Halotolerance is a relative term that refers to the ability to tolerate salt concentrations higher than those necessary for growth, and microorganisms are considered halotolerant if they survive at high salt concentrations but do not require these conditions for growth (Anton, 2014). With advances in technology, halotolerance mechanisms have been investigated using omics approaches. For instance, comparative transcriptomic and physiological analysis revealed that the halotolerant bacterium Egicoccus halophilus EGI 80432^T increased inorganic ions uptake and accumulated trehalose and glutamate in response to moderate salinity condition, while the high salt condition led to up-regulated transcription of genes required for the synthesis of compatible solutes, such as glutamate, histidine, threonine, proline, and ectoine (Chen et al., 2021). The role of glutamate as a key compatible solute for halotolerance was also reported in a halotolerant strain of Staphylococcus saprophyticus based on transcriptome comparison of cells cultivated in media containing different concentrations of NaCl (0, 10, and 20%; Jo et al., 2022). In the exoproteome of the halotolerant bacterium Tistlia consotensis grown at high salinity, proteins associated with osmosensing, exclusion of Na⁺ and transport of compatible solutes, such as glycine betaine or proline are abundant (Rubiano-Labrador et al., 2015). Similarly, the proteomic analysis of halotolerant nodule endophytes, Rahnella aquatilis strain Ra4 and Serratia plymuthica strain Sp2 identified that different trans-membrane ABC transporters (ATP-binding cassettes) were the most represented among the up-regulated proteins in response to salt stress (Novello et al., 2022). Moreover, the proteome comparison of halotolerant bacterium Staphylococcus aureus under different osmotic stress conditions revealed the differentially expressed proteins (DEPs) involved in fatty acid synthesis, proline/glycine betaine biosynthesis and transportation, stress tolerance, cell wall biosynthesis, and the TCA cycle, which may contribute to the osmotic stress tolerance of S. aureus (Ming et al., 2019). These findings shed light on halotolerance mechanisms. However, halotolerance-related genes may be ignored in transcriptomic and proteomic comparison if there is no significant change in their expression under the experimental conditions.

Genomic comparisons of closely related halotolerant microorganisms can identify genes conserved among species as well as genes that may give an organism its unique characteristics, which helps to understand the mechanisms of salt tolerance. For example, through comparative genome analysis it was uncovered that the members of Acidihalobacter genus contained similar genes for the synthesis and transport of ectoine, as well as genes encoding low affinity potassium pumps. Variations were observed in genes encoding high affinity potassium pumps and proteins involved in the synthesis and/or transport of periplasmic glucans, sucrose, proline, taurine, and glycine betaine (Khaleque et al., 2019). To elucidate salt adaptation strategies in Nitriliruptoria, the genomes of five members from group Nitriliruptoria were analyzed. The results showed that Nitriliruptoria harbor similar synthesis systems of solutes, such as trehalose, glutamine, glutamate, and proline, and on the other hand each member of Nitriliruptoria species possesses specific mechanisms, K⁺ influx and efflux, betaine and ectoine synthesis, and compatible solutes transport (Chen et al., 2020). Using whole-genome analysis, the halotolerant strains of Martelella soudanensis, NC18^T and NC20, were predicted to harbor various halotolerant-associated genes, including K⁺ uptake protein, K⁺ transport system, ectoine transport system, glycine betaine transport system, and glycine betaine uptake protein, indicating that strains NC18^T and NC20 might tolerate high salinity through the accumulation of potassium ions, ectoine, glycine betaine (Lee and Kim, 2022). Although these findings help to understand the versatile mechanisms of halotolerance existing in halotolerant microbes, genomic comparisons are usually based on genome-wide searches for homologs of known halotolerance-related genes, such as those involved in K⁺ and Na⁺ influx and efflux and the synthesis and transport of compatible solutes.

The aim of this perspective is to provide new insights into the development of novel hypotheses and promote further studies on the halotolerance mechanisms. Therefore, co-occurrence analysis between halotolerance and open reading frames (ORFs) was performed to provide intuitive information on halotolerance.

