Strategies of Environmental Adaptation in the Haloarchaeal Genera Haloarcula and Natrinema

Academic Editor: Ricardo Amils Received: 27 February 2025 Revised: 19 March 2025 Accepted: 23 March 2025 Published: 27 March 2025 Citation: Straková, D.; Sánchez-Porro, C.; de la Haba, R.R.; Ventosa, A. Strategies of Environmental Adaptation in the Haloarchaeal Genera Haloarcula and Natrinema.Microorganisms 2025,13, 761. https://doi.org/10.3390/ microorganisms13040761 Copyright: © 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/). Article Strategies of Environmental Adaptation in the Haloarchaeal Genera Haloarcula and Natrinema Dáša Straková , Cristina Sánchez-Porro , Rafael R. de la Haba * and Antonio Ventosa * Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain; [email protected] (D.S.); [email protected] (C.S.-P.) *Correspondence: [email protected] (R.R.d.l.H.); [email protected] (A.V.) Abstract: Haloarchaea, a group of extremophilic archaea, thrive in hypersaline environments characterized not only by high salinity but also by other extreme conditions, such as intense UV radiation, high osmotic pressure, heavy metal contamination, oxidative stress, and fluctuating temperatures. This study investigates the environmental adaptation strategies of species of two genera, Haloarcula and Natrinema, the second and third largest haloarchaeal genera, respectively, after Halorubrum. Comparative genomic analyses were conducted on 48 species from both genera to elucidate their genomic diversity, metabolic potential, and stress-tolerance mechanisms. The genomes revealed diverse metabolic pathways, including rhodopsin-mediated phototrophy, nitrogen assimilation, and thiamine biosynthesis, which support their survival and adaptation to extreme conditions. The analysis identified mechanisms for oxidative stress mitigation, DNA repair, “salt-in” and “salt-out” osmoregulatory strategies, adaptations to temperature shifts and heavy metal exposure, and immune defense. Experimental validation of four representative species, Haloarcula terrestris S1AR25-5A T ,Haloarcula saliterrae S1CR25-12 T ,Haloarcula onubensis S3CR25-11 T , and Natrinema salsiterrestre S1CR25-10 T , isolated from the heavymetal-rich hypersaline soils in the Odiel Saltmarshes (Huelva, Spain), demonstrated their tolerance, especially to arsenic, corroborating genomic predictions. This study advances our understanding of the resilience of haloarchaea under poly-extreme conditions and underscores their ecological significance and promise for biotechnological applications, such as the bioremediation of heavy-metal-polluted environments and the production of valuable biomolecules. Keywords: environmental adaptation; haloarchaea; Haloarcula; heavy metal tolerance; hypersaline environments; Natrinema; thiamine biosynthesis 1. Introduction Haloarchaea, members of the class Halobacteria within the domain Archaea, are microorganisms thriving at the limits of life’s extreme conditions. These extremophiles inhabit hypersaline environments, such as salt flats, saline lakes, and salt mines, which are characterized by high salinity, intense solar radiation, fluctuating temperatures, oxygen availability, and toxic compounds [ 1 , 2 ]. These harsh conditions pose significant challenges to microbial survival, yet haloarchaea have evolved a range of unique adaptations that enable them to endure and prosper. Predominantly aerobic and chemoorganotrophic haloarchaea derive energy from the degradation of organic compounds [ 3 ]. However, they also exhibit metabolic versatility, using light energy through rhodopsin-based phototrophy or performing anaerobic respiration using alternative electron acceptors such as Microorganisms 2025,13, 761 https://doi.org/10.3390/microorganisms13040761 Microorganisms 2025,13, 761 2 of 24 nitrate [ 4 , 5 ]. Their ecological roles extend beyond hypersaline habitats, as they contribute to biogeochemical cycles, nutrient turnover, and ecosystem stability [ 6 , 7 ]. Resilience of haloarchaea to environmental stressors, including heavy metal toxicity, oxidative stress, and UV radiation, has positioned them as valuable candidates for biotechnological applications, such as bioremediation and bioplastics production [8–11]. Among haloarchaea, the genus Halorubrum is the largest, encompassing 40 species widely distributed across hypersaline environments [ 12 ]. Following Halorubrum, Haloarcula and Natrinema are the second and third largest genera within the class Halobacteria and are the focus of this study. Both genera exhibit diversity at multiple levels: species, genetic, metabolic, and ecological. The genus Haloarcula, belonging to the family Haloarculaceae , order Halobacteriales, was established in 1986 when Halobacterium vallismortis was reclassified as Haloarcula vallismortis [ 13 ]. Recent phylogenomic study [ 14 ] merged the genera Haloarcula and Halomicroarcula into a single genus, Haloarcula, to better reflect evolutionary relationships. Currently, the genus comprises 29 species ; however, recent revisions proposed synonymizing three species based on genomic evidence [ 15 , 16 ]. The genus Natrinema , part of the family Natrialbaceae, order Halobacteriales, was described in 1998 by reclassifying two strains previously assigned to Halobacterium [ 17 ]. However, almost simultaneously, another related genus, Haloterrigena , was proposed [ 18 ]. Taxonomic ambiguity between Natrinema and Haloterrigena was recently resolved through comparative genomic studies, leading to the reclassification of several Haloterrigena species into Natrinema [ 19 ]. With 22 recognized species, Natrinema demonstrates ecological and phylogenetic diversity, inhabiting hypersaline environments such as salt lakes, salterns, and hypersaline soils. Despite their adaptability, the mechanisms underlying haloarchaeal responses to extreme environmental stressors, such as high salinity, heavy metal exposure, and oxidative stress, remain areas of active research. The growing impacts of climate change, combined with pollution from industrialization, are contributing to elevated salinity levels mainly in water systems [ 8 ]. Harnessing the bioremediation potential of halophilic microorganisms presents a viable and innovative approach to addressing these challenges. The objective of this study was to investigate the adaptive mechanisms of two haloarchaeal genera, with a particular focus on their heavy metal tolerance. By experimental analysis of four representative species isolated from the hypersaline soils of the Odiel Saltmarshes—a habitat characterized by high salinity and elevated heavy metal concentrations—the research aimed to determine their ability to cope with heavy metal stress, particularly with arsenic. Additionally, this study was conducted to explore the metabolic functions, osmoregulatory strategies, and other environmental adaptations that enable these haloarchaea to thrive under poly-extreme conditions. Furthermore, the objective was to examine the potential for de novo thiamine biosynthesis in Haloarcula and Natrinema species, providing insights into their metabolic complexity. 