Halocin H4 is activated through cleavage by halolysin HlyR4.

Halocins are antimicrobial peptides secreted by haloarchaea capable of inhibiting the growth of other haloarchaea or bacteria. Halocin H4 (HalH4) is secreted by the model halophilic archaeon Haloferax mediterranei ATCC 33500. Despite attempts to express halH4 heterologously in Escherichia coli and subsequent careful renaturation procedures commonly employed for haloarchaeal proteins, no active halocin was obtained. However, it was discovered that the antihaloarchaeal activity of this halocin could be activated through cleavage by halolysin R4 (HlyR4), a serine protease also secreted by Hfx. mediterranei ATCC 33500. Replacement of the cysteine at the number 115 amino acid with glycine and deletion of the internal trans-membrane region (15 aa) markedly abolished HalH4's antihaloarchaeal activity. Compared to the N-terminus, the C-terminal amino acid sequence was found to be more crucial for HalH4 to exert its antihaloarchaeal activity. Mass spectrometry analysis revealed that the biologically active antihaloarchaeal peptide produced after hydrolytic cleavage by HlyR4 was the C-terminus of HalH4, suggesting a potential mechanism of action involving pore formation within competitor species' cell membranes. Taken together, this study offers novel insights into the interplay between halocins and secreted proteases, as well as their contribution to antagonistic interaction within haloarchaea. IMPORTANCE: The antihaloarchaeal function of halocin H4 (HalH4) can be activated by extracellular proteases from haloarchaea, as demonstrated in this study. Notably, we report the first instance of halocin activation through proteolytic cleavage, highlighting its significance in the field. The C-terminus of HalH4 (CTH4) has been identified as the antihaloarchaeal peptide present in hydrolysates generated by HlyR4. The CTH4 exhibited inhibitory activity against a range of haloarchaeal species (Haloarchaeobius spp., Haloarcula spp., Haloferax spp., Halorubellus spp., and Halorubrum spp.), as well as selected bacterial species (Aliifodinibius spp. and Salicola spp.), indicating its broad-spectrum inhibitory potential across domains. The encoding gene of halocin HalH4, halH4, from the model halophilic archaeon Haloferax mediterranei ATCC 33500 can be expressed in Escherichia coli without codon optimization.

A TrmBL2-like transcription factor mediates the growth phase-dependent expression of halolysin SptA in a concentration-dependent manner in Natrinema gari J7-2.

To cope with a high-salinity environment, haloarchaea generally employ the twin-arginine translocation (Tat) pathway to transport secretory proteins across the cytoplasm membrane in a folded state, including Tat-dependent extracellular subtilases (halolysins) capable of autocatalytic activation. Some halolysins, such as SptA of Natrinema gari J7-2, are produced at late-log phase to prevent premature enzyme activation and proteolytic damage of cellular proteins in haloarchaea; however, the regulation mechanism for growth phase-dependent expression of halolysins remains largely unknown. In this study, a DNA-protein pull-down assay was performed to identify the proteins binding to the 5'-flanking sequence of sptA encoding halolysin SptA in strain J7-2, revealing a TrmBL2-like transcription factor (NgTrmBL2). The ΔtrmBL2 mutant of strain J7-2 showed a sharp decrease in the production of SptA, suggesting that NgTrmBL2 positively regulates sptA expression. The purified recombinant NgTrmBL2 mainly existed as a dimer although monomeric and higher-order oligomeric forms were detected by native-PAGE analysis. The results of electrophoretic mobility shift assays (EMSAs) showed that NgTrmBL2 binds to the 5'-flanking sequence of sptA in a non-specific and concentration-dependent manner and exhibits an increased DNA-binding affinity with the increase in KCl concentration. Moreover, we found that a distal cis-regulatory element embedded in the neighboring upstream gene negatively regulates trmBL2 expression and thus participates in the growth phase-dependent biosynthesis of halolysin SptA. IMPORTANCE: Extracellular proteases play important roles in nutrient metabolism, processing of functional proteins, and antagonism of haloarchaea, but no transcription factor involved in regulating the expression of haloaechaeal extracellular protease has been reported yet. Here we report that a TrmBL2-like transcription factor (NgTrmBL2) mediates the growth phase-dependent expression of an extracellular protease, halolysin SptA, of haloarchaeon Natrinema gari J7-2. In contrast to its hyperthermophilic archaeal homologs, which are generally considered to be global transcription repressors, NgTrmBL2 functions as a positive regulator for sptA expression. This study provides new clues about the transcriptional regulation mechanism of extracellular protease in haloarchaea and the functional diversity of archaeal TrmBL2.

