Production, characterization and biomedical potential of biosurfactants produced by haloalkaliphilic archaea from Wadi El-Natrun, Egypt.

Extreme halophilic archaea that can live in high saline environments can offer potential applications in different biotechnological fields. This study delves into the fascinating field of halophilic archaea and their ability to produce biosurfactants. Some strains of haloarchaea were isolated from Wadi El-Natrun and were screened for biosurfactants production in a standard basal medium using emulsification index assay. Two strains were chosen as the potential strains for surface tension reduction. They were identified as Natrialba sp. BG1 and N3. The biosurfactants production was optimized and the produced emulsifiers were partially purified and identified using FTIR and NMR. Sequential statistical optimization, Plackett-Burman (PB) and Box-Behnken Designs (BBD) were carried out using 5 factors: oil, NaCl, casamino acids, pH, and inoculum size. The most significant factors were used for the next Response Surface Methodology experiment. The final optimal conditions for biosurfactants production were the inoculum size 2% pH 11 and NaCl 250 g/L, for Natrialba sp. BG1 and inoculum size 2.2%, pH 10 and NaCl 100 g/L for Natrialba sp. N3. The produced biosurfactants were tested for wound healing and the results indicated that Natrialba sp. BG1 biosurfactants is more efficient than Natrialba sp. N3 biosurfactants. Biosurfactants extracts were tested for their cytotoxic effects on normal cell line as well as on different cancer cells using MTT assay. The findings demonstrated that varying concentrations of the biosurfactants (31.25, 62.5, 125, 250, 500 and 1000 µg/mL) exhibited cytotoxic effects on the cell lines being tested. Additionally, the outcomes unveiled the presence of anti-inflammatory and antioxidant properties for both biosurfactants. Consequently, they could potentially serve as natural, safe, and efficient novel agents for combating cancer, promoting wound healing, and providing anti-inflammatory and antioxidant benefits.

Key determinants for signaling in the sensory rhodopsin II/transducer complex are different between Halobacterium salinarum and Natronomonas pharaonis.

The cell membrane of Halobacterium salinarum contains a retinal-binding photoreceptor, sensory rhodopsin II (HsSRII), coupled with its cognate transducer (HsHtrII), allowing repellent phototaxis behavior for shorter wavelength light. Previous studies on SRII from Natronomonas pharaonis (NpSRII) pointed out the importance of the hydrogen bonding interaction between Thr204NpSRII and Tyr174NpSRII in signal transfer from SRII to HtrII. Here, we investigated the effect on phototactic function by replacing residues in HsSRII corresponding to Thr204NpSRII and Tyr174NpSRII . Whereas replacement of either residue altered the photocycle kinetics, introduction of any mutations at Ser201HsSRII and Tyr171HsSRII did not eliminate negative phototaxis function. These observations imply the possibility of the presence of an unidentified molecular mechanism for photophobic signal transduction differing from NpSRII-NpHtrII.

Dissecting the Arginine and Lysine Biosynthetic Pathways and Their Relationship in Haloarchaeon Natrinema gari J7-2 via Endogenous CRISPR-Cas System-Based Genome Editing.

The evolutionary relationship between arginine and lysine biosynthetic pathways has been well established in bacteria and hyperthermophilic archaea but remains largely unknown in haloarchaea. Here, the endogenous CRISPR-Cas system was harnessed to edit arginine and lysine biosynthesis-related genes in the haloarchaeon Natrinema gari J7-2. The ΔargW, ΔargX, ΔargB, and ΔargD mutant strains display an arginine auxotrophic phenotype, while the ΔdapB mutant shows a lysine auxotrophic phenotype, suggesting that strain J7-2 utilizes the ArgW-mediated pathway and the diaminopimelate (DAP) pathway to synthesize arginine and lysine, respectively. Unlike the ArgD in Escherichia coli acting as a bifunctional aminotransferase in both the arginine biosynthesis pathway and the DAP pathway, the ArgD in strain J7-2 participates only in arginine biosynthesis. Meanwhile, in strain J7-2, the function of argB cannot be compensated for by its evolutionary counterpart ask in the DAP pathway. Moreover, strain J7-2 cannot utilize α-aminoadipate (AAA) to synthesize lysine via the ArgW-mediated pathway, in contrast to hyperthermophilic archaea that employ a bifunctional LysW-mediated pathway to synthesize arginine (or ornithine) and lysine from glutamate and AAA, respectively. Additionally, the replacement of a 5-amino-acid signature motif responsible for substrate specificity of strain J7-2 ArgX with that of its hyperthermophilic archaeal homologs cannot endow the ΔdapB mutant with the ability to biosynthesize lysine from AAA. The in vitro analysis shows that strain J7-2 ArgX acts on glutamate rather than AAA. These results suggest that the arginine and lysine biosynthetic pathways of strain J7-2 are highly specialized during evolution. IMPORTANCE Due to their roles in amino acid metabolism and close evolutionary relationship, arginine and lysine biosynthetic pathways represent interesting models for probing functional specialization of metabolic routes. The current knowledge with respect to arginine and lysine biosynthesis is limited for haloarchaea compared to that for bacteria and hyperthermophilic archaea. Our results demonstrate that the haloarchaeon Natrinema gari J7-2 employs the ArgW-mediated pathway and the DAP pathway for arginine and lysine biosynthesis, respectively, and the two pathways are functionally independent of each other; meanwhile, ArgX is a key determinant of substrate specificity of the ArgW-mediated pathway in strain J7-2. This study provides new clues about haloarchaeal amino acid metabolism and confirms the convenience and efficiency of endogenous CRISPR-Cas system-based genome editing in haloarchaea.

