Halophilic Archaeon Research Articles

Natronomonas salsuginis sp. nov., a New Inhabitant of a Marine Solar Saltern.

A halophilic archaeon, strain F20-122T, was isolated from a marine saltern of Isla Bacuta (Huelva, Spain). Cells were Gram-stain-negative, aerobic, and coccoid in morphology. It grew at 25–50 °C (optimum 37 °C), pH 6.5–9.0 (optimum pH 8.0), and 10–30% (w/v) total salts (optimum 25% salts). The phylogenetic analyses based on the 16S rRNA and rpoB’ genes showed its affiliation with the genus Natronomonas and suggested its placement as a new species within this genus. The in silico DNA–DNA hybridization (DDH) and average nucleotide identity (ANI) analyses of this strain against closely related species supported its placement in a new taxon. The DNA G + C content of this isolate was 63.0 mol%. The polar lipids of strain F20-122T were phosphatidylglycerol phosphate methyl ester (PGP-Me), phosphatidylglycerol (PG), and phosphatidylglycerol sulfate (PGS). Traces of biphosphatidylglycerol (BPG) and other minor phospholipids and unidentified glycolipids were also present. Based on the phylogenetic, genomic, phenotypic, and chemotaxonomic characterization, we propose strain F20-122T (= CCM 8891T = CECT 9564T = JCM 33320T) as the type strain of a new species within the genus Natronomonas, with the name Natronomonas salsuginis sp. nov. Rhodopsin-like sequence analysis of strain F20-122T revealed the presence of haloarchaeal proton pumps, suggesting a light-mediated ATP synthesis for this strain and a maximum wavelength absorption in the green spectrum.

Salinibaculum litoreum gen. nov., sp. nov., isolated from salted brown alga Laminaria.

A novel Gram-stain-negative, aerobic and rod-shaped halophilic archaeon, designated HD8-45T, was isolated from the red brine of salted brown alga Laminaria produced at Dalian, PR China. According to the results of 16S rRNA gene and rpoB' gene sequence comparisons, strain HD8-45T showed the highest sequence similarity to the corresponding genes of Salinirussus salinus YGH44T (95.1 and 85.2 % similarities, respectively), Halovenus aranensis EB27T (91.2 and 86.0 % similarities, respectively). The low sequence similarity and the phylogeny implied the novel generic status of strain HD8-45T. Genomic relatedness analyses showed that strain HD8-45T were clearly distinguished from other species in the order Halobacteriales, with average nucleotide identity, amino acid identity and in silico DNA-DNA hybridization values not more than 75.1, 65.6 and 21.5 %. The polar lipid pattern contained phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, two major glycolipids and two minor glycolipids. The two major glycolipids and a minor glycolipid were chromatographically identical to disulfated mannosyl glucosyl diether, sulfated mannosyl glucosyl diether and mannosyl glucosyl diether, respectively. The major respiratory quinones were menaquinone MK-8 and MK-8(H2). The DNA G+C content was 62.0 mol% (Tm) and 61.9 mol% (genome). All these results showed that strain HD8-45T represents a novel species of a new genus in the order Halobacteriales, for which the name Salinibaculum litoreum gen. nov., sp. nov. is proposed. The type strain of Salinibaculum litoreum is HD8-45T (=CGMCC 1.15328T=JCM 31107T).

New proposal of nitrogen metabolism regulation by small RNAs in the extreme halophilic archaeon Haloferax mediterranei.

The regulatory networks involved in the uptake and metabolism of different nitrogen sources in response to their availability are crucial in all organisms. Nitrogen metabolism pathways have been studied in detail in archaea such as the extreme halophilic archaeon Haloferax mediterranei. However, knowledge about nitrogen metabolism regulation in haloarchaea is very scarce, and no transcriptional regulators involved in nitrogen metabolism have been identified to date. Advances in the molecular biology field have revealed that many small RNAs (sRNAs) are involved in the regulation of a diverse metabolic pathways. Surprisingly, no studies on regulation mediated by sRNAs have focused on the response to environmental fluctuations in nitrogen in haloarchaea. To identify sRNAs involved in the transcriptional regulation of nitrogen assimilation genes in Haloferax mediterranei and, thus, propose a novel regulatory mechanism, RNA-Seq was performed using cells grown in the presence of two different nitrogen sources. The differential transcriptional expression analysis of the RNA-Seq data revealed differences in the transcription patterns of 102 sRNAs according to the nitrogen source, and the molecular functions, cellular locations and biological processes with which the target genes were associated were predicted. These results enabled the identification of four sRNAs that could be directly related to the regulation of genes involved in nitrogen metabolism. This work provides the first proposed regulatory mechanism of nitrogen assimilation-related gene expression by sRNAs in haloarchaea as an alternative to transcriptional regulation mediated by proteins.