2. Materials and Methods 2.1. Genomic and Metabolic Profiling A total of 48 genomic sequences of type strains, comprising 26 from Haloarcula species and 22 from Natrinema species, were retrieved from the NCBI GenBank database for analysis. The accession numbers of the genomes used are provided in Supplementary Table S1. The pangenome analysis was performed using the Enveomics toolkit v.1.0 [ 20 ]. To visualize core, variable (“shell”), and strain-specific genes, a flower plot was generated using the SRplot server [ 21 ], with the variable genes manually integrated into the visualization. Graphical depictions illustrating the evolution of both the pan-genomes and core-genomes for the genera Haloarcula and Natrinema were generated using the Pan-Genome Profile Microorganisms 2025,13, 761 3 of 24 Analyze Tool (PanGP) v.1.0.1, following the recommended algorithms [ 22 ]. Functional annotation of orthologous genes was carried out using the BlastKOALA tool v.3.0 [ 23 ], assigning KEGG Orthology (KO) numbers and mapping them to KEGG pathways and modules for reconstructing metabolic pathways. In addition, CRISPR-Cas systems were identified using the CRISPRCasFinder tool v. 4.2.21 [ 24 ]. The amino acid frequency and isoelectric points were determined from reading frames translated into protein sequences. The isoelectric points of predicted proteomes were computed with the “iep” tool from the EMBOSS package v.6.5.7.0 [ 25 ] and visualized with the R package “ggplot2” v.3.5.1 [ 26 ]. Amino acid frequencies were analyzed with the “Biostrings” v.2.74.1 package in R [ 27 ], and a radar (spider) plot representing the amino acid frequencies was generated using the R package “fmsb” v.0.7.6 [28]. 2.2. Assessment of Heavy Metal Tolerance The distribution of genes associated with arsenic tolerance within the genera Haloarcula and Natrinema was highlighted using approximately maximum-likelihood phylogenomic trees. They were reconstructed using FastTreeMP v.2.1.8 [ 29 ] based on 1599 and 1503 coreorthologous protein sequences, respectively. Branch support values (%) were computed with the Shimodaira–Hasegawa test. The susceptibility of four selected representatives (Haloarcula terrestris S1AR25-5AT , Haloarcula saliterrae S1CR25-12 T ,Haloarcula onubensis S3CR25-11 T , and Natrinema salsiterrestre S1CR25-10 T ), isolated from hypersaline soils in the Odiel Saltmarshes, located in Huelva, Southwest Spain, to five heavy metals (arsenic, cadmium, copper, lead, and zinc) was assessed. Cultures of these strains—incubated for seven days in Reasoner’s 2A (R2A) broth medium (Difco, Franklin Lakes, NJ, USA) supplemented with a 25% (w/v) seawater salt solution, prepared by diluting a 30% (w/v) stock solution following Subov [ 30 ]—were used to inoculate Petri dishes containing R2A 25% (w/v) agar medium. The seawater salt solution consisted of (g/L): NaCl, 195; MgCl 2· 6H 2 O, 32.5; MgSO 4· 7H 2 O, 50.8; CaCl2, 0.83 ; KCl, 5.0; NaHCO 3 , 0.17; and NaBr, 0.58. The agar medium was supplemented with varying concentrations of heavy metal salts, including C 2 H 6 AsNaO 2· 3H 2 O, CdCl2·H2O , CuSO 4 , Pb(C 2 H 3 O 2 ) 2· 3H 2 O, and ZnSO 4· 7H 2 O. Concentrations ranged from 0.01 mM to 700 mM (0.01, 0.05, 0.5, 1, 2.5, 4, 5, 10, 20, 50, 80, 100, 150, 200, 300, 500, 600, and 700 mM , respectively) [ 31 ]. The heavy metal solutions were sterilized through 0.2 µ m filter membranes before being added to the medium. The tolerance assays were conducted in duplicates. Following incubation at 37 ◦ C for 1 to 4 weeks, colony growth in media with each heavy metal was evaluated. The lowest concentration of each heavy metal that completely inhibited haloarchaeal growth was recorded as the minimum inhibitory concentration (MIC). R2A 25% (w/v) agar media without heavy metals were used as controls for each isolate. 3. Results and Discussion 3.1. Comparative Genomic Analysis and Pan-Genome Dynamics of the Genera Haloarcula and Natrinema We analyzed 26 genomes from species of the genus Haloarcula and 22 genomes from those of the genus Natrinema. Within the Haloarcula genomes, a total of 108,027 translated coding sequences (CDSs) were identified and categorized into 8835 orthologous gene clusters (OGs), comprising 1592 core OGs and 7243 variable OGs. Additionally, 8198 singleton gene clusters were detected, resulting in a pan-genome encompassing 17,033 distinct gene clusters. In the Natrinema genomes, 94,900 protein CDSs were identified and grouped into 8235 orthologous gene clusters, including 1664 core OGs and 6571 variable OGs. Furthermore, 9101 singleton gene clusters were identified, con- Microorganisms 2025,13, 761 4 of 24 tributing to a pan-genome comprising 17,336 different gene clusters. The flower plots in Figure 1illustrate the distribution of core, variable (shared by multiple but not all strains), and singleton (strain-specific) gene clusters across the species within the genera Haloarcula and Natrinema. Microorganisms 2025, 13, x FOR PEER REVIEW 4 of 26 Furthermore, 9101 singleton gene clusters were identified, contributing to a pan-genome comprising 17,336 different gene clusters. The flower plots in Figure 1 illustrate the distribution of core, variable (shared by multiple but not all strains), and singleton (strain-specific) gene clusters across the species within the genera Haloarcula and Natrinema. Figure 1. Flower plot showing the core (in the center), variable (in the annulus), and strain-specific (in the petals) genes of (A) the 26 Haloarcula species and (B) the 22 Natrinema species. The pan-genomes of Haloarcula and Natrinema exhibit continuous expansions with each additional genome, as demonstrated by the steep upward trend in the pan-genome size curves (Figure 2A,C). The ongoing identification of novel genes (Figure 2B,D) further supports this pattern. This indicates that both