Transcriptomic profiling of haloarchaeal denitrification through RNA-Seq analysis.

Denitrification, a crucial biochemical pathway prevalent among haloarchaea in hypersaline ecosystems, has garnered considerable attention in recent years due to its ecological implications. Nevertheless, the underlying molecular mechanisms and genetic regulation governing this respiration/detoxification process in haloarchaea remain largely unexplored. In this study, RNA-sequencing was used to compare the transcriptomes of the haloarchaeon Haloferax mediterranei under oxic and denitrifying conditions, shedding light on the intricate metabolic alterations occurring within the cell, such as the accurate control of the metal homeostasis. Furthermore, the investigation identifies several genes encoding transcriptional regulators and potential accessory proteins with putative roles in denitrification. Among these are bacterioopsin-like transcriptional activators, proteins harboring a domain of unknown function (DUF2249), and cyanoglobin. In addition, the study delves into the genetic regulation of denitrification, finding a regulatory motif within promoter regions that activates numerous denitrification-related genes. This research serves as a starting point for future molecular biology studies in haloarchaea, offering a promising avenue to unravel the intricate mechanisms governing haloarchaeal denitrification, a pathway of paramount ecological importance.IMPORTANCEDenitrification, a fundamental process within the nitrogen cycle, has been subject to extensive investigation due to its close association with anthropogenic activities, and its contribution to the global warming issue, mainly through the release of N2O emissions. Although our comprehension of denitrification and its implications is generally well established, most studies have been conducted in non-extreme environments with mesophilic microorganisms. Consequently, there is a significant knowledge gap concerning extremophilic denitrifiers, particularly those inhabiting hypersaline environments. The significance of this research was to delve into the process of haloarchaeal denitrification, utilizing the complete denitrifier haloarchaeon Haloferax mediterranei as a model organism. This research led to the analysis of the metabolic state of this microorganism under denitrifying conditions and the identification of regulatory signals and genes encoding proteins potentially involved in this pathway, serving as a valuable resource for future molecular studies.

Halobacterium hubeiense sp. nov., a haloarchaeal species isolated from a bore core drilled in Hubei Province, PR China.

Eight colonies of live microbes were isolated from an extensively surface-sterilized halite sample which had been retrieved from a depth of 2000 m from a salt mine in the Qianjiang Depression, Hubei Province, PR China. The eight colonies, obtained after 4 weeks of incubation, were named JI20-1T-JI20-8 and JI20-1T was selected as the type strain. The strains have been previously described, including a genomic analysis based on the complete genome for strain JI20-1T and draft genomes for the other strains. In that study, the name Halobacterium hubeiense was suggested, based on the location of the drilling site. Previous phylogenomic analysis showed that strain JI20-1T is most closely related to the Permian isolate Halobacterium noricense from Alpine rock salt. The orthologous average nucleotide identity (orthoANI) and digital DNA-DNA hybridization (dDDH) percentages between the eight strains are 100-99.6 % and 99.8-96.4 %, respectively. The orthoANI and dDDH values of these strains with respect to the type strains of species of the genus Halobacterium are 89.9-78.2 % and 37.3-21.6 %, respectively, supporting their placement in a novel extremely halophilic archaeal species. The phylogenomic tree based on the comparison of sequences of 632 core-orthologous proteins confirmed the novel species status for these haloarchaea. The polar lipid profile includes phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, and sulfated galactosyl mannosyl galactosyl glucosyl diether, a profile compatible with that of Halobacterium noricense. Based on genomic, phenotypic, and chemotaxonomic characterization, we propose strain JI20-1T (=DSM 114402T = HAMBI 3616T) as the type strain of a novel species in the genus Halobacterium, with the name Halobacterium hubeiense sp. nov.

Characterization of carotenoids extracted from Haloferax larsenii NCIM 5678 isolated from Pachpadra salt lake, Rajasthan.