A conserved Trp residue in HwBR contributes to its unique tolerance toward acidic environments.

Bacteriorhodopsin (BR) is a light-driven outward proton pump found mainly in halophilic archaea. A BR from an archaeon Haloquadratum walsbyi (HwBR) was found to pump protons under more acidic conditions compared with most known BR proteins. The atomic structural study on HwBR unveiled that a pair of hydrogen bonds between the BC and FG loop in its periplasmic region may be a factor in such improved pumping capability. Here, we further investigated the retinal-binding pocket of HwBR and found that Trp94 contributes to the higher acid tolerance. Through single mutations in a BR from Halobacterium salinarum and HwBR, we examined the conserved tryptophan residues in the retinal-binding pocket. Among these residues of HwBR, mutagenesis at Trp94 facing the periplasmic region caused the most significant disruption to optical stability and proton-pumping capability under acidic conditions. The other tryptophan residues of HwBR exerted little impact on both maximum absorption wavelength and pH-dependent proton pumping. Our findings suggest that the residues from Trp94 to the hydrogen bonds at the BC loop confer both optical stability and functionality on the overall protein in low-pH environments.

Structural and Functional Analysis of DndE Involved in DNA Phosphorothioation in the Haloalkaliphilic Archaea Natronorubrum bangense JCM10635.

Phosphorothioate (PT) modification, a sequence-specific modification that replaces the nonbridging oxygen atom with sulfur in a DNA phosphodiester through the gene products of dndABCDE or sspABCD, is widely distributed in prokaryotes. DNA PT modification functions together with gene products encoded by dndFGH, pbeABCD, or sspE to form defense systems that can protect against invasion by exogenous DNA particles. While the functions of the multiple enzymes in the PT system have been elucidated, the exact role of DndE in the PT process is still obscure. Here, we solved the crystal structure of DndE from the haloalkaliphilic archaeal strain Natronorubrum bangense JCM10635 at a resolution of 2.31 Å. Unlike the tetrameric conformation of DndE in Escherichia coli B7A, DndE from N. bangense JCM10635 exists in a monomeric conformation and can catalyze the conversion of supercoiled DNA to nicked or linearized products. Moreover, DndE exhibits preferential binding affinity to nicked DNA by virtue of the R19- and K23-containing positively charged surface. This work provides insight into how DndE functions in PT modification and the potential sulfur incorporation mechanism of DNA PT modification. IMPORTANCE DndABCDE proteins have been demonstrated to catalyze DNA PT modification with the nonbridging oxygen in the DNA sugar-phosphate backbone replaced by sulfur. In the PT modification pathway, DndA exerts cysteine desulfurase activity capable of catalyzing the mobilization of sulfur from l-cysteine, which involves the ion-sulfur cluster assembly of DndC. This is regarded as the initial step of the DNA PT modification. Moreover, DndD has ATPase activity in vitro, which is believed to provide energy for the oxygen-sulfur swap, while the function of DndE is unknown. However, the exact function of the key enzyme DndE remains to be elucidated. By determining the structure of DndE from the haloalkaliphilic strain Natronorubrum bangense JCM10635, we showed that the archaeal DndE adopts a monomer conformation. Notably, DndE can introduce nicks to supercoiled DNA and exhibits a binding preference for nicked DNA; the nicking is believed to be the initial step for DNA to facilitate the sulfur incorporation.