genera possess an open pan-genome, wherein the discovery of new genes persists as additional genomes are analyzed, reflecting a high degree of genetic diversity and a dynamic capacity for gene acquisition. Figure 1. Flower plot showing the core (in the center), variable (in the annulus), and strain-specific (in the petals) genes of (A) the 26 Haloarcula species and (B) the 22 Natrinema species. The pan-genomes of Haloarcula and Natrinema exhibit continuous expansions with each additional genome, as demonstrated by the steep upward trend in the pan-genome size curves (Figure 2A,C). The ongoing identification of novel genes (Figure 2B,D) further supports this pattern. This indicates that both genera possess an open pan-genome, wherein the discovery of new genes persists as additional genomes are analyzed, reflecting a high degree of genetic diversity and a dynamic capacity for gene acquisition. Microorganisms 2025, 13, x FOR PEER REVIEW 5 of 26 Figure 2. Gene accumulation curves of the pan-genomes (blue) and core-genomes (green) for species of the genera Haloarcula (A,B) and Natrinema (C,D). Panels A and C display the empirical gene accumulation curves, while panels B and D illustrate the least squares fit of the power law applied to the average values. The power law fit parameters and their respective coefficients of determination (R2) are shown for each dataset. 3.2. Metabolic Potential and Pathway Analysis 3.2.1. Carbohydrate Metabolism The primary metabolic pathways of Haloarcula and Natrinema species were elucidated through genome analysis and functional annotation using the BlastKOALA tool. The species of both genera have key carbohydrate metabolism pathways, including gluconeogenesis and the semi-phosphorylative Entner–Doudoroff glycolytic pathway, enabling the conversion of glucose and other hexoses into pyruvate for entry into the tricarboxylic acid cycle. The Embden–Meyerhof–Parnas glycolytic pathway was absent in most species, with the exception of four Natrinema species (Supplementary Table S2). In addition, the species of both genera exhibit metabolic versatility, utilizing alternative carbon sources, like glycerol. Glycerol metabolism can proceed through phosphorylation by glycerol kinase to form sn-glycerol-3-phosphate (G3P), followed by its oxidation to dihydroxyacetone phosphate (DHAP) via G3P dehydrogenase, or through oxidation by glycerol dehydrogenase to dihydroxyacetone (DHA), which is subsequently phosphorylated by DHA kinase to form DHAP [32]. The G3P pathway was present in all Haloarcula and Natrinema species, except for Natrinema salifodinaeae CGMCC 1.12284T (Supplementary Table Figure 2. Cont. Microorganisms 2025,13, 761 5 of 24 Microorganisms 2025, 13, x FOR PEER REVIEW 5 of 26 Figure 2. Gene accumulation curves of the pan-genomes (blue) and core-genomes (green) for species of the genera Haloarcula (A,B) and Natrinema (C,D). Panels A and C display the empirical gene accumulation curves, while panels B and D illustrate the least squares fit of the power law applied to the average values. The power law fit parameters and their respective coefficients of determination (R2) are shown for each dataset. 3.2. Metabolic Potential and Pathway Analysis 3.2.1. Carbohydrate Metabolism The primary metabolic pathways of Haloarcula and Natrinema species were elucidated through genome analysis and functional annotation using the BlastKOALA tool. The species of both genera have key carbohydrate metabolism pathways, including gluconeogenesis and the semi-phosphorylative Entner–Doudoroff glycolytic pathway, enabling the conversion of glucose and other hexoses into pyruvate for entry into the tricarboxylic acid cycle. The Embden–Meyerhof–Parnas glycolytic pathway was absent in most species, with the exception of four Natrinema species (Supplementary Table S2). In addition, the species of both genera exhibit metabolic versatility, utilizing alternative carbon sources, like glycerol. Glycerol metabolism can proceed through phosphorylation by glycerol kinase to form sn-glycerol-3-phosphate (G3P), followed by its oxidation to dihydroxyacetone phosphate (DHAP) via G3P dehydrogenase, or through oxidation by glycerol dehydrogenase to dihydroxyacetone (DHA), which is subsequently phosphorylated by DHA kinase to form DHAP [32]. The G3P pathway was present in all Haloarcula and Natrinema species, except for Natrinema salifodinaeae CGMCC 1.12284T (Supplementary Table Figure 2. Gene accumulation curves of the pan-genomes (blue) and core-genomes (green) for species of the genera Haloarcula (A,B) and Natrinema (C,D). Panels A and C display the empirical gene accumulation curves, while panels B and D illustrate the least squares fit of the power law applied to the average values. The power law fit parameters and their respective coefficients of determination (R2) are shown for each dataset. 3.2. Metabolic Potential and Pathway Analysis 3.2.1. Carbohydrate Metabolism The primary metabolic pathways of Haloarcula and Natrinema species were elucidated through genome analysis and functional annotation using the BlastKOALA tool. The species of both genera have key carbohydrate metabolism pathways, including gluconeogenesis and the semi-phosphorylative Entner–Doudoroff glycolytic pathway, enabling the conversion of glucose and other hexoses into pyruvate for entry into the tricarboxylic acid cycle. The Embden–Meyerhof–Parnas glycolytic pathway was absent in most species, with the exception of four Natrinema species (Supplementary Table S2). In addition, the species of both genera exhibit metabolic versatility, utilizing alternative carbon sources, like glycerol. Glycerol metabolism can proceed through phosphorylation by glycerol kinase to form sn-glycerol-3phosphate (G3P), followed by its oxidation to dihydroxyacetone phosphate (DHAP) via G3P dehydrogenase, or through oxidation by glycerol dehydrogenase to dihydroxyacetone (DHA), which is subsequently phosphorylated by DHA kinase to form DHAP [ 32 ]. The G3P pathway was present in all Haloarcula and Natrinema species, except for Natrinema salifodinaeae CGMCC 1.12284 T (Supplementary Table S2). Furthermore, a fructose-specific phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) was found in most Haloarcula species and a few Natrinema species, facilitating fructose uptake and metabolism [ 33 , 34 ]. Moreover, gat genes involved in galactitol (dulcitol) metabolism as part of the bacterial-type PTS system [ 35 ] were identified in Haloarcula amylovorans LR21 T (Supplementary Table S2). Both genera also harbored genes for synthesizing polyhydroxyalkanoates (PHAs), biopolymers produced during nutrient-limited conditions that serve as carbon storage, offering potential for sustainable plastic alternatives [36–40]. 