Cadmium and proton adsorption onto a halophilic archaeal species: The role of cell envelope sulfhydryl sites

The adsorption of metal onto archaea could affect metal behavior and bioavailability in hypersaline environments where archaea represent the dominant life form. However, the mechanism and thermodynamics of metal adsorption onto halophilic archaea are not well understood. In this study, Cd adsorption experiments and potentiometric titrations were conducted to model metal adsorption by the halophilic archaeon Halobacterium sp.. Cd adsorption onto the archaeal cells exhibits slow adsorption kinetics, taking 8 h to reach steady-state. The extent of Cd adsorption depends strongly on pCH+ and Cd loading. Desorption of Cd is even slower than adsorption, but exposure of the Cd-adsorbed biomass to high concentrations of cysteine promotes desorption and demonstrates that the distribution of Cd is controlled by reversible adsorption reactions, likely involving sulfhydryl binding of Cd on the cell surface. We conducted potentiometric titration experiments with and without sulfhydryl site blocking to yield a total sulfhydryl site concentration of 91 ± 28 μmol/g within the archaeal cell envelope. Using a combination of the X-ray absorption fine structure analysis results from Showalter et al. (2016) and the Cd adsorption measurements and titration data from this study, we construct a surface complexation model of the Cd adsorption behavior. Because the Cd adsorption behavior is likely similar to that of a range of chalcophilic micronutrients, the importance of Cd-sulfhydryl binding suggests that the production of high affinity sulfhydryl binding sites by the archaea may represent an adaptive strategy for halophiles to obtain metal nutrients in environments in which aqueous metal-chloride complexation dominates the speciation for many metals.

Haloterrigena sp. Strain SGH1, a Bacterioruberin-Rich, Perchlorate-Tolerant Halophilic Archaeon Isolated From Halite Microbial Communities, Atacama Desert, Chile.

An extreme halophilic archaeon, strain SGH1, is a novel microorganism isolated from endolithic microbial communities colonizing halites at Salar Grande, Atacama Desert, in northern Chile. Our study provides structural, biochemical, genomic, and physiological information on this new isolate living at the edge of the physical and chemical extremes at the Atacama Desert. SGH1 is a Gram-negative, red-pigmented, non-motile unicellular coccoid organism. Under the transmission electron microscope, strain SGH1 showed an abundant electro-dense material surrounding electron-lucent globular structures resembling gas vacuoles. Strain SGH1 showed a 16S rRNA gene sequence with a close phylogenetic relationship to the extreme halophilic archaea Haloterrigena turkmenica and Haloterrigena salina and has been denominated Haloterrigena sp. strain SGH1. Strain SGH1 grew at 20–40°C (optimum 37°C), at salinities between 15 and 30% (w/v) NaCl (optimum 25%) and growth was improved by addition of 50 mM KCl and 0.5% w/v casamino acids. Growth was severely restricted at salinities below 15% NaCl and cell lysis is avoided at a minimal 10% NaCl. Maximal concentrations of magnesium chloride and sodium or magnesium perchlorates that supported SGH1 growth were 0.5 and 0.15M, respectively. Haloterrigena sp. strain SGH1 accumulates bacterioruberin (BR), a C50 xanthophyll, as the major carotenoid. Total carotenoids in strain SGH1 amounted to nearly 400 μg BR per gram of dry biomass. Nearly 80% of total carotenoids accumulated as geometric isomers of BR: all-trans-BR (50%), 5-cis-BR (15%), 9-cis-BR (10%), 13-cis-BR (4%); other carotenoids were dehydrated derivatives of BR. Carotenogenesis in SGH1 was a reversible and salt-dependent process; transferring BR-rich cells grown in 25% (w/v) NaCl to 15% (w/v) NaCl medium resulted in depigmentation, and BR content was recovered after transference and growth of unpigmented cells to high salinity medium. Methanol extracts and purified BR isomers showed an 8–9-fold higher antioxidant activity than Trolox or β-carotene. Both, plasma membrane integrity and mitochondrial membrane potential measurements under acute 18-h assays showed that purified BR isomers were non-toxic to cultured human THP-1 cells.

