Haloferax Research Articles

Techno-Economic Analysis on an Industrial-Scale Production System of Polyhydroxyalkanoates (PHA) from Cheese By-Products by Halophiles

Polyhydroxyalkanoates (PHA) are a family of biodegradable plastics used as an ecofriendly alternative for conventional plastics in various applications. In this study, an industrial-scale PHA production system was designed and analyzed for the material flows and economics with the use of SuperPro Designer. Haloferax mediterranei was utilized to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). Byproduct streams from a local cheese plant, with an input of 168.7 metric ton/day (MT/day) lactose, were used as the feedstock. Three scenarios with different processes for the treatments of used enzyme and spent medium were investigated and the major factors that influence the overall economics were identified. The simulated system produces 9700 MT/year PHBV with a yield of 0.2 g PHBV/g lactose and an overall process efficiency of 87%. The breakeven price was found to be more sensitive to the lactose price than enzyme price. The scenario with enzyme reuse and spent medium recycling achieved the lowest breakeven price among others, which can be less than 4 $/kg PHA based on the delactosed permeate (DLP) unit price. The study suggests utilizing dairy derived feedstocks has the potential to make PHA competitive in the bioplastic market, which could be beneficial to both dairy and bioplastic industries.

Haloarchaea have a high genomic diversity for the biosynthesis of carotenoids of biotechnological interest

Haloarchaea are mostly components of the microbial biomass of saline aquatic environments, where they can be a dietary source of heterotrophic metazoans or contribute to flamingo's plumage coloration. The diversity of secondary metabolites (SMs) produced by haloarchaea, which might play multiple ecological roles and have diverse biotechnological applications has been largely understudied. Herein, 67 haloarchaeal complete genomes were analyzed and 182 SMs biosynthetic gene clusters (BGCs) identified that encode the production of terpenes (including carotenoids), RiPPs and siderophores. Terpene BGCs were further analysed and it was concluded that all haloarchaea might produce squalene and bacterioruberin, which one a strong antioxidant. Most of them have other carotenoid BGCs that include a putative β-carotene ketolase that was not characterized so far in haloarchaea, but may be involved with canthaxanthin's biosynthesis. The production of bacterioruberin by Haloferax mediterranei ATCC 33500 was found to be not related to its antimicrobial activity.

Enhanced biosynthesis of polyhydroxyalkanoates by continuous feeding of volatile fatty acids in Haloferax mediterranei