3.2.2. Nitrogen Metabolism Genes encoding glutamine synthetase, glutamate synthase, and glutamate dehydrogenase, which are involved in ammonia assimilation [ 41 ], were identified in all genomes. Additionally, urease gene clusters (ureABC and ureDEFGH), responsible for urea hydrolysis into ammonia and CO 2 [ 42 ], were present in most Haloarcula species and a few Natrinema species. Some species possessed assimilatory nitrate and nitrite reductases, enabling nitrate utilization when ammonia or urea are scarce, enhancing adaptability in nitrogen-fluctuating Microorganisms 2025,13, 761 6 of 24 environments. Furthermore, the majority of Natrinema species and half of the Haloarcula species encoded nitroalkane oxidase, catalyzing nitroalkanes to nitrite [ 43 ], which can be further reduced to ammonia. Nitrilase was identified in some Haloarcula species and Natrinema zhouii YPL30 T , while formamidase and cyanate lyase were found in specific Haloarcula species (Supplementary Table S2), reflecting metabolic flexibility in utilizing various nitrogen sources and detoxifying harmful compounds. 3.2.3. Transporters The metabolic versatility is also supported by a variety of transporters. Multiple ABC transporters for sugars, phosphates, amino acids, and metals were prevalent across both genera (Supplementary Table S2). Transport systems for glucose/mannose and arabinogalactan oligomers were particularly abundant in Haloarcula. Ammonia transporters were detected across all genomes. A urea transporter (UrtABCDE) was identified in Haloarcula species containing urease genes, although only three Natrinema species encoded an active urea transport system. Natrinema species containing urease but lacking the urtABCDE gene cluster may have adapted to environments with high urea concentrations, allowing urea to enter the cell through passive diffusion [ 44 ]. Phosphate ABC transporters were present across all genomes, often appearing in multiple copies. Approximately half of the Natrinema and Haloarcula genomes also contained genes encoding a phosphonate ABC transporter. The TupABC tungstate transport system was present in all Natrinema species except for Natrinema marinum DT87 T . No Haloarcula species originally classified under Halomicroarcula and later reassigned to Haloarcula possessed an ABC tungstate transport system (Supplementary Table S2). This system is crucial for the high-affinity uptake of tungstate ions (WO 42− ), which are essential for tungsten-dependent enzymes, such as aldehyde ferredoxin oxidoreductase and formate dehydrogenase, involved in anaerobic metabolism and energy conservation [ 45 ]. Additionally, zinc ABC transporters were detected in all Natrinema genomes and in most Haloarcula genomes. In contrast, the CbiMNQO cobalt/nickel transport system was less common, found in some Haloarcula species and only two Natrinema species (Supplementary Table S2). 3.2.4. Phototrophy In addition, rhodopsins, light-driven proteins critical for phototrophic growth, osmoregulation, and environmental sensing, were highly represented in both genera, especially Haloarcula (Figure 3, Supplementary Table S2). Bacteriorhodopsin, a light-driven proton pump, facilitates energy generation, while halorhodopsins help maintain osmotic balance by importing chloride ions. Sensory rhodopsins enable phototaxis, guiding movement toward or away from light sources [ 46 ]. The presence of the rhodopsins highlights the capability of these haloarchaea to harness light energy, supporting survival in hypersaline habitats and offering potential for various biotechnological applications, including optogenetics, bioelectronics, and bioenergy [47,48]. 3.3. Osmoregulatory Mechanisms and Proteomic Adaptations 3.3.1. “Salt-In” Strategy Haloarchaea employ a variety of mechanisms to maintain cellular homeostasis in response to fluctuating osmotic conditions. A key strategy involves the regulation of ion transporters, which control the movement of ions across the cell membrane. By adjusting intracellular ion concentrations, haloarchaea mitigate osmotic stress and maintain optimal cellular function. Both Natrinema and Haloarcula possessed specialized transporters that mediate K + uptake and Na + expulsion, crucial components of the “salt-in” strategy for maintaining osmotic balance in hypersaline environments (Figure 4). Additionally, Cl − transporters, including chloride channels and halorhodopsins, play complementary roles. Microorganisms 2025,13, 761 7 of 24 While chloride channels passively regulate chloride influx to maintain osmotic balance, halorhodopsins actively pump chloride ions into the cytoplasm using light energy, contributing to energy production [ 49 , 50 ]. Halorhodopsins were found in the majority of species from both genera (Figure 3), while a chloride channel was identified exclusively in Haloarcula limicola JCM 18640 T (Supplementary Table S2). To cope with hypoosmotic shock, small-conductance mechanosensitive channels (MscS) release ions and small compatible solutes to prevent cell lysis [ 51 , 52 ]. The mscS genes involved in this mechanism were identified in some species of Natrinema and Haloarcula (Figure 4, Supplementary Table S2), allowing them to survive sudden decreases in salinity. Microorganisms 2025, 13, x FOR PEER REVIEW 7 of 26 by importing chloride ions. Sensory rhodopsins enable phototaxis, guiding movement toward or away from light sources [46]. The presence of the rhodopsins highlights the capability of these haloarchaea to harness light energy, supporting survival in hypersaline habitats and offering potential for various biotechnological applications, including optogenetics, bioelectronics, and bioenergy [47,48]. Figure 3. Distribution of genes encoding bacteriorhodopsin, halorhodopsin, and sensory rhodopsin in species of the genera Haloarcula and Natrinema. 