. Most bacteria and eukaryotes store or synthesize organic solutes, such as glycine betaine, ectoine, and other amino acids and sugar derivatives, to deal with high salinity (Oren, 2002; Roberts, 2005). Halophiles have developed basic biochemical adaptations in their proteins, osmoregulation systems, nucleic acids, and lipids to thrive and reproduce in such high-salt, low-water activity conditions (Javor, 2012).

Getbol, tidal flats in Korean, are located along the west coast of the Republic of Korea and include several manmade salterns, such as Taepyeong salt farms in Sinan-gun, Jeung-do (Lee et al., 2018), and Yubu-do solar saltern in Taean-gun. The coastal tidal flats have been extensively studied, and numerous microorganisms have been reported and isolated from this ecosystem (Yeon et al., 2005). However, it is worth noting that microorganisms are also being isolated, particularly from abandoned salterns. Among them, the genus Halomonas is frequently isolated from high-salt environments such as abandoned salterns (Lim et al., 2004; Lee et al., 2005; Lee et al., 2016). The genus Halomonas belongs to the phylum Gammaproteobacteria, class Oceanospirillales, family Halomonadaceae, and is one of the largest halophilic genus so far with over 100 species and diverse salinity growth range.

In a previous study, the comparative evaluation of bacterial diversity and community structure was conducted on three abandoned salterns with different natural restoration periods (Lee et al., 2020). As the salterns were abandoned for longer periods of time, the microbial community structure became increasingly similar to that of the adjacent natural tidal flats. In a recent study, a novel halophilic bacterial species named “Halomonas getboli YJPS3-2” was isolated and reported from the sediment of an abandoned saltern in Yongyu-do, where the difference in microbial community structure between the saltern and the adjacent natural tidal flat was the greatest in previous studies (Lee et al., 2020; Yoo et al., 2022). Therefore, this study was conducted as a follow-up to the previous research to evaluate the ecological role of halophilic bacteria in abandoned saltern, using newly identified halophilic bacteria “YJPS3-2” as a research subject. The specific objectives of this study were: 1) to reveal adaptation strategies to the hypersaline environment by exposing the strain to various salt conditions and 2) to report halotolerant-related genes through the newly identified species.

This study focused on halophilic bacteria isolated from abandoned solar salterns, aiming to reveal adaptation strategies under hypersaline environments and designating this halophilic bacterium as a pioneering species in the field. The study utilized the halophilic bacterial strain YJPS3-2, which was found to primarily adapt to the halophilic environment by modulating the ABC transporter to maintain osmotic balance, regulating flagellar assembly, and enhancing the DNA repair pathway ( Figure 6 ). Additionally, the strain also acted as a pioneering species, allowing other organisms to thrive by settling first in hypersaline environments and producing a variety of secondary metabolites ( Figure 4 ).

ABC transporters, which should be initially discussed when studying the halophilic adaptive mechanism of strain YJPS3-2, use the energy of ATP hydrolysis for the uptake and efflux of solutes across the cell membrane. These transporters are crucial for halophiles, which produce or acquire compatible solutes (such as ectoine, trehalose, glycine betaine, and choline) and anions into the cytoplasm to balance the osmotic pressure (van der Heide and Poolman, 2000; Lewis et al., 2012; Das et al., 2015). Genes related to the ABC transporter for maltose (malK), trehalose (thuF, thuG, and thuK), mannitol (smoK), and L-arabinose (araG) were upregulated in a 5% NaCl environment. However, genes associated with negatively charged ions such as phosphate (PO₄ ^3-), glutamate, and amino acids were downregulated, indicating that the bacterium avoids importing them to adapt to osmotic pressure.

Motility is a common strategy used by various types of bacteria to respond to adversity (Li et al., 1993), and it has been found that it is higher in 25% NaCl environments compared to 0% NaCl (Remonsellez et al., 2018). The bacterial flagellum functions as a motor, protein exporter, and assembly apparatus (Macnab, 2003), and is composed of an MS ring formed by the inner-membrane protein FliF (Kubori et al., 1992), T3SS (type III secretion system), and a hook-filament junction. Chemotaxis is a mechanism by which bacteria efficiently and rapidly respond to changes in chemical composition using methyl-accepting chemotaxis proteins (MCPs) and cheA and cheY genes (Bren and Eisenbach, 2000). In response to changes, bacteria use flagella to attach to and invade targets (Bange et al., 2010), promote plant growth (Glick, 1995), and remove contaminants (Omotayo et al., 2013). In the study, genes related to flagellar assembly, including MS ring formation (filF and fliI), T3SS (flhA and filK), and attachment (cheA), were overexpressed, with the most highly overexpressed gene being cheA, which regulates gene expression in response to changing environmental conditions (Stock et al., 1988). The cheA was expressed more in 15% NaCl than in the 5% NaCl conditions, which might be attributed to its role in the bacterial chemotactic system.

The overexpression of ligA and ruvB, which plays an important role in DNA processes (Alomari, 2018) and recombination, respectively, was upregulated in both conditions. Nucleotide repair-related genes in bacteria have previously been reported to be important for bacterial survival under salt stress (Mirete et al., 2015), and their over and under-expression is expected to be common in extreme environments (Puig et al., 2021).