Microorganisms develop abilities that enable them to deal with evolutionary pressure from the environment, such as salinity, temperature, and the power of hydrogen (pH). The phylogenetically closely related strains, which showed similar growth temperature and pH range but different halotolerance, would simplify the analysis. Furthermore, considering the ionic strength of different media may affect the cell growth, the tolerance to NaCl used for co-occurrence analysis should be determined by using same medium. Herein six type strains from two phylogenetically close genera, Pontixanthobacter and Allopontixanthobacter, were chosen for this study. Because of their close phylogenetic relationship, Allopontixanthobacter sediminis and Allopontixanthobacter confluentis have been previously classified as Pontixanthobacter species (Xu et al., 2020; Liu et al., 2021b), and later were reclassified as Allopontixanthobacter species (Xu et al., 2020; Liu et al., 2021a,b). Notably, all the type strains belonging to the two genera were isolated from the Yellow Sea and surrounding areas, but from diverse salty environments, such as Pontixanthobacter aestiaquae KCTC 42006^T and Pontixanthobacter rizhaonensis KCTC 62828^T from seawater (Jung et al., 2014; Liu et al., 2021b), Pontixanthobacter gangjinensis JCM 17802^T and Pontixanthobacter luteolus KCTC 12311^T from tidal flat (Yoon et al., 2005; Jeong et al., 2013), Pontixanthobacter aquaemixtae KCTC 52763^T from the junction between ocean and fresh spring (Park et al., 2017), A. sediminis KCTC 42453^T from lagoon sediments (Kim et al., 2016), and A. confluentis KCTC 52259^T from water of estuary environment (Park et al., 2017). These strains showed similar optimum NaCl concentrations for growth (1–3%, w/v), but displayed different halotolerances, from 3 to 10% (w/v; Table 1), indicating that these strains adapt to their diverse habitats, including lagoon, junction between ocean and fresh spring, tidal flat, and seawater. The availability of their genomes provides remarkable opportunity to understand their different halotolerances by comparative genome analysis. Here, co-occurrence between halotolerance and the open reading frames (ORFs) was calculated among six strains of Pontixanthobacter and Allopontixanthobacter, and the ORFs showing high co-occurrence were discussed for possible contribution to halotolerance.

Open reading frames in the six genomes were predicted and clustered based on similarity using R package micropan (Snipen and Liland, 2015). Analysis of co-occurrence between ORFs and the maximum NaCl concentration tolerated among the six strains was conducted, and 113 clusters of ORFs were identified with co-occurrence greater than 0.8 (Table 2). The co-occurrence for the remaining clusters is listed in Supplementary material, as well as ORFs predicted in the six genomes and the index for clusters and ORFs. ORFs were annotated by searching standard database using protein–protein BLAST.¹

Salinity is one of the most important environmental factors for aquatic microorganisms and varies among habitats. Therefore, halotolerant microorganisms have developed versatile strategies to cope with saline stress. Based on the findings of co-occurrence analysis, possible explanations for mechanisms resulting in different salt tolerances among six strains are discussed above, which provided hypotheses for further investigations. Moreover, among the highly co-occurred clusters, there are several uncharacterized or hypothetical proteins (Table 2), which may contribute to halotolerance. It should be noted that the genes related to resistance to salts other than sodium chloride could also be discovered by co-occurrence analysis, since various salts co-exist in high ionic environments. For instance, ORFs of Cluster_2171 (co-occurrence as 0.81, Table 2) were annotated as divalent-cation tolerance protein CutA, which is required for copper tolerance in E. coli and affects tolerance levels to zinc, nickel, cobalt, and cadmium salts (Fong et al., 1995). This study sheds light on the mechanisms through which microorganisms cope with environmental stress. With the increasing number of isolated halotolerant strains and their genomes being sequenced, analyzing genome-wide co-occurrence between genetic diversity and physiological characteristics would expand the knowledge of the salinity adaptation strategies and provide comprehensive information on how microorganisms adapt to the environment, together with findings at the transcriptomic and proteomic levels.

Publicly available datasets were analyzed in this study. This data can be found here: https://www.ncbi.nlm.nih.gov. Accession Numbers are as follows: GCF_009827455.1_ASM982745v1, GCF_009827545.1_ASM982754v1, GCF_009828095.1_ASM982809v1, GCF_009827395.1_ASM982739v1, GCF_009828115.1_ASM982811v1, and GCF_009827615.1_ASM982761v1.

PZ contributed to study concept and design and performed data acquisition, analysis and visualization, and interpretation of results. PZ and Y-XB drafted the manuscript. LX, X-WX, and H-BS revised the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by grants from the National Science and Technology Fundamental Resources Investigation Program of China (2021FY100900), the Scientific Research Fund of the Second Institute of Oceanography, MNR (No. JZ1901 and JB2003), Natural Science Foundation of China (32000001), and the Oceanic Interdisciplinary Program of Shanghai Jiao Tong University (SL2022ZD108).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2023.1111472/full#supplementary-material