Carotenoids are a diverse group of pigments known for their broad range of biological functions and applications. This study delves into multifaceted potential of carotenoids extracted from Haloferax larsenii NCIM 5678 previously isolated from Pachpadra Salt Lake in Rajasthan, India. H. larsenii NCIM 5678 was able to grow up to OD600 1.77 ± 0.03 with carotenoid concentration, 3.3 ± 0.03 µg/ml. The spectrophotometric analysis of carotenoid extract indicated the presence of three-fingered peak (460, 490 and 520 nm) which is a characteristic feature of bacterioruberin and its derivatives. The bacterioruberin was purified using silica gel column chromatography and thin layer chromatography. The carotenoid extract showed 12.3 ± 0.09 mm zone of growth inhibition with a minimum inhibitory concentration 546 ng/ml against indicator strain, H. larsenii HA4. The percentage antioxidant activity of carotenoid was found to be 84% which was higher as compared to commercially available ascorbic acid (56.74%). Thus, carotenoid extract from H. larsenii NCIM 5678 possesses unique attributes with compelling evidence of antimicrobial and antioxidant potential for the development of novel pharmaceuticals and nutraceuticals.

Multi-omics association study of hexadecane degradation in haloarchaeal strain Halogranum rubrum RO2-11.

Haloarchaea with the capacity to degrade alkanes is promising to deal with petroleum pollution in hypersaline environments. However, only a limited number of haloarchaeal species are investigated, and their pathway and mechanism for alkane degradation remain unclear. In this study, Halogranum rubrum RO2-11, a haloarchaeal strain, verified the ability to degrade kerosene and hexadecane in 184 g/L NaCl, with 53% and 52% degradation rates after 9 and 4 days, respectively. Genome sequencing and gene annotation indicated that strain RO2-11 possesses a complete potential alkane-degrading pathway, of which alkane hydroxylases may include CYP450, AlmA, and LadA. Transcriptome and metabolome analyses revealed that the upregulation of related genes in TCA cycle, lysine biosynthesis, and acetylation may help improve hexadecane degradation. Additionally, an alternative degrading pathway of hexadecane based on dual-terminal ß-oxidation may occur in strain RO2-11. It is likely to be the first report of alkane degradation by the genus Halogranum, which may be helpful for applications of oil-pollution bioremediation under high-salt conditions.

Halophilic archaea as tools for bioremediation technologies.

Haloarchaea are extremophilic microorganisms belonging to the Archaea domain that require high salt concentrations to be alive, thus inhabiting ecosystems like salty ponds, salty marshes, or extremely salty lagoons. They are more abundantly and widely distributed worldwide than initially expected. Most of them are grouped into two families: Halobacteriaceae and Haloferacaceae. The extreme conditions under which haloarchaea survive contribute to their metabolic and molecular adaptations, thus making them good candidates for the design of bioremediation strategies to treat brines, salty water, and saline soils contaminated with toxic compounds such as nitrate, nitrite, oxychlorates such as perchlorate and chlorate, heavy metals, hydrocarbons, and aromatic compounds. New advances in understanding haloarchaea physiology, metabolism, biochemistry, and molecular biology suggest that biochemical pathways related to nitrogen and carbon, metals, hydrocarbons, or aromatic compounds can be used for bioremediation proposals. This review analyses the novelty of the most recent results showing the capability of some haloarchaeal species to assimilate, modify, or degrade toxic compounds for most living beings. Several examples of the role of these microorganisms in the treatment of polluted brine or salty soils are also discussed in connection with circular economy-based processes. KEY POINTS: • Haloarchaea are extremophilic microorganisms showing genuine metabolism • Haloarchaea can metabolise compounds that are highly toxic to most living beings • These metabolic capabilities are useful for designing soil and water bioremediation strategies.