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.

Conditional Alternative Protein Splicing Promoted by Inteins from Haloquadratum walsbyi.

Protein splicing is a post-translational process by which an intervening protein, or an intein, catalyzes its own excision from flanking polypeptides, or exteins, coupled to extein ligation. Four inteins interrupt the MCM helicase of the halophile Haloquadratum walsbyi, two of which are mini-inteins that lack a homing endonuclease. Both inteins can be overexpressed in Escherichia coli and purified as unspliced precursors; splicing can be induced in vitro by incubation with salt. However, one intein can splice in 0.5 M NaCl in vitro, whereas the other splices efficiently only in buffer containing over 2 M NaCl; the organism also requires high salt to grow, with the standard growth media containing over 3 M NaCl and about 0.75 M magnesium salts. Consistent with this difference in salt-dependent activity, an intein-containing precursor protein with both inteins promotes conditional alternative protein splicing (CAPS) to yield different spliced products dependent on the salt concentration. Native Trp fluorescence of the inteins suggests that the difference in activity may be due to partial unfolding of the inteins at lower salt concentrations. This differential salt sensitivity of intein activity may provide a useful mechanism for halophiles to respond to environmental changes.

Production of Polyhydroxyalkanoates in Unsterilized Hyper-Saline Medium by Halophiles Using Waste Silkworm Excrement as Carbon Source.

The chlorophyll ethanol-extracted silkworm excrement was hardly biologically reused or fermented by most microorganisms. However, partial extremely environmental halophiles were reported to be able to utilize a variety of inexpensive carbon sources to accumulate polyhydroxyalkanoates. In this study, by using the nile red staining and gas chromatography assays, two endogenous haloarchaea strains: Haloarcula hispanica A85 and Natrinema altunense A112 of silkworm excrement were shown to accumulate poly(3-hydroxybutyrate) up to 0.23 g/L and 0.08 g/L, respectively, when using the silkworm excrement as the sole carbon source. The PHA production of two haloarchaea showed no significant decreases in the silkworm excrement medium without being sterilized compared to that of the sterilized medium. Meanwhile, the CFU experiments revealed that there were more than 60% target PHAs producing haloarchaea cells at the time of the highest PHAs production, and the addition of 0.5% glucose into the open fermentation medium can largely increase both the ratio of target haloarchaea cells (to nearly 100%) and the production of PHAs. In conclusion, our study demonstrated the feasibility of using endogenous haloarchaea to utilize waste silkworm excrement, effectively. The introduce of halophiles could provide a potential way for open fermentation to further lower the cost of the production of PHAs.

The Inducible Intein-Mediated Self-Cleaving Tag (IIST) System: A Novel Purification and Amidation System for Peptides and Proteins.

An efficient self-cleavable purification tag could be a powerful tool for purifying recombinant proteins and peptides without additional proteolytic processes using specific proteases. Thus, the intein-mediated self-cleavage tag was developed and has been commercially available as the IMPACT™ system. However, uncontrolled cleavages of the purification tag by the inteins in the IMPACT™ system have been reported, thereby reducing final yields. Therefore, controlling the protein-splicing activity of inteins has become critical. Here we utilized conditional protein splicing by salt conditions. We developed the inducible intein-mediated self-cleaving tag (IIST) system based on salt-inducible protein splicing of the MCM2 intein from the extremely halophilic archaeon, Halorhabdus utahensis and applied it to small peptides. Moreover, we described a method for the amidation using the same IIST system and demonstrated 15N-labeling of the C-terminal amide group of a single domain antibody (VHH).

In Vivo Neocortical [K]o Modulation by Targeted Stimulation of Astrocytes.

A normally functioning nervous system requires normal extracellular potassium ion concentration ([K]o). Throughout the nervous system, several processes, including those of an astrocytic nature, are involved in [K]o regulation. In this study we investigated the effect of astrocytic photostimulation on [K]o. We hypothesized that in vivo photostimulation of eNpHR-expressing astrocytes leads to a decreased [K]o. Using optogenetic and electrophysiological techniques we showed that stimulation of eNpHR-expressing astrocytes resulted in a significantly decreased resting [K]o and evoked K responses. The amplitude of the concomitant spreading depolarization-like events also decreased. Our results imply that astrocytic membrane potential modification could be a potential tool for adjusting the [K]o.

Evaluation of DIBMA nanoparticles of variable size and anionic lipid content as tools for the structural and functional study of membrane proteins.