Surviving salt fluctuations: stress and recovery in Halobacterium salinarum, an extreme halophilic Archaeon

Halophilic proteins subjected to below about 15% salt in vitro denature through misfolding, aggregation and/or precipitation. Halobacteria, however, have been detected in environments of fluctuating salinity such as coastal salterns and even around fresh water springs in the depths of the Dead Sea. In order to identify the underlying mechanisms of low salt survival, we explored the reactivation capacity of Halobacterium (Hbt) salinarum sub-populations after incubation in low salt media and recovery in physiological salt. Respiratory oxygen consumption was assessed in stressed cells and cell viability was estimated by Live/Dead staining and flow cytometry. In vivo neutron scattering experiments showed that the recovery of Hbt salinarum sub-populations exposed to severe low salt conditions is related to a rapid retrieval of functional molecular dynamics in the proteome. In the hypothesis that the observations on Hbt salinarum have wider relevance, they could be of key ecological significance for the dispersion of extremophiles when environmental fluctuations become severe.

Genome Sequence of Salarchaeum sp. Strain JOR-1, an Extremely Halophilic Archaeon from the Dead Sea.

An extremely halophilic archaeon, Salarchaeum sp. strain JOR-1, was isolated from the east coast of the Dead Sea, Kingdom of Jordan, and sequenced using single-molecule real-time (SMRT) sequencing. The GC-rich 2.5-Mbp genome was composed of a circular chromosome and a megaplasmid. The genome contained 2,633 genes and was incorporated into HaloWeb (https://halo.umbc.edu/).

How to Cope With Heavy Metal Ions: Cellular and Proteome-Level Stress Response to Divalent Copper and Nickel in Halobacterium salinarum R1 Planktonic and Biofilm Cells.

Halobacterium salinarum R1 is an extremely halophilic archaeon capable of adhesion and forming biofilms, allowing it to adjust to a range of growth conditions. We have recently shown that living in biofilms facilitates its survival under Cu2+ and Ni2+ stress, with specific rearrangements of the biofilm architecture observed following exposition. In this study, quantitative analyses were performed by SWATH mass spectrometry to determine the respective proteomes of planktonic and biofilm cells after exposition to Cu2+ and Ni2+.Quantitative data for 1180 proteins were obtained, corresponding to 46% of the predicted proteome. In planktonic cells, 234 of 1180 proteins showed significant abundance changes after metal ion treatment, of which 47% occurred in Cu2+ and Ni2+ treated samples. In biofilms, significant changes were detected for 52 proteins. Only three proteins changed under both conditions, suggesting metal-specific stress responses in biofilms. Deletion strains were generated to assess the potential role of selected target genes. Strongest effects were observed for ΔOE5245F and ΔOE2816F strains which exhibited increased and decreased biofilm mass after Ni2+ exposure, respectively. Moreover, EPS obviously plays a crucial role in H. salinarum metal ion resistance. Further efforts are required to elucidate the molecular basis and interplay of additional resistance mechanisms.

Unique Features of a New Baeyer–Villiger Monooxygenase from a Halophilic Archaeon

Type I Baeyer–Villiger monooxygenases (BVMOs) are flavin-dependent monooxygenases that catalyze the oxidation of ketones to esters or lactones, a reaction otherwise performed in chemical processes by employing hazardous and toxic peracids. Even though various BVMOs are extensively studied for their promising role in industrial biotechnology, there is still a demand for enzymes that are able to retain activity at high saline concentrations. To this aim, and based on comparative in silico analyses, we cloned HtBVMO from the extremely halophilic archaeon Haloterrigena turkmenica DSM 5511. When expressed in standard mesophilic cell factories, proteins adapted to hypersaline environments often behave similarly to intrinsically disordered polypeptides. Nevertheless, we managed to express HtBVMO in Escherichia coli and could purify it as active enzyme. The enzyme was characterized in terms of its salt-dependent activity and resistance to some water–organic-solvent mixtures. Although HtBVMO does not seem suitable for industrial applications, it provides a peculiar example of an alkalophilic and halophilic BVMO characterized by an extremely negative charge. Insights into the behavior and structural properties of such salt-requiring may contribute to more efficient strategies for engineering the tuned stability and solubility of existing BVMOs.