Unveiling the repressive mechanism of a PPS-like regulator (PspR) in polyhydroxyalkanoates biosynthesis network.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a type of polyhydroxyalkanoates (PHA) that exhibits numerous outstanding properties and is naturally synthesized and elaborately regulated in various microorganisms. However, the regulatory mechanism involving the specific regulator PhaR in Haloferax mediterranei, a major PHBV production model among Haloarchaea, is not well understood. In our previous study, we showed that deletion of the phosphoenolpyruvate (PEP) synthetase-like (pps-like) gene activates the cryptic phaC genes in H. mediterranei, resulting in enhanced PHBV accumulation. In this study, we demonstrated the specific function of the PPS-like protein as a negative regulator of phaR gene expression and PHBV synthesis. Chromatin immunoprecipitation (ChIP), in situ fluorescence reporting system, and in vitro electrophoretic mobility shift assay (EMSA) showed that the PPS-like protein can bind to the promoter region of phaRP. Computational modeling revealed a high structural similarity between the rifampin phosphotransferase (RPH) protein and the PPS-like protein, which has a conserved ATP-binding domain, a His domain, and a predicted DNA-binding domain. Key residues within this unique DNA-binding domain were subsequently validated through point mutation and functional evaluations. Based on these findings, we concluded that PPS-like protein, which we now renamed as PspR, has evolved into a repressor capable of regulating the key regulator PhaR, and thereby modulating PHBV synthesis. This regulatory network (PspR-PhaR) for PHA biosynthesis is likely widespread among haloarchaea, providing a novel approach to manipulate haloarchaea as a production platform for high-yielding PHA. KEY POINTS: • The repressive mechanism of a novel inhibitor PspR in the PHBV biosynthesis was demonstrated • PspR is widespread among the PHA accumulating haloarchaea • It is the first report of functional conversion from an enzyme to a trans-acting regulator in haloarchaea.

Bacterioruberin: Biosynthesis, Antioxidant Activity, and Therapeutic Applications in Cancer and Immune Pathologies.

Halophilic archaea, also termed haloarchaea, are a group of moderate and extreme halophilic microorganisms that constitute the major microbial populations in hypersaline environments. In these ecosystems, mainly aquatic, haloarchaea are constantly exposed to ionic and oxidative stress due to saturated salt concentrations and high incidences of UV radiation (mainly in summer). To survive under these harsh conditions, haloarchaea have developed molecular adaptations including hyperpigmentation. Regarding pigmentation, haloarchaeal species mainly synthesise the rare C50 carotenoid called bacterioruberin (BR) and its derivatives, monoanhydrobacterioruberin and bisanhydrobacterioruberin. Due to their colours and extraordinary antioxidant properties, BR and its derivatives have been the aim of research in several research groups all over the world during the last decade. This review aims to summarise the most relevant characteristics of BR and its derivatives as well as describe their reported antitumoral, immunomodulatory, and antioxidant biological activities. Based on their biological activities, these carotenoids can be considered promising natural biomolecules that could be used as tools to design new strategies and/or pharmaceutical formulas to fight against cancer, promote immunomodulation, or preserve skin health, among other potential uses.

Looking through the Lens of the Ribosome Biogenesis Evolutionary History: Possible Implications for Archaeal Phylogeny and Eukaryogenesis.

Our understanding of microbial diversity and its evolutionary relationships has increased substantially over the last decade. Such an understanding has been greatly fueled by culture-independent metagenomics analyses. However, the outcome of some of these studies and their biological and evolutionary implications, such as the origin of the eukaryotic lineage from the recently discovered archaeal Asgard superphylum, is debated. The sequences of the ribosomal constituents are amongst the most used phylogenetic markers. However, the functional consequences underlying the analysed sequence diversity and their putative evolutionary implications are essentially not taken into consideration. Here, we propose to exploit additional functional hallmarks of ribosome biogenesis to help disentangle competing evolutionary hypotheses. Using selected examples, such as the multiple origins of halophily in archaea or the evolutionary relationship between the Asgard archaea and Eukaryotes, we illustrate and discuss how function-aware phylogenetic framework can contribute to refining our understanding of archaeal phylogeny and the origin of eukaryotic cells.

Physiological and genomic insights into abiotic stress of halophilic archaeon Natrinema altunense 4.1R isolated from a saline ecosystem of Tunisian desert.