3.3. Osmoregulatory Mechanisms and Proteomic Adaptations 3.3.1. “Salt-In” Strategy Haloarchaea employ a variety of mechanisms to maintain cellular homeostasis in response to fluctuating osmotic conditions. A key strategy involves the regulation of ion transporters, which control the movement of ions across the cell membrane. By adjusting intracellular ion concentrations, haloarchaea mitigate osmotic stress and maintain optimal cellular function. Both Natrinema and Haloarcula possessed specialized transporters that mediate K⁺ uptake and Na⁺ expulsion, crucial components of the “salt-in” strategy for maintaining osmotic balance in hypersaline environments (Figure 4). Additionally, Cl⁻ transporters, including chloride channels and halorhodopsins, play complementary roles. While chloride channels passively regulate chloride influx to maintain osmotic balance, halorhodopsins actively pump chloride ions into the cytoplasm using light energy, contributing to energy production [49,50]. Halorhodopsins were found in the majority of species from both genera (Figure 3), while a chloride channel was identified exclusively in Haloarcula limicola JCM 18640T (Supplementary Table S2). To cope with hypoosmotic shock, small-conductance mechanosensitive channels (MscS) release ions and small compatible solutes to prevent cell lysis [51,52]. The mscS genes involved in this mechanism were identified in some species of Natrinema and Haloarcula (Figure 4, Supplementary Table S2), allowing them to survive sudden decreases in salinity. 0 1 2 3 4 5 6 7 T H. saliterrae S1CR25-12 T H. onubensis S3CR25-11 T H. laminariae LYG-108 T H. rara SHR3 T H. salinisoli F24A T H. marina DT1 T H. pellucida CECT 7537 T H. rubra F13 T H. litorea GDY20 T H. amylovorans LR21 T H. limicola JCM 18640 T H. nitratireducens F27 T H. pelagica YJ-61-S T H. halophila DFY41 T H. halobia XH51 T H. ordinaria ZS-22-S1 T H. salina JCM 18369 T H. terrestris S1AR25-5A T H. mannanilytica MD130-1 T H. vallismortis ATCC 29715 T H. amylolytica JCM 13557 T H. hispanica ATCC 33960 T H. marismortui ATCC 43049 T H. japonica DSM 6131 T H. sebkhae JCM 19018 T H. argentinensis DSM 12282 T N. salifodinae CGMCC 1.12284 T N. limicola JCM 13563 T N. hispanicum DSM 18328 T N. marinum DT87 T N. halophilum YPL8 T N. gelatinilyticum BND6 T N. caseinilyticum ZJ2 T N. soli DC36 T N. zhouii YPL30 T N. mahii H13 T N. saccharevitans AB14 T N. pellirubrum DSM 15624 T N. thermotolerans DSM 11552 T N. salaciae DSM 25055 T N. longum ABH32 T N. salinisoli SLN56 T N. salsiterrestre S1CR25-10 T N. versiforme JCM 10478 T N. amylolyticum LT61 T N. gari JCM 14663 T N. altunense AJ2 T N. pallidum DSM 3751 Ortholog copy numbers bacteriorhodopsin halorhodopsin sensory rhodopsin Figure 3. Distribution of genes encoding bacteriorhodopsin, halorhodopsin, and sensory rhodopsin in species of the genera Haloarcula and Natrinema. Microorganisms 2025, 13, x FOR PEER REVIEW 9 of 26 Figure 4. Heatmap of genes associated with “salt-in” and “salt-out” osmoregulatory mechanisms in Haloarcula and Natrinema species. 3.3.3. Proteome and Genomic Adaptation Adaptations of haloarchaea to high-salt environments further involve both acidification of their proteome and an increased genomic G+C content [61]. Proteome acidification is achieved through a higher proportion of acidic amino acids, particularly aspartate (D) and glutamate (E). These acidic residues contribute to the surface charge of proteins, enhancing water retention and counteracting the destabilizing effects of high ionic concentrations. This adaptation ensures proper protein folding and functionality under extreme salinity, preventing aggregation and maintaining stability under osmotic stress [9]. In the studied genomes, aspartate and glutamate were prominently represented in both genera, particularly in Natrinema species, following alanine and leucine as the most abundant amino acids (Figure 5). Furthermore, both genera exhibited a high genomic G+C content, ranging from 60.1 to 65.9 mol%. While high G+C content is primarily associated with thermophilic adaptation, it also contributes to genomic stability in haloarchaea, which often face elevated temperatures and intense solar radiation in hypersaline environments. Additionally, G+C content can indirectly influence protein stability by shaping codon usage and amino acid composition [62]. Proteins encoded by G+C-rich genes may exhibit a higher prevalence of specific amino acids that enhance structural stability, aiding survival in high-salt conditions. Figure 4. Heatmap of genes associated with “salt-in” and “salt-out” osmoregulatory mechanisms in Haloarcula and Natrinema species. Microorganisms 2025,13, 761 8 of 24 3.3.2. “Salt-Out” Strategy Another key adaptation to osmotic stress is the accumulation of compatible solutes—part of the “salt-out” strategy—which stabilizes proteins and maintains cellular function without interfering with metabolic processes [ 53 ]. Our analysis showed that both genera contained genes for transport and de novo synthesis of compatible solutes. Previous research has shown that the organic solutes trehalose and glycine betaine are universally present in extremely halophilic archaea, obtained either through de novo biosynthesis or uptake from external sources [ 54 ]. Our genomic analysis revealed transport systems for a variety of solutes, including proline, choline, glycine betaine, proline betaine, and trehalose (Figure 4, Supplementary Table S2). The trehalose biosynthesis pathway (OtsAB) was detected in all Natrinema species, although it was less prevalent in Haloarcula. Conversely, a trehalose transporter was present in all Natrinema and Haloarcula species except Haloarcula ordinaria ZS-22-S1 T . The proline biosynthesis pathway from glutamate was identified in all Natrinema species and in twelve Haloarcula species [ 55 ]. The opuABC gene cluster—which encodes an ABC transport system involved in the uptake of glycine betaine, proline betaine, and other small organic molecules—was present in Natrinema versiforme JCM 10478 T and several Haloarcula species, including H. amylovorans LR21 T ,H. pelagica YJ-61-S T ,H. halophila DFY41 T , H. salina JCM 18369 T ,H. terrestris S1AR25-5A T , and H. mannanilytica MD130-1 T (Figure 4, Supplementary Table S2). The biosynthesis pathway of glycine betaine from choline, mediated by BetAB [ 56 ], was not identified in any of the Haloarcula or Natrinema species. Similarly, the de novo ectoine biosynthesis pathway appeared incomplete. Aspartate kinase and aspartate semialdehyde dehydrogenase, which are involved in the initial steps of ectoine production, were present in all the studied genomes. However, these enzymes also participate in the formation of threonine, methionine, and isoleucine [ 57 ]. The ectB gene was detected in ten Haloarcula species and all Natrinema species, with multiple