Amphiphilic maleic acid-containing polymers allow for the direct extraction of membrane proteins into stable, homogenous, water-soluble copolymer/lipid nanoparticles without the use of detergents. By adjusting the polymer/lipid ratio, the size of the nanoparticles can be tuned at convenience for the incorporation of protein complexes of different size. However, an increase in the size of the lipid nanoparticles may correlate with increased sample heterogeneity, thus hampering their application to spectroscopic and structural techniques where highly homogeneous samples are desirable. In addition, size homogeneity can be affected by low liposome solubilization efficiency by DIBMA, which carries a negative charge, in the presence of high lipid charge density. In this work, we apply biophysical tools to characterize the size and size heterogeneity of large (above 15 nm) lipid nanoparticles encased by the diisobutylene/maleic acid (DIBMA) copolymer at different DIBMA/lipid ratios and percentages of anionic lipids. Importantly, for nanoparticle preparations in the diameter range of 40 nm or below, the size homogeneity of the DIBMA/lipid nanoparticles (DIBMALPs) remains unchanged. In addition, we show that anionic lipids do not affect the production, size and size homogeneity of DIBMALPs. Furthermore, they do not affect the overall lipid dynamics in the membrane, and preserve the functionality of an enclosed membrane protein. This work strengthens the suitability of DIBMALPs as universal, native-like lipid environments for functional studies of membrane proteins and provide useful insight on the suitability of these systems for those structural techniques requiring highly homogeneous sample preparations.

Bacterioruberin from Haloarchaea plus dexamethasone in ultra-small macrophage-targeted nanoparticles as potential intestinal repairing agent.

Oral administration of antioxidant and anti-inflammatory drugs have the potential to improve the current therapy of inflammatory bowel disease. Success of oral treatments, however, depends on the capacity of drugs to remain structurally stable along the gastrointestinal tract, and on the feasibility of accessing the target cells. Delivering anti-inflammatory and antioxidant drugs to macrophages using targeted nanoparticles, could make treatments more efficient. In this work structural features and in vitro activity of macrophage-targeted nanostructured archaeolipid carriers (NAC) containing the high antioxidant dipolar C50 carotenoid bacterioruberin (BR) plus dexamethasone (Dex): NAC-Dex, are described. Ultra-small (66 nm), -32 mV ζ potential, 1200 µg Dex /ml NAC-Dex, consisted of a compritol and BR core, covered by a shell of sn 2,3 ether linked archaeolipids and Tween 80 (2: 2: 1.2: 3 % w/w) were obtained. NAC-Dex were extensively captured by macrophages and Caco-2 cells and displayed high anti-inflammatory and antioxidant activities on a gut inflammation model made of Caco-2 cells and lipopolysaccharide stimulated THP-1 derived macrophages reducing 65 % and 55 % TNF-α and IL-8 release, respectively and 60 % reactive oxygen species production. NAC-Dex also reversed the morphological changes induced by inflammation and increased the transepithelial electrical resistance, partly reconstituting the barrier function. Activity of BR and Dex in NAC-Dex was partially protected after simulated gastrointestinal digestion, improving the chances of BR-Dex joint activity. Results suggest that oral NAC-Dex deserve further exploration as intestinal barrier repairing agent.

Light-induced silencing of neural activity in Rosa26 knock-in and BAC transgenic mice conditionally expressing the microbial halorhodopsin eNpHR3.

An engineered light-inducible chloride pump, Natronomonas pharaonis halorhodopsin 3 (eNpHR3) enables temporally and spatially precise inhibition of genetically defined cell populations in the intact nervous tissues. In this report, we show the generation of new mouse strains that express eNpHR3-EYFP fusion proteins after Cre- and/or Flp-mediated recombination to optically inhibit neuronal activity. In these mouse strains, Cre/Flp recombination induced high levels of opsin expression. We confirmed their light-induced activities by brain slice whole-cell patch clamp experiments. eNpHR3-expressing neurons were optically hyperpolarized and silenced from firing action potentials. In prolonged silencing of action potentials, eNpHR3 was superior to eNpHR2, a former version of the engineered pump. Thus, these eNpHR3 mouse strains offer reliable genetic tools for light-induced inhibiting of neuronal activity in defined sets of neurons.

MBSP1: a biosurfactant protein derived from a metagenomic library with activity in oil degradation.