Halophilic archaea are polyextremophiles with the ability to withstand fluctuations in salinity, high levels of ultraviolet radiation, and oxidative stress, allowing them to survive in a wide range of environments and making them an excellent model for astrobiological research. Natrinema altunense 4.1R is a halophilic archaeon isolated from the endorheic saline lake systems, Sebkhas, located in arid and semi-arid regions of Tunisia. It is an ecosystem characterized by periodic flooding from subsurface groundwater and fluctuating salinities. Here, we assess the physiological responses and genomic characterization of N. altunense 4.1R to UV-C radiation, as well as osmotic and oxidative stresses. Results showed that the 4.1R strain is able to survive up to 36% of salinity, up to 180 J/m2 to UV-C radiation, and at 50 mM of H2O2, a resistance profile similar to Halobacterium salinarum, a strain often used as UV-C resistant model. In order to understand the genetic determinants of N. altunense 4.1R survival strategy, we sequenced and analyzed its genome. Results showed multiple gene copies of osmotic stress, oxidative stress, and DNA repair response mechanisms supporting its survivability at extreme salinities and radiations. Indeed, the 3D molecular structures of seven proteins related to responses to UV-C radiation (excinucleases UvrA, UvrB, and UvrC, and photolyase), saline stress (trehalose-6-phosphate synthase OtsA and trehalose-phosphatase OtsB), and oxidative stress (superoxide dismutase SOD) were constructed by homology modeling. This study extends the abiotic stress range for the species N. altunense and adds to the repertoire of UV and oxidative stress resistance genes generally known from haloarchaeon.

Natrinema salsiterrestre sp. nov., an extremely halophilic archaeon isolated from a hypersaline soil.

An extremely halophilic archaeal strain, designated S1CR25-10T, was isolated from hypersaline soil sampled in the Odiel Saltmarshes Natural Area in Southwestern Spain (Huelva) and subjected to a polyphasic taxonomic characterization. The cells were Gram-stain-negative, motile and their colonies were pink-pigmented. It was a strictly aerobic haloarchaeon that could grow at 25-55 °C (optimum, 37 °C), at pH 6.0-9.0 (optimum, pH 7.0-8.0) and in the presence of 12-30 % (w/v) total salts (optimum, 20-25 %, w/v). The phylogenetic analysis based on the comparison of the 16S rRNA gene sequences revealed that strain S1CR25-10T belongs to the genus Natrinema, with 98.9 % similarity to Natrinema salinisoli SLN56T. In addition, the values of orthologous average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity were below the threshold limits accepted for prokaryotic species delineation, with N. salinisoli SLN56T showing the highest relatedness values (92.6 % and 48.4 %, respectively). The major polar lipids were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and a glycolipid chromatographically identical to sulfated diglycosyl diether. The DNA G+C content of the isolate was 63.8 mol%. Based on the phylogenetic, phenotypic and chemotaxonomic characterization and the whole genome results, strain S1CR25-10T represents a new species within the genus Natrinema, for which the name Natrinema salsiterrestre sp. nov., with type strain S1CR25-10T (=CECT 30623T=CCM 9251T), is proposed.

Characterization of Haloarcula terrestris sp. nov. and reclassification of a Haloarcula species based on a taxogenomic approach.

An extremely halophilic archaeon, strain S1AR25-5AT, was isolated from a hypersaline soil sampled in Odiel Saltmarshes Natural Area (Huelva, Spain). The cells were Gram-stain-negative, motile, pleomorphic rods. Cell growth was observed in the presence of 15-30 % (w/v) NaCl [optimum, 25 % (w/v) NaCl], at pH 6.0-9.0 (optimum, pH 6.5-7.5) and at 25-50 °C (optimum, 37 °C). Based on the 16S rRNA and rpoB' gene sequence comparisons, strain S1AR25-5AT was affiliated to the genus Haloarcula. Taxogenomic analysis, including comparison of the genomes and the phylogenomic tree based on the core-orthologous proteins, together with the genomic indices, i.e., orthologous average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity, confirmed that strain S1AR25-5AT (=CCM 9249T=CECT 30619T) represents a new species of the genus Haloarcula, for which we propose the name Haloarcula terrestris sp. nov. The major polar lipids were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulphate and an unidentified glycolipid, which correlated with the lipid profile of species of the genus Haloarcula. In addition, based on the modern approach in description of species in taxonomy of prokaryotes, the above mentioned genomic indexes indicated that the species Haloarcula tradensis should be considered as a heterotypic synonym of Haloarcula argentinensis.