copies found in most Natrinema genomes. In contrast, the ectA and ectC genes were absent in all the studied genomes, along with ectD, which is responsible for converting ectoine into 5-hydroxyectoine, often considered a superior compatible solute due to its enhanced hydration and antioxidant properties [ 58 , 59 ]. In terms of glucosylglycerate biosynthesis, all the Natrinema species and approximately half of the Haloarcula species carried the gpgS gene (Figure 4, Supplementary Table S2) which encodes glucosyl-3-phosphoglycerate synthase, responsible for the first step of the pathway. However, none of the species contained the gpgP gene, encoding glucosyl-3-phosphoglycerate phosphatase, which converts glucosyl3-phosphoglycerate to glucosylglycerate, another compatible solute [ 60 ]. This absence suggests the potential use of alternative enzymes, non-specific phosphatases, or reliance on other osmoregulatory mechanisms. Comprehensive genomic analysis suggests that Haloarcula and Natrinema demonstrate adaptability in osmoregulatory mechanisms, likely employing both “salt-in” and “salt-out” strategies in response to varying osmotic stress levels and resource availability. 3.3.3. Proteome and Genomic Adaptation Adaptations of haloarchaea to high-salt environments further involve both acidification of their proteome and an increased genomic G+C content [ 61 ]. Proteome acidification is achieved through a higher proportion of acidic amino acids, particularly aspartate (D) and glutamate (E). These acidic residues contribute to the surface charge of proteins, enhancing water retention and counteracting the destabilizing effects of high ionic concentrations. This adaptation ensures proper protein folding and functionality under extreme salinity, preventing aggregation and maintaining stability under osmotic stress [ 9 ]. In the studied genomes, aspartate and glutamate were prominently represented in both genera, particularly in Natrinema species, following alanine and leucine as the most abundant amino Microorganisms 2025,13, 761 9 of 24 acids (Figure 5). Furthermore, both genera exhibited a high genomic G+C content, ranging from 60.1 to 65.9 mol%. While high G+C content is primarily associated with thermophilic adaptation, it also contributes to genomic stability in haloarchaea, which often face elevated temperatures and intense solar radiation in hypersaline environments. Additionally, G+C content can indirectly influence protein stability by shaping codon usage and amino acid composition [ 62 ]. Proteins encoded by G+C-rich genes may exhibit a higher prevalence of specific amino acids that enhance structural stability, aiding survival in high-salt conditions. Microorganisms 2025, 13, x FOR PEER REVIEW 10 of 26 Figure 5. Amino acid frequency of the species of the genera (A) Haloarcula and (B) Natrinema. The acid–base balance of the proteome is further visualized by the isoelectric point (pI) distribution. A shift towards acidic pI values (~4) indicates a predominance of acidic residues, consistent with the “salt-in” strategy for osmoregulation, while higher pI values (above 7) correspond to basic proteins [61]. The isoelectric point profiles of species of both Haloarcula and Natrinema revealed a predominantly acidic proteome, with major peaks around pI 4 (Figure 6); however, the extended distribution toward higher pI values indicates the presence of basic proteins, such as DNA-binding proteins, transporters, and membrane-associated proteins, which require a positive charge for proper function or interaction with cellular components. In summary, the bimodal distribution of isoelectric points reflects a balance between the adaptation of haloarchaea to hypersaline environments via proteome acidification and the functional necessity for basic proteins in specific cellular processes. Figure 5. Amino acid frequency of the species of the genera (A)Haloarcula and (B)Natrinema. The acid–base balance of the proteome is further visualized by the isoelectric point (pI) distribution. A shift towards acidic pI values (~4) indicates a predominance of acidic residues, consistent with the “salt-in” strategy for osmoregulation, while higher pI values (above 7) correspond to basic proteins [ 61 ]. The isoelectric point profiles of species of both Haloarcula and Natrinema revealed a predominantly acidic proteome, with major peaks around pI 4 (Figure 6); however, the extended distribution toward higher pI values indicates the presence of basic proteins, such as DNA-binding proteins, transporters, and membraneassociated proteins, which require a positive charge for proper function or interaction with cellular components. In summary, the bimodal distribution of isoelectric points reflects a balance between the adaptation of haloarchaea to hypersaline environments via proteome acidification and the functional necessity for basic proteins in specific cellular processes. Microorganisms 2025,13, 761 16 of 24 the activity of Thi4, a eukaryotic-like protein homolog, along with a presumed NUDIX hydrolase. Once synthesized, the thiamine ring precursors, 4-methyl-5-[ β -hydroxyethyl] thiazole phosphate (THZ-P) and HMP-PP, are combined to form thiamine monophosphate (ThMP) through the catalytic action of a ThMP synthase, either of the ThiE or ThiN type [ 105 – 107 ]. ThMP is subsequently phosphorylated by ThiL to produce the biologically active coenzyme, ThDP [108]. The functional genomic analysis revealed that the thiC gene, encoding the enzyme responsible for the initial step in aminopyrimidine moiety synthesis (Figure 9), was present in all Natrinema species and in the majority of Haloarcula members ( Supplementary Table S2 ). The absence of thiC in some Haloarcula species results in an incomplete thiamine biosynthetic pathway. In contrast, the thiDN gene was identified in all the studied species. Haloarchaea possess Thi4 homologs containing a conserved active-site cysteine residue, which is essential for the synthesis of adenylated thiazole (ADT) through a single-turnover reaction involving the conversion of nicotinamide adenine dinucleotide (NAD) and glycine [ 109 ]. These functional homologs were detected across all the species under study ( Supplementary Tables S2 and S4 ). The formation of ThMP is catalyzed by either ThiE or ThiN, with the ThiN domain often occurring as part of the fusion protein ThiDN. The archaeal ThiDN protein is a multifunctional enzyme capable of catalyzing three consecutive steps in the