Microorganisms represent the most abundant biomass on the planet; however, because of several cultivation technique limitations, most of this genetic patrimony has been inaccessible. Due to the advent of metagenomic methodologies, such limitations have been overcome. Prevailing over these limitations enabled the genetic pool of non-cultivable microorganisms to be exploited for improvements in the development of biotechnological products. By utilising a metagenomic approach, we identified a new gene related to biosurfactant production and hydrocarbon degradation. Environmental DNA was extracted from soil samples collected on the banks of the Jundiaí River (Natal, Brazil), and a metagenomic library was constructed. Functional screening identified the clone 3C6, which was positive for the biosurfactant protein and revealed an open reading frame (ORF) with high similarity to sequences encoding a hypothetical protein from species of the family Halobacteriaceae. This protein was purified and exhibited biosurfactant activity. Due to these properties, this protein was named metagenomic biosurfactant protein 1 (MBSP1). In addition, E. coli RosettaTM (DE3) strain cells transformed with the MBSP1 clone showed an increase in aliphatic hydrocarbon degradation. In this study, we described a single gene encoding a protein with marked tensoactive properties that can be produced in a host cell, such as Escherichia coli, without substrate dependence. Furthermore, MBSP1 has been demonstrated as the first protein with these characteristics described in the Archaea or Bacteria domains.

Natronorubrum halophilum sp. nov. isolated from two inland salt lakes.

Two halophilic archaeal strains, SHR37T and NEN6, were isolated from salt lakes located in the Tibet and Xinjiang regions of China. The two strains were found to form a single cluster (99.9% and 99.3% similarity, respectively) separating them from the six current members of Natronorubrum (94.7-96.9% and 86.1-90.8% similarity, respectively) on the basis of the 16S rRNA and rpoB' gene sequence similarities and phylogenetic analysis. Diverse phenotypic characteristics differentiate strains SHR37T and NEN6 from current Natronorubrum members. Their polar lipids are C20C20 and C20C25glycerol diether derivatives of PG, PGP-Me, and a major gycolipid chromatographically identical to disulfated mannosyl glucosyl diether (S2-DGD). Four minor unidentified gycolipids are also present. The OrthoANI and in silico DDH values of the two strains were 97.3% and 76.1%, respectively, which were much higher than the threshold values proposed as a species boundary (ANI 95-96% and in silico DDH 70%), which revealed that the two strains represent one species; the two values (ANI 79.0-81.9% and in silico DDH 23.5-25.7%) of the strains examined in this study and the current members of Natronorubrum are much lower than the recommended threshold values, suggesting that strains SHR37T and NEN6 represent a genomically different species of Natronorubrum. These results showed that strains SHR37T (= CGMCC 1.15233T = JCM 30845T) and NEN6 (= CGMCC 1.17161) represent a novel species of Natronorubrum, for which the name Natronorubrum halophilum sp. nov. is proposed.

Production of Polyhydroxyalkanoates by Two Halophilic Archaeal Isolates from Chott El Jerid Using Inexpensive Carbon Sources.

The large use of conventional plastics has resulted in serious environmental problems. Polyhydroxyalkanoates represent a potent replacement to synthetic plastics because of their biodegradable nature. This study aimed to screen bacteria and archaea isolated from an extreme environment, the salt lake Chott El Jerid for the accumulation of these inclusions. Among them, two archaeal strains showed positive results with phenotypic and genotypic methods. Phylogenetic analysis, based on the 16S rRNA gene, indicated that polyhydroxyalkanoate (PHA)-producing archaeal isolates CEJGTEA101 and CEJEA36 were related to Natrinema altunense and Haloterrigena jeotgali, respectively. Gas chromatography and UV-visible spectrophotometric analyses revealed that the PHA were identified as polyhydroxybutyrate and polyhydroxyvalerate, respectively. According to gas chromatography analysis, the strain CEJGTEA101 produced maximum yield of 7 wt % at 37 °C; pH 6.5; 20% NaCl and the strain CEJEA36 produced 3.6 wt % at 37 °C; pH 7; 25% NaCl in a medium supplemented with 2% glucose. Under nutritionally optimal cultivation conditions, polymers were extracted from these strains and were determined by gravimetric analysis yielding PHA production of 35% and 25% of cell dry weight. In conclusion, optimization of PHA production from inexpensive industrial wastes and carbon sources has considerable interest for reducing costs and obtaining high yield.

Halobellus captivus sp. nov., an extremely halophilic archaeon isolated from a subterranean salt mine.

An extremely halophilic archaeon, strain ZY21T, was isolated from a subterranean rock salt sample in Yunnan, China. Colonies of strain ZY21T on nutrient-rich agar plates are orange, wet and transparent. Cells are pleomorphic, motile, Gram-stain negative and lyse in distilled water. Cells can grow at 20-55 °C (optimum 42 °C), in the presence of 15-30% (w/v) NaCl (optimum 18-20%) and at pH 6.0-9.5 (optimum 7.5). Mg2+ is required for growth (optimum 0.3 M). The major polar lipids of strain ZY21T are phosphatidylglycerol, phosphatidylglycerol sulfate and phosphatidylglycerol phosphate methyl ester, sulfated mannosyl-glucosyl-glycerol diether-1 and seven unidentified glycolipids. Sequence similarity searches with the 16S rRNA gene and rpoB' gene showed that strain ZY21T is closely related to Halobellus rufus CBA1103T (sequence similarities: 97.5% for 16S rRNA gene and 93.3% for rpoB' gene). The DNA G+C content of strain ZY21T was determined to be 63.0 mol% based on the draft genome sequence. Genome-based sequence similarity analysis showed that the values of the ANI, AAI, and DDH were far below the boundary for delineation of new species. Phenotypic, chemotaxonomic characteristics and phylogenetic properties suggest that strain ZY21T represents a novel species in the genus Halobellus, for which the name Halobellus captivus sp. nov. is proposed. The type strain is ZY21T (= CGMCC 1.16343T = NBRC 113439T).

Heterologous and Homologous Expression of Proteins from Haloarchaea: Denitrification as Case of Study.

Haloarchaea (halophilic microbes belonging to the Archaea domain) are microorganisms requiring mid or even high salt concentrations to be alive. The molecular machinery of these organisms is adapted to such conditions, which are stressful for most life forms. Among their molecular adaptations, halophilic proteins are characterized by their high content of acidic amino acids (Aspartate (Asp) and glumate (Glu)), being only stable in solutions containing high salt concentration (between 1 and 4 M total salt concentration). Recent knowledge about haloarchaeal peptides, proteins, and enzymes have revealed that many haloarchaeal species produce proteins of interest due to their potential applications in biotechnology-based industries. Although proteins of interest are usually overproduced in recombinant prokaryotic or eukaryotic expression systems, these procedures do not accurately work for halophilic proteins, mainly if such proteins contain metallocofactors in their structures. This work summarizes the main challenges of heterologous and homologous expression of enzymes from haloarchaea, paying special attention to the metalloenzymes involved in the pathway of denitrification (anaerobic reduction of nitrate to dinitrogen), a pathway with significant implications in wastewater treatment, climate change, and biosensor design.

Physiological response to silver toxicity in the extremely halophilic archaeon Halomicrobium mukohataei.

Adaptive strategies responsible for heavy metal tolerance were explored in the extremely halophilic archaeon Halomicrobium mukohataei DSM 12286. The tested strain was seemingly able to overcome silver-induced oxidative stress (assessed by malondialdehyde quantification, catalase assay and total antioxidant capacity measurement) mainly through non-enzymatic antioxidants. Energy dispersive spectrometry analysis illustrated the presence of colloidal silver in Hmc. mukohataei cultures exposed to AgNO3. Bright-field and transmission electron microscopy images, as well as dynamic light scattering analysis, demonstrated the presence of intracellular nanoparticles, mostly spherical, within a size range of 20-100 nm. As determined by the zeta potential measurement, the biosynthesized nanoparticles were highly stable, with a negative surface charge. Our research is a first attempt in the systematic study of the oxidative stress and intracellular silver nanoparticle accumulation, generated by exposure to silver ions, in members of Halobacteria class, thus broadening our knowledge on mechanisms supporting heavy metal tolerance of microbial cells living under saline conditions.

Discovery of fructans in Archaea.

Fructans are fructose-based oligo- and polysaccharides derived from sucrose that occur in a plethora of Eubacteria and plants. While fructan-producing (fructanogenic) Eubacteria are abundant in hypersaline environments, fructan production by Archaea has never been reported before. Exopolysaccharides accumulated by various Archaea from the Halobacteria class (belonging to the genera of Halomicrobium, Haloferax and Natronococcus) originating from different locations on Earth were structurally characterized as either levans or inulins with varying branching degrees (10%-16%). Thus, we show for the first time in the literature that fructans are produced in all three domains of life, including Archaea. This proof of concept will not only provide insight into Archaeal glycans and evolution but it may also open new frontiers for innovative strategies to overcome the ever-increasing threat of excessive salinization.