Evaluation of Haloferax mediterranei Strain R4 Capabilities for Cadmium Removal from Brines.

Haloferax mediterranei has revealed a high bioremediation potential for several inorganic anions (e.g., nitrates and nitrites) and metals from hypersaline waters and brines. However, it is unclear, to date, whether this microorganism allows Cd (II) bioremediation. Consequently, the main objective of this work was to assess the Cd (II) bioremediation potential of Hfx. mediterranei R4. To this end, Hfx. mediterranei cell growth rate and metal bioaccumulation were investigated using different culture media (complex, CM, and defined medium, DM) containing Cd (II) up to 1 mM. In addition, the elemental profile of the biomass (i.e., Al, Ba, Ca, Co, Cu, Fe, K, Mg, Mn, Na, Ni, Sr and Zn) has also been monitored to gain insight into the metabolic processes that may be taking place at the intracellular level for Cd (II) removal. Because of the formation of CdS precipitate, CM is not a suitable culture media for evaluating Cd bioremediation since metal concentration could not be appropriately controlled. When operating in DM, it was observed that the cell doubling time increases three times in the presence of Cd (II). Hfx. mediterranei can bioaccumulate Cd, showing the highest significant accumulation at concentrations of 0.4 mM (108 ± 12 mg Cd/g dry tissue). Finally, the presence of Cd (II) affects the content of K, Mg, Mn and Zn in the biomass, by increasing K levels up to 27 ± 18% and Mn up to 310 ± 140% and reducing Mg levels up to 55 ± 36% and Zn up to 30 ± 4%. These results suggest that different mechanisms are involved in Cd (II) tolerance by Hfx. mediterranei, resulting in increasing the cell concentration of stress-tolerant elements in the biomass (K and Mn), while lowering the concentration of elements which Cd (II) competes with (Mg and Zn), and that all affects the physiological response of the organism by decreasing its growth rate.

Symbiosis between nanohaloarchaeon and haloarchaeon is based on utilization of different polysaccharides.

Nano-sized archaeota, with their small genomes and limited metabolic capabilities, are known to associate with other microbes, thereby compensating for their own auxotrophies. These diminutive and yet ubiquitous organisms thrive in hypersaline habitats that they share with haloarchaea. Here, we reveal the genetic and physiological nature of a nanohaloarchaeon-haloarchaeon association, with both microbes obtained from a solar saltern and reproducibly cultivated together in vitro. The nanohaloarchaeon Candidatus Nanohalobium constans LC1Nh is an aerotolerant, sugar-fermenting anaerobe, lacking key anabolic machinery and respiratory complexes. The nanohaloarchaeon cells are found physically connected to the chitinolytic haloarchaeon Halomicrobium sp. LC1Hm. Our experiments revealed that this haloarchaeon can hydrolyze chitin outside the cell (to produce the monosaccharide N-acetylglucosamine), using this beta-glucan to obtain carbon and energy for growth. However, LC1Hm could not metabolize either glycogen or starch (both alpha-glucans) or other polysaccharides tested. Remarkably, the nanohaloarchaeon's ability to hydrolyze glycogen and starch to glucose enabled growth of Halomicrobium sp. LC1Hm in the absence of a chitin. These findings indicated that the nanohaloarchaeon-haloarchaeon association is both mutualistic and symbiotic; in this case, each microbe relies on its partner's ability to degrade different polysaccharides. This suggests, in turn, that other nano-sized archaeota may also be beneficial for their hosts. Given that availability of carbon substrates can vary both spatially and temporarily, the susceptibility of Halomicrobium to colonization by Ca Nanohalobium can be interpreted as a strategy to maximize the long-term fitness of the host.

Toxic heavy metal/oxyanion tolerance in haloarchaea from some saline and hypersaline ecosystems.