de novo synthesis of vitamin B 1 [ 105 ] (Figure 9). Interestingly, certain extremophilic microorganisms, such as Pyrococcus furiosus and Sulfolobus solfataricus, possess both thiDN and thiE genes, indicating coexisting pathways for thiamine monophosphate synthesis [ 105 ]. In the current study, both thiDN and thiE genes were detected in all the analyzed species, suggesting evolutionary advantages such as enhanced metabolic flexibility and adaptation to fluctuating environmental conditions ( Supplementary Table S2 ). The enzyme ThiL, responsible for catalyzing the final step in ThDP formation, was found in all the studied haloarchaea. Additionally, adenylate kinase (Adk) facilitates the phosphorylation of ThDP to produce thiamine triphosphate (ThTP) (Figure 9). ThTP, a multifunctional molecule associated with energy metabolism, stress response, and cellular signaling [ 110 , 111 ], however, remains poorly understood in archaea. ThiR, identified in all species of both genera, is a central regulatory protein ensuring balanced thiamine biosynthesis and transport depending on environmental and intracellular conditions [ 104 , 106 ]. Taken together, the findings indicate that all the studied Natrinema species and the majority of Haloarcula species possessing the thiC gene have the potential for de novo thiamine biosynthesis. In addition to the biosynthetic pathway, the thiamine salvage pathway plays a critical role in minimizing energy consumption and preventing the incorporation of thiamine degradation products into ThDP-dependent enzymes. This pathway is anticipated to involve enzymes from de novo synthesis (ThiDN, ThiE, and ThiL) alongside salvage-specific enzymes such as ThiM and TenA [ 107 ]. The thiM gene, responsible for the phosphorylation of 4-methyl-5-( β -hydroxyethyl) thiazole (THZ), was detected in all Natrinema species but only in two Haloarcula members (Supplementary Table S2). This absence in most Haloarcula species might indicate a reliance on an external thiamine supply or de novo synthesis. In contrast, the tenA gene, involved in salvaging base-degraded thiamine derivatives [ 107 , 112 ], was more prevalent in Haloarcula species than in Natrinema representatives (Supplementary Table S2). Additionally, alkaline phosphatase (PhoA), which supports thiamine recycling and the salvage pathway, was detected in only a few species, predominantly within the genus Natrinema. The transport of thiamine, its phosphorylated derivatives, and intermediates is facilitated by the thiamine transport system, comprising ThiB (K02064), ThiP (K02063), and ThiQ (K02062) subunits [ 113 ] (Figure 9), ensuring adaptability to environmental nutrient availability. The thiB gene was present in all Haloarcula and Natrinema Microorganisms 2025,13, 761 17 of 24 species; while thiP was not annotated for H. rara SHR3 T ; and thiQ was absent in N. limicola JCM 13563 T ,N. hispanicum DSM 18328 T , and N. marinum DT87 T (Supplementary Table S2). Microorganisms 2025, 13, x FOR PEER REVIEW 18 of 26 synthesis. In contrast, the tenA gene, involved in salvaging base-degraded thiamine derivatives [107,112], was more prevalent in Haloarcula species than in Natrinema representatives (Supplementary Table S2). Additionally, alkaline phosphatase (PhoA), which supports thiamine recycling and the salvage pathway, was detected in only a few species, predominantly within the genus Natrinema. The transport of thiamine, its phosphorylated derivatives, and intermediates is facilitated by the thiamine transport system, comprising ThiB (K02064), ThiP (K02063), and ThiQ (K02062) subunits [113] (Figure 9), ensuring adaptability to environmental nutrient availability. The thiB gene was present in all Haloarcula and Natrinema species; while thiP was not annotated for H. rara SHR3T; and thiQ was absent in N. limicola JCM 13563T, N. hispanicum DSM 18328T, and N. marinum DT87T (Supplementary Table S2). Figure 9. Biosynthesis and salvage pathways of thiamine (vitamin B1) in Haloarcula and Natrinema species based on functional genomic analysis. The presence of enzymes in the studied Haloarcula and Natrinema species is represented by colored symbols, with designations provided in the figure legend. ADP-thiazole, ADP-5-ethyl-4-methylthiazole-2-carboxylate; AIR, 5′-phosphoribosyl-5-aminoimidazole; Cys, cysteine; DHAla, dehydroalanine; Gly, glycine; HMP, 4-amino-5-hydroxymethyl-2-methylpyrimidine; HMP-P, 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate; HMP-PP, 4-amino-5-hydroxymethyl-2-methylpyrimidine diphosphate; NAD, nicotinamide adenine dinucleotide; NAM, nicotinamide; ThMP, thiamine monophosphate; ThDP, thiamine diphosphate; ThTP, thiamine triphosphate; THZ, 4-methyl-5-(β-hydroxyethyl) thiazole; THZ-P, 4-methyl5-(β-hydroxyethyl) thiazole phosphate. The enzymes are discussed in the text. Figure 9. Biosynthesis and salvage pathways of thiamine (vitamin B 1 ) in Haloarcula and Natrinema species based on functional genomic analysis. The presence of enzymes in the studied Haloarcula and Natrinema species is represented by colored symbols, with designations provided in the figure legend. ADP-thiazole, ADP-5-ethyl-4-methylthiazole-2-carboxylate; AIR, 5′-phosphoribosyl - 5-aminoimidazole ; Cys, cysteine; DHAla, dehydroalanine; Gly, glycine; HMP, 4-amino - 5-hydroxymethyl - 2-methylpyrimidine ; HMP-P , 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate; HMP-PP, 4-amino - 5-hydroxymethyl - 2-methylpyrimidine diphosphate; NAD, nicotinamide adenine dinucleotide; NAM, nicotinamide; ThMP, thiamine monophosphate; ThDP, thiamine diphosphate; ThTP, thiamine triphosphate; THZ, 4-methyl - 5-(β-hydroxyethyl) thiazole; THZ-P, 4-methyl-5-( β -hydroxyethyl) thiazole phosphate. The enzymes are discussed in the text. 3.7. Research Implications This study provides valuable insights into stress adaptation and metabolic versatility in Haloarcula and Natrinema, while also emphasizing their potential for various biotechnological applications. The characterization of heavy metal resistance mechanisms, particularly the exceptional arsenic tolerance observed in some species, with MIC values reaching up to 700 mM, highlights the potential of these haloarchaea for the bioremediation of arseniccontaminated environments. These microorganisms also show promise for the production of valuable biomolecules, offering sustainable alternatives to traditional methods. For instance, they harbor genes involved in the biosynthesis of polyhydroxyalkanoates (PHAs), a