Understanding the mechanisms of halotolerance in members of Pontixanthobacter and Allopontixanthobacter by comparative genome analysis

Halotolerant microorganisms have developed versatile mechanisms for coping with saline stress. With the increasing number of isolated halotolerant strains and their genomes being sequenced, comparative genome analysis would help understand the mechanisms of salt tolerance. Six type strains of Pontixanthobacter and Allopontixanthobacter, two phylogenetically close genera, were isolated from diverse salty environments and showed different NaCl tolerances, from 3 to 10% (w/v). Based on the co-occurrence greater than 0.8 between halotolerance and open reading frame (ORF) among the six strains, possible explanations for halotolerance were discussed regarding osmolyte, membrane permeability, transportation, intracellular signaling, polysaccharide biosynthesis, and SOS response, which provided hypotheses for further investigations. The strategy of analyzing genome-wide co-occurrence between genetic diversity and physiological characteristics sheds light on how microorganisms adapt to the environment.


Introduction
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
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.

Strains used for analysis
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.

Clusters highly co-occurred with 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 Frontiers in Microbiology 03 frontiersin.org

Osmolyte
The ORFs of Cluster_111 (co-occurrence of 0.97, Table 2) were annotated as TauD/TfdA family dioxygenase. TauD is involved in the utilization of taurine (VanderPloeg et al., 1996), an organic osmolyte involved in cell volume regulation (Harris and Wen, 2012). Taurine is used as an osmoprotectant, such as in Escherichia coli at high osmolarity (McLaggan and Epstein, 1991) and in microbial communities from biofilms in metal-rich environment (Mosier et al., 2013). The ORFs of Cluster_111 only exist in three halotolerant strains, suggesting that taurine may be accumulated as an osmoprotectant. Interestingly, halotolerant strains harbor genes involved in various pathways related to glutamate generation. For instance, according to annotation, ORFs of Cluster_113 (co-occurrence of 0.81, Table 2) belong to the hydantoinase B/ oxoprolinase family, which includes 5-oxoprolinase, catalyzing the formation of L-glutamate from 5-oxo-L-proline (Niehaus et al., 2017). Besides, ORFs of Cluster_1328 (co-occurrence of 0.81, Table 2) possess similarity to p-aminobenzoyl-glutamate (PABA-GLU) hydrolase subunit from Altererythrobacter insulae (GenBank Accession Number: RGP41665.1). PABA-GLU is a folate catabolite found in bacteria, and the enzyme PABA-GLU hydrolase breaks down PABA-GLU by cleaving glutamate (Larimer et al., 2014). Additionally, ORFs of Cluster_1747 (co-occurrence of 0.81 Table 2) showed similarity to asparagine synthase from Salinigranum halophilum (GenBank Accession Number: WP_136601134.1). Asparagine synthetase catalyzes an ATP-dependent amidotransferase reaction between aspartate and glutamine, which produces asparagine and glutamate (Richards and Kilberg, 2006).

Permeability
To ensure a physiologically acceptable level of cellular hydration and turgor at high osmolarity, many bacteria accumulate compatible solutes as osmoprotectants (Ziegler et al., 2010). ORFs of Cluster_875 (co-occurrence of 0.81, Table 2) were annotated as proteins of Betaine/ Carnitine/Choline Transporter (BCCT) family. The BCCT family includes transporters for carnitine, choline and glycine betaine, and some of which exhibit osmosensory and osmoregulatory properties (Ziegler et al., 2010). Furthermore, the ORFs of Cluster_1740, annotated as ABC transporter ATP-binding proteins, were present only in these three halotolerant strains. The salt-induced ABC transporter Ota from Methanosarcina mazei Gö1 acts as a glycine betaine transporter (Schmidt et al., 2007). Another ABC transporter in Listeria, OpuC, is shown to be necessary for glycine betaine and choline chloride uptake (Verheul et al., 1997). Compared to the wild type of S. aureus, mutating OpuC did reduce their ability to grow under osmotic stress (10% NaCl; Kiran et al., 2009). The function of ORFs of Cluster_1740 and their contribution to halotolerance can be further characterized. Additionally, previous studies have shown that water permeability is clearly affected by the number of double bonds in the fatty acid conjugates of lipids, the higher the degree of unsaturation, the greater the water permeability (Graziani and Livne, 1972), and sterol type is one of the determining factors in the permeability of membranes to small solutes (Frallicciardi et al., 2022). The genomes of three halotolerant strains contain ORFs of Cluster_1548, annotated as sterol desaturase family proteins, indicating that sterols might be used to change permeability.

Cell signaling
Cluster_1549 also consists of three ORFs present in the three halotolerant strains, which showed similarity to the domain superfamily found in a large number of proteins including magnesium dependent endonucleases and phosphatases involved in intracellular signaling (Dlakic, 2000). Its role in the regulation of gene expression, such as triggering the salt-stress response, is worth of further study.

Polysaccharide
It has been reported that extracellular polysaccharides (EPS) may influence the salt tolerance of certain rhizobial strains (Samir and Kanak, 1997) and the lipopolysaccharide pattern could alter according to different salinities in a salt-tolerant strain of Mesorhizobium cicero (Soussi et al., 2001). All three halotolerant strains harbor ORFs annotated with polysaccharide/lipopolysaccharide biosynthesis (Cluster_2062, 2065(Cluster_2062, , 2067(Cluster_2062, , 2069(Cluster_2062, , 2071(Cluster_2062, , 2074(Cluster_2062, , and 2076 in Table 2), such as 3-deoxy-d-manno-octulosonate cytidylyltransferase, a key enzyme in the biosynthesis of lipopolysaccharide (LPS) in Gramnegative organisms (Yi et al., 2011). Furthermore, ORFs of Cluster_2473 (co-occurrence as 0.81 Table 2) were annotated to The tolerance of NaCl for all the six strains were investigated based on marine broth (MB). The strain Pontixanthobacter rizhaonensis KCTC 62828 T was excluded from this study, because it is tested on different medium (Liu et al., 2020(Liu et al., , 2021b. Frontiers in Microbiology 04 frontiersin.org  encode proteins of the GtrA family, whose members are often involved in the synthesis of cell surface polysaccharides (Kolly et al., 2015).

DNA repair
Open reading frames of Cluster_2677 are annotated encoding SOS response-associated peptidase family protein. The bacterial SOS response induced under stress conditions is recruited to DNA repair and adaptive mutagenesis (Shinagawa, 1996;Aravind et al., 2013). Hence, ORFs of Cluster_2677 could be further investigated for its importance to halotolerance.

Discussion
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.

Author contributions
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.