Toxic heavy metal/oxyanion contamination has increased severely through the last decades. In this study, 169 native haloarchaeal strains were isolated from different saline and hypersaline econiches of Iran. After providing pure culture and performing morphological, physiological, and biochemical tests, haloarchaea resistance toward arsenate, selenite, chromate, cadmium, zinc, lead, copper, and mercury were surveyed using an agar dilution method. On the basis of minimum inhibitory concentrations (MICs), the least toxicities were found with selenite and arsenate, while the haloarchaeal strains revealed the highest sensitivity for mercury. On the other hand, the majority of haloarchaeal strains exhibited similar responses to chromate and zinc, whereas the resistance level of the isolates to lead, cadmium, and copper was very heterogeneous. 16 S ribosomal RNA (rRNA) gene sequence analysis revealed that most haloarchaeal strains belong to the Halorubrum and Natrinema genera. The obtained results from this study showed that among the identified isolates, Halococcus morrhuae strain 498 had an exceptional resistance toward selenite and cadmium (64 and 16 mM, respectively). Also, Halovarius luteus strain DA5 exhibited a remarkable tolerance against copper (32 mM). Moreover, strain Salt5, identified as Haloarcula sp., was the only strain that could tolerate all eight tested heavy metals/oxyanions and had a significant tolerance of mercury (1.5 mM).

Unveiling novel insights into haloarchaea (Halolamina pelagica CDK2) for alleviation of drought stress in wheat.

Plant growth promoting microorganisms have various implications for plant growth and drought stress alleviation; however, the roles of archaea have not been explored in detail. Herein, present study was aimed for elucidating potential of haloarchaea (Halolamina pelagica CDK2) on plant growth under drought stress. Results showed that haloarchaea inoculated wheat plants exhibited significant improvement in total chlorophyll (100%) and relative water content (30.66%) compared to the uninoculated water-stressed control (30% FC). The total root length (2.20-fold), projected area (1.60-fold), surface area (1.52-fold), number of root tips (3.03-fold), number of forks (2.76-fold) and number of links (1.45-fold) were significantly higher in the inoculated plants than in the uninoculated water stressed control. Additionally, the haloarchaea inoculation resulted in increased sugar (1.50-fold), protein (2.40-fold) and activity of antioxidant enzymes such as superoxide dismutase (1.93- fold), ascorbate peroxidase (1.58-fold), catalase (2.30-fold), peroxidase (1.77-fold) and glutathione reductase (4.70-fold), while reducing the accumulation of proline (46.45%), glycine betaine (35.36%), lipid peroxidation (50%), peroxide and superoxide radicals in wheat leaves under water stress. Furthermore, the inoculation of haloarchaea significantly enhanced the expression of stress-responsive genes (DHN, DREB, L15, and TaABA-8OH) and wheat vegetative growth under drought stress over the uninoculated water stressed control. These results provide novel insights into the plant-archaea interaction for plant growth and stress tolerance in wheat and pave the way for future research in this area.

Resurrection of Ancestral Malate Dehydrogenases Reveals the Evolutionary History of Halobacterial Proteins: Deciphering Gene Trajectories and Changes in Biochemical Properties.

Extreme halophilic Archaea thrive in high salt, where, through proteomic adaptation, they cope with the strong osmolarity and extreme ionic conditions of their environment. In spite of wide fundamental interest, however, studies providing insights into this adaptation are scarce, because of practical difficulties inherent to the purification and characterization of halophilic enzymes. In this work, we describe the evolutionary history of malate dehydrogenases (MalDH) within Halobacteria (a class of the Euryarchaeota phylum). We resurrected nine ancestors along the inferred halobacterial MalDH phylogeny, including the Last Common Ancestral MalDH of Halobacteria (LCAHa) and compared their biochemical properties with those of five modern halobacterial MalDHs. We monitored the stability of these various MalDHs, their oligomeric states and enzymatic properties, as a function of concentration for different salts in the solvent. We found that a variety of evolutionary processes, such as amino acid replacement, gene duplication, loss of MalDH gene and replacement owing to horizontal transfer resulted in significant differences in solubility, stability and catalytic properties between these enzymes in the three Halobacteriales, Haloferacales, and Natrialbales orders since the LCAHa MalDH. We also showed how a stability trade-off might favor the emergence of new properties during adaptation to diverse environmental conditions. Altogether, our results suggest a new view of halophilic protein adaptation in Archaea.

Sec-Dependent Secretion of Subtilase SptE in Haloarchaea Facilitates Its Proper Folding and Heterocatalytic Processing by Halolysin SptA Extracellularly.

In response to high-salt conditions, haloarchaea export most secretory proteins through the Tat pathway in folded states; however, it is unclear why some haloarchaeal proteins are still routed to the Sec pathway. SptE is an extracellular subtilase of Natrinema sp. strain J7-2. Here, we found that SptE precursor comprises a Sec signal peptide, an N-terminal propeptide, a catalytic domain, and a long C-terminal extension (CTE) containing seven domains (C1 to C7). SptE is produced extracellularly as a mature form (M180) in strain J7-2 and a proform (ΔS) in the ΔsptA mutant strain, indicating that halolysin SptA mediates the conversion of the secreted proform into M180. The proper folding of ΔS is more efficient in the presence of NaCl than KCl. ΔS requires SptA for cleavage of the N-terminal propeptide and C-terminal C6 and C7 domains to generate M180, accompanied by the appearance of autoprocessing product M120 lacking C5. At lower salinities or elevated temperatures, M180 and M120 could be autoprocessed into M90, which comprises the catalytic and C1 domains and has a higher activity than M180. When produced in Haloferax volcanii, SptE could be secreted as a properly folded proform, but its variant (TSptE) with a Tat signal peptide does not fold properly and suffers from severe proteolysis extracellularly; meanwhile, TSptE is more inclined to aggregate intracellularly than SptE. Systematic domain deletion analysis reveals that the long CTE is an important determinant for secretion of SptE via the Sec rather than Tat pathway to prevent enzyme aggregation before secretion. IMPORTANCE While Tat-dependent haloarchaeal subtilases (halolysins) have been extensively studied, the information about Sec-dependent subtilases of haloarchaea is limited. Our results demonstrate that proper maturation of Sec-dependent subtilase SptE of Natrinema sp. strain J7-2 depends on the action of halolysin SptA from the same strain, yielding multiple hetero- and autocatalytic mature forms. Moreover, we found that the different extra- and intracellular salt types (NaCl versus KCl) of haloarchaea and the long CTE are extrinsic and intrinsic factors crucial for routing SptE to the Sec rather than Tat pathway. This study provides new clues about the secretion and adaptation mechanisms of Sec substrates in haloarchaea.

Halorhabdus amylolytica sp. nov. and Halorhabdus salina sp. nov., isolated from hypersaline environments.

Two novel extremely halophilic archaeal strains, designated H27T and FL145T, were isolated from a salt mine and a kelp salt sample, respectively. Cells of both strains were Gram-stain-negative, motile and pleomorphic. The 16S rRNA and rpoB' gene sequence similarities between strains H27T and FL145T were 96.60 and 88.77%. Strains H27T and FL145T were both closely related to Halorhabdus rudnickae WSM-64T, Halorhabdus tiamatea SARL4BT and Halorhabdus utahensis AX-2T, with a 16S rRNA gene sequence similarities of 98.14, 96.34 and 96.27% for strain H27T and 96.42, 95.82 and 96.17% for strain FL145T. The genome-based average nucleotide identity (ANI) values between strains H27T and FL145T, and these three species were 83.93, 79.79 and 79.09% (for strain H27T), and 78.32, 77.95 and 77.05% (for strain FL145T), respectively. The ANI value between strains H27T and FL145T was 78.65 %. The digital DNA-DNA hybridization values between strains H27T and FL145T, and these three species were less than 27.40%, which were below the recommended threshold for membership of the same species. The major polar lipids of both strains were found to consist of sulfated diglycosyl diether, triglycosyl diether, phosphatidylglycerol phosphate methyl ester and phosphatidylglycerol. The DNA G+C content was determined from genome to be 62.10 mol% for strain H27T and 61.51 mol% for strain FL145T. Based on phylogenetic, phenotypic, chemotaxonomic and genomic analyses, these two new isolates should be classified as representing two novel species in the genus Halorhabdus, with strain H27T (=CGMCC 1.16342T=NBRC 113589T) as the type strain of a new species for which we propose the name Halorhabdus amylolytica sp. nov., and strain FL145T (=CGMCC 1.13888T=NBRC 114260T) as the type strain of another new species for which we propose the name Halorhabdus salina sp. nov.