From Gene to Function: A Safe Laboratory Exercise Promoting the Learning of the Central Dogma of Biology for the Undergraduate Biology Curriculum.

The central dogma of biology, which explains how the information in genes flows into proteins, can be challenging to teach in the undergraduate classroom. To allow students to understand the correlation between genes and protein functions, we have developed a practical laboratory activity that complements classroom learning. By using PCR analysis targeting the flagellin gene flgA1, students will investigate the genotype of an unknown strain of Haloferax volcanii, a halophilic archaeon appropriate for activities with undergraduates as it is nonpathogenic, inexpensive, easy to grow using “grocery store ingredients,” and offers many genetic tools. This species swims by means of flagella, and its motility can be tested using modified agar plates. Motile colonies will form swimming halos; meanwhile motility mutants will show only a dot on the motility plate, at the point of inoculation. First students will extract DNA for PCR amplification. They will not be told whether the strain they are assigned is motile or not. They will learn how to design primers for the target gene and set up the PCR reaction. Subsequently they will set up a motility assay and control of the provided strain. In the next laboratory period, students perform gel electrophoresis on the products of their PCR reactions and analyze the results. They compare these to the results of the motility assay to confirm the motility of the strain. In summary, this inquiry-based lab is easy and safe to perform and allows students to follow the information flow from a gene to a protein product.

Catalytic mechanism and evolutionary characteristics of thioredoxin from Halobacterium salinarum NRC-1

Thioredoxin (TRX) is an important antioxidant against oxidative stress. TRX from the extremely halophilic archaeon Halobacterium salinarum NRC-1 (HsTRX-A), which has the highest acidic residue content [(Asp + Glu)/(Arg + Lys + His) = 9.0] among known TRXs, was chosen to elucidate the catalytic mechanism and evolutionary characteristics associated with haloadaptation. X-ray crystallographic analysis revealed that the main-chain structure of HsTRX-A is similar to those of homologous TRXs; for example, the root-mean-square deviations on Cα atoms were <2.3 Å for extant archaeal TRXs and <1.5 Å for resurrected Precambrian TRXs. A unique water network was located near the active-site residues (Cys45 and Cys48) in HsTRX-A, which may enhance the proton transfer required for the reduction of substrates under a high-salt environment. The high density of negative charges on the molecular surface (3.6 × 10-3 e Å-2) should improve the solubility and haloadaptivity. Moreover, circular-dichroism measurements and enzymatic assays using a mutant HsTRX-A with deletion of the long flexible N-terminal region (Ala2-Pro17) revealed that Ala2-Pro17 improves the structural stability and the enzymatic activity of HsTRX-A under high-salt environments (>2 M NaCl). The elongation of the N-terminal region in HsTRX-A accompanies the increased hydrophilicity and acidic residue content but does not affect the structure of the active site. These observations offer insights into molecular evolution for haloadaptation and potential applications in halophilic protein-related biotechnology.

Verification of the Stabilized Protein Design Based on the Prediction of Intrinsically Disordered Regions: Ribosomal Proteins L1.

In our previous papers, we proposed the idea that programs predicting intrinsically disordered regions in amino acid sequences can be used for finding weakened sites in proteins. The regions predicted by such programs are suitable targets for the introduction of protein-stabilizing mutations. However, for each specific protein, it remains unclear what determines protein stabilization - the amino acid sequence (and accordingly, prediction of weakened sites) or the 3D structure. To answer this question, it is necessary to study two proteins with similar structures but different amino acid sequences and, consequently, different predictions of weakened regions. By introducing identical mutations into identical elements of the two proteins, we will be able to reveal whether predictions of the weakened sites or the 3D protein structure are the key factors in the protein stability increase. Here, we have chosen ribosomal proteins L1 from the halophilic archaeon Haloarcula marismortui (HmaL1) and extremophilic bacterium Aquifex aeolicus (AaeL1). These proteins are identical in their structure but different in amino acid sequences. A disulfide bond introduced into the region predicted as the structured one in AaeL1 did not lead to the increase in the protein melting temperature. At the same time, a disulfide bond introduced into the same region in HmaL1 that was predicted as a weakened one, resulted in the increase in the protein melting temperature by approximately 10°C.