In contrast to T3SS, T6SS (type VI secretion system) is a versatile mechanism that can transfer toxins to both eukaryotic and bacterial cells (Douzi et al., 2016), increasing the survival rate of bacteria by removing risk factors from their surrounding environment. T6SS component genes, namely clpV and lcmF were found to be upregulated in 5% and 15% NaCl conditions, respectively. The genus Halomonas has been studied for its biochemical potential, but some studies have reported its pathogenicity (Rojas et al., 2009; Tsuji et al., 2022) and potential harm to humans (Stevens et al., 2009; Kim et al., 2012). HELO_2920 gene encodes the T6SS cluster protein VasA, which is a macromolecular machine that plays an important role in the pathogenicity of Gram-negative bacteria (Wang et al., 2021) by transferring and secreting effectors (Mariano et al., 2019) and injecting a toxin into eukaryotic cells to hydrolyze the cell walls. The existence of such a potential halophilic human pathogen in abandoned salterns near tidal flats that are easily accessible to people may pose a danger.

During the process of adapting to high salinity conditions, it is crucial to investigate not only the overexpressed genes but also to genes that are downregulated or underexpressed.

In this study, we observed a substantial underexpression of genes associated with nitrogen metabolism, including nirB, nirD, and nasA Notably, nirD exhibited the most pronounced underexpression among all the downregulated genes. This finding is consistent with previous studies reporting a decrease in nitrogen metabolism in bacteria under high salinity environments (Li et al., 2021). The observed decrease in nitrogen metabolism suggests a strategic energy conservation strategy employed by bacteria to optimize their physiological responses to high salinity stress and allocate resources primarily to essential metabolic pathways required for their adaptation and survival.

The intriguing similarity in gene expression patterns across major pathways is noteworthy, wherein genes that were overexpressed in 5% NaCl conditions were under-expressed in 15% NaCl conditions, and vice versa. Nevertheless, some genes displayed higher expression in 15% NaCl conditions compared to the 5% NaCl conditions ( Figure 5 ). This suggested that the genes that enable adaption to the halophilic environment may also contribute to survival in a subsequent hypersaline environment.

Halophilic microorganisms, while adapting to hypersaline environments, produce various secondary metabolites that aid the survival of other organisms (Chung et al., 2020). The genomic analysis of strain YJPS3-2 by antiSMASH predicted the production of ectoine and siderophore, which was consistent with the transcriptome data. Ectoine helps the organism tolerate osmotic pressure (Hahn et al., 2016), while the compatible solutes produced by the bacteria are absorbed by algae and plants in a halophilic environment, enabling them to withstand osmotic stress (Rontein et al., 2002; Moghaieb et al., 2006). Moreover, siderophores, which are peptidic chelates excreted by microorganisms to uptake iron (Scavino and Pedraza, 2013), contribute to plant growth and phytosanitary protection (Compant et al., 2005; Maheshwari, 2011). Thus, halophilic strains living in an extremely halophilic environment produce various secondary metabolites that assist other organisms in surviving in saline environments, thereby promoting the recovery these areas through the bloom of algae and plant growth and the increase in biodiversity (Scavino and Pedraza, 2013). This highlights the significance of the “halophiles settlement” phenomenon.

The genus Halomonas is known for its ability to degrade aromatic compounds (García et al., 2005; Oie et al., 2007; Mnif et al., 2009), as well as its high tolerance to hypersaline environments. Strain YJPS3-2 also contains genes involved in the degradation of quinate, biphenyl, benzoate, p-hydroxybenzene, and generate. However, these genes were not expressed in either 5% or 15% NaCl conditions, suggesting that aromatic compounds were not supplied to the medium during transcriptomic analysis. This finding implies that this strain has the potential to act as a pioneer species in both salt and oil contamination.

In the present study, we confirmed low microbial diversity in hypersaline environments and investigated the adaptive genes of a halophilic bacterium, with a focus on its potential to serve as a pioneering species in harsh environments. Genomic and transcriptomic analysis of strain YJPS3-2 revealed that this strain adapts to halophilic environments by secreting substances that can be used by surrounding organisms, aiding in the thriving of algae and plants in hypersaline environments.

We investigated the adaptative strategies of halophilic bacteria in hypersaline environments, using a newly identified strain YJPS3-2 as a model approach. The transmembrane protein in the cell membrane seemed to play a key role in withstanding osmotic pressure and this bacterium could aid other living organisms to survive in harsh environment by adapting to a salt-stress environment. Although whole-genome sequencing analysis and transcriptomic analysis determined the gene expression levels under various environmental conditions, only known and annotated genes were interpreted. Further research would be necessary to evaluate the newly identified salt-stress-related gene and, particularly identification of the secondary metabolites. Our findings suggest YJPS3-2 would be considered as a pioneer in improving the surrounding by first settling in hypersaline areas near the coast, and elsewhere.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

YY: Conceptualization, formal analysis, statistical analyses, visualization, writing – original draft, writing - review & editing. HL: Conceptualization, formal analysis, statistical analyses, visualization, writing – original draft, writing - review & editing. JL: Visualization, writing - review & editing. JK: Conceptualization, data curation, writing - review & editing, project administration. J-JK: Conceptualization, writing – review & editing, project administration, funding acquisition, supervision. All authors contributed to the article and approved the submitted version.