class of biodegradable and biocompatible biopolymers with thermoplastic properties. Microorganisms 2025,13, 761 18 of 24 PHAs have gained attention as sustainable alternatives to conventional plastics, with potential applications in packaging, medical implants, and drug delivery systems [ 39 , 40 ]. While challenges remain in optimizing PHA production at an industrial scale, haloarchaea offer advantages due to their ability to grow in high-salinity conditions, reducing the risk of contamination. Furthermore, the presence of rhodopsin-related genes in the studied Haloarcula and Natrinema species highlights their potential in optogenetics, bioelectronics, and bioenergy [ 47 , 48 ]. Carotenoids such as bacterioruberin and β -carotene, identified in the studied species through functional genomic analysis, have broad applications in the food and cosmetic industries, as well as in biomedicine, where they serve as antioxidants, anti-tumor agents, cardiovascular protectants, and vitamin A precursors [ 114 ]. Furthermore, the discovery of de novo thiamine synthesis in most of the studied species presents potential applications in biotechnology. Thiamine is essential for supporting the nervous and cardiovascular systems, strengthening the immune response, and enhancing cognitive functions [115]. It has potential in drug development, particularly in targeting cancer and fungal infections [ 116 ]. While vitamin B 1 is predominantly produced through chemical synthesis on an industrial scale, elucidating its biosynthetic pathway in haloarchaea may enable future biotechnological production. Additionally, thiamine plays a role in nutrient cycling and could contribute to improving crop resistance [ 107 ]. Finally, the present work provides a foundation for future studies, integrating transcriptomics and proteomics approaches to validate the genomic predictions and further elucidate the regulatory networks underlying these adaptive mechanisms. Gaining deeper insights into these processes will not only expand our fundamental understanding of haloarchaeal physiology but also contribute to the development of sustainable biotechnological applications. 4. Conclusions This study provides a comprehensive analysis of the environmental adaptation strategies in the haloarchaeal genera Haloarcula and Natrinema, focusing on their genomic diversity, metabolic capabilities, and mechanisms for stress resistance. The findings reveal adaptive mechanisms, such as DNA repair systems, antioxidant and immune defenses, “salt-in” and “salt-out” osmoregulatory strategies, protection against thermal shifts, and heavy metal detoxification pathways, which enable these extremophiles to thrive under poly-extreme conditions. Comparative genomic analysis highlights their metabolic versatility, including pathways for phototrophy, nitrogen assimilation, and thiamine biosynthesis, which demonstrate their ecological resilience and potential for biotechnological applications. In particular, the elucidation of heavy metal resistance mechanisms offers a valuable basis for exploring haloarchaea in sustainable bioremediation strategies for heavy metalcontaminated environments. The identification of genes encoding efflux transporters, reductases, regulatory proteins, and other components of heavy metal resistance pathways underscores the genomic plasticity and adaptability of the Haloarcula and Natrinema genera. Experimental validation of four representative species confirmed their exceptional heavy metal tolerance, especially to arsenic. These findings provide a foundation for future research on haloarchaeal physiology and their optimization for potential biotechnological applications. Integrating transcriptomics and proteomics approaches to validate these genomic predictions can further elucidate the regulatory networks governing these adaptive mechanisms. Furthermore, this knowledge may contribute to the development of sustainable strategies to address environmental challenges, including heavy metal contamination, plastic pollution, increasing salinization, and the need for sustainable production of valuable biomolecules. Microorganisms 2025,13, 761 19 of 24 Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/microorganisms13040761/s1, Table S1: Genome accession numbers of species of Haloarcula and Natrinema used in the study; Table S2: Overview of KEGG Modules and KEGG Orthology (KO) numbers and their associated definitions as annotated within the genomes of Haloarcula (A) and Natrinema (B) species; Table S3: CRISPR-Cas system in species of the genera Haloarcula and Natrinema; Table S4: Alignment of the active-site region of protein sequences of Thi4 homologs encoded by thi4 genes across species of the genera Haloarcula and Natrinema. Author Contributions: Conceptualization, A.V., R.R.d.l.H. and C.S.-P.; methodology, D.S., C.S.-P. and R.R.d.l.H.; software, D.S. and R.R.d.l.H.; validation, D.S.; formal analysis, investigation, resources, and data curation, D.S., C.S.-P., R.R.d.l.H. and A.V.; writing—original draft preparation, D.S.; writing—review and editing, D.S., C.S.-P., R.R.d.l.H. and A.V.; visualization, D.S.; supervision, R.R.d.l.H. and A.V.; project administration, C.S.-P. and A.V.; funding acquisition, C.S.-P. and A.V. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by grant numbers PID2020-118136GB-I00 and PID2023148654NB-I00 (to A.V. and C.S.-P.) funded by MICIU/AEI/10.13039/501100011033 and by ERDF/EU. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are openly available in GenBank/EMBL/DDBJ databases at https://www.ncbi.nlm.nih.gov/genbank/ (accessed on 14 August 2024 ), reference numbers included in the Supplementary Materials. Conflicts of Interest: The authors declare no conflicts of interest. References 1. Ventosa, A. Unusual micro-organisms from unusual habitats: Hypersaline environments. In Prokaryotic Diversity: Mechanisms and Significance; Logan, N.A., Lappin-Scott, H.M., Oyston., P.C.F., Eds.; Cambridge University Press: Cambridge, UK, 2006; pp. 223–254. [CrossRef] 2. DasSarma, S.; DasSarma, P.; Laye, V.; Schwieterman, E. Extremophilic models for astrobiology: Haloarchaeal survival strategies and pigments for remote sensing. Extremophiles 2019,24, 31–41. [CrossRef] [PubMed] 3. Falb, M.; Müller, K.; Königsmaier, L.; Oberwinkler, T.; Horn, P.; von Gronau, S.; Gonzalez, O.; Pfeiffer, F.; Bornberg-Bauer, E.; Oesterhelt, D. Metabolism of halophilic archaea. 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