Whole-genome comparison between the type strain of Halobacterium salinarum (DSM 3754T ) and the laboratory strains R1 and NRC-1.

Halobacterium salinarum is an extremely halophilic archaeon that is widely distributed in hypersaline environments and was originally isolated as a spoilage organism of salted fish and hides. The type strain 91‐R6 (DSM 3754T) has seldom been studied and its genome sequence has only recently been determined by our group. The exact relationship between the type strain and two widely used model strains, NRC‐1 and R1, has not been described before. The genome of Hbt. salinarum strain 91‐R6 consists of a chromosome (2.17 Mb) and two large plasmids (148 and 102 kb, with 39,230 bp being duplicated). Cytosine residues are methylated (m4C) within CTAG motifs. The genomes of type and laboratory strains are closely related, their chromosomes sharing average nucleotide identity (ANIb) values of 98% and in silico DNA–DNA hybridization (DDH) values of 95%. The chromosomes are completely colinear, do not show genome rearrangement, and matching segments show <1% sequence difference. Among the strain‐specific sequences are three large chromosomal replacement regions (>10 kb). The well‐studied AT‐rich island (61 kb) of the laboratory strains is replaced by a distinct AT‐rich sequence (47 kb) in 91‐R6. Another large replacement (91‐R6: 78 kb, R1: 44 kb) codes for distinct homologs of proteins involved in motility and N‐glycosylation. Most (107 kb) of plasmid pHSAL1 (91‐R6) is very closely related to part of plasmid pHS3 (R1) and codes for essential genes (e.g. arginine‐tRNA ligase and the pyrimidine biosynthesis enzyme aspartate carbamoyltransferase). Part of pHS3 (42.5 kb total) is closely related to the largest strain‐specific sequence (164 kb) in the type strain chromosome. Genome sequencing unraveled the close relationship between the Hbt. salinarum type strain and two well‐studied laboratory strains at the DNA and protein levels. Although an independent isolate, the type strain shows a remarkably low evolutionary difference to the laboratory strains.

A new GH13 subfamily represented by the α-amylase from the halophilic archaeon Haloarcula hispanica.

α-Amylase catalyzes the endohydrolysis of α-1,4-glucosidic linkages in starch and related α-glucans. In the CAZy database, most α-amylases have been classified into the family GH13 counting at present more than 80,000 sequences and ~ 30 different enzyme specificities. The family has already been divided into 42 subfamilies, but additional subfamilies are still emerging. The present bioinformatics study was undertaken in an effort to propose a novel GH13 subfamily around the experimentally characterized α-amylase from the halophilic archaeon Haloarcula hispanica, which until now has not been assigned to any GH13 subfamily. The in silico analysis resulted in collecting a convincing group of putative haloarchaeal α-amylase homologues sharing sequence similarities mainly in their conserved sequence regions (CSRs) and forming a cluster in the evolutionary tree, which is well separated from representatives of established GH13 subfamilies. One of the most exclusive sequence features of the novel GH13 subfamily is the tyrosine (Tyr79 in H. hispanica α-amylase numbering) succeeding the glycine at the beginning of the CSR-VI at the β2 strand of the catalytic TIM-barrel. Evolutionarily, the novel GH13 α-amylase subfamily was most closely related to two clusters of GH13 subfamilies with the specificity of α-amylase, i.e. subfamilies GH13_5, 6 and 7 as well as GH13_15, 24, 27 and 28.

Draft Genome Sequence of Haloferax volcanii SS0101, Isolated from Salt Farms in Samut Sakhon, Thailand.

Haloferax volcanii SS0101 is a halophilic archaeon isolated from salt farms in Thailand. The genome sequence of H. volcanii SS0101 contains a gene encoding capreomycidine synthase, a key enzyme for capreomycidine biosynthesis. This 3.8-Mb draft genome sequence of H. volcanii SS0101 will provide the tools for investigating genes involved in capeomycidine production in haloarchaea.

Halorientalis pallida sp. nov., an extremely halophilic archaeon isolated from a marine saltern.

An extremely halophilic archaeon, strain F13-25T, was isolated from a marine saltern located in Isla Cristina, Huelva, on the south-west coast of Spain. The novel strain had pink-pigmented, non-motile, coccoid cells. Optimal growth was achieved at 25 % (w/v) NaCl, pH 7.5 and 37 °C. Strain F13-25T possessed two heterogeneous 16S rRNA genes (rrnA and rrnB) most closely related to Halorientalis persicus D108T (97.6-99.2 % sequence similarity) and Halorientalis regularis TNN28T (95.9-98.8 %). On the basis of the results of rpoB' gene sequence analysis, strain F13-25T was also closely related to Halorientalis persicus IBRC-M 10043T (89.9 %) and Halorientalis regularis TNN28T (92.3 %). Relatedness values, computed using the Genome-to-Genome Distance Calculator, between strain F13-25T and Halorientalis persicus IBRC-M 10043T and Halorientalis regularis IBRC-M 10760T were 34.6 and 36.2 %, respectively. Average nucleotide identity values based on orthoANI, ANIb and ANIm of strain F13-25T and Halorientalis persicus IBRC-M 10043T and Halorientalisregularis IBRC-M 10760T were 88.0 and 88.8, 87.1 and 87.6 %, and 89.2 and 89.6 %, respectively. All values were far below the threshold accepted for prokaryotic species delineation. The major polar lipids were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and one glycolipid chromatographically identical to sulfated diglycosyl diether. The DNA G+C content was 65.7 mol% (genome). The results of phylogenetic, phenotypic and chemotaxonomic analyses indicated that strain F13-25T represents a novel species of the genus Halorientalis, for which the name Halorientalis pallida sp. nov., with type strain F13-25T (=CECT 9384T=IBRC-M 11176T), is proposed.

The extremely halophilic archaeon Halobacterium salinarum ETD5 from the solar saltern of Sfax (Tunisia) produces multiple halocins

The extremely halophilic archaeon Halobacterium salinarum strain ETD5 was previously isolated from the solar saltern of Sfax (Tunisia) and shown to encode and express halocin S8. The Hbt. salinarum ETD5 culture supernatant was shown here to exhibit high antimicrobial activity against several halophilic archaea and bacteria of different genera, showing a cross-domain inhibition. The antimicrobial activity was destroyed by proteases, thus pointing to halocins. A bioguided purification procedure was applied using two chromatography steps and antimicrobial assays directed against Halorubrum chaoviator ETR14. In-gel screening assay showed the presence of two antimicrobial bands of approximately 8 and 14 kDa, for which characterization was investigated by N-terminal sequencing and mass spectrometry. The full-length form of halocin S8 that contains 81 amino acids and differs from the 36 amino acid short-length halocin S8 previously described from an uncharacterized haloarchaeon S8a, was identified in the 8 kDa halocin band. A novel halocin that we termed halocin S14 was found in the 14 kDa band. It exhibits amino acid sequence identities with the N-terminally truncated region of the archaeal Mn-superoxide dismutase. These results show that Hbt. salinarum ETD5 produces multiple halocins, a feature that had not been described until now in the domain Archaea.

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 0.3M). 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 63.0mol% 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).

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 20-100nm. 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.

A CUGGU/UUGGU-specific MazF homologue from Methanohalobium evestigatum

MazF is a sequence-specific endoribonuclease or mRNA interferase, which cleaves RNA at a specific sequence. Since the expression of a specific gene or a group of specific genes can be regulated by MazF, expanding the repertoire of recognition sequences by MazF mRNA interferases is highly desirable for biotechnological and medical applications. Here, we identified a gene for a MazF homologue (MazFme) from Methanohalobium evestigatum, an extremely halophilic archaeon. In order to suppress the toxicity of MazFme to the E. coli cells, the C-terminal half of the cognate antitoxin MazEme was fused to the N-terminal end of MazFme. Since the fusion of the C-terminal half of MazEme to MazFme was able to neutralize MazFme toxicity, the MazEme-MazFme fusion protein was expressed in a large amount without any toxic effects. After purification of the MazEme, the free MazFme RNA cleavage specificity was determined by primer extension and synthetic ribonucleotides, revealing that MazFme is a CUGGU/UUGGU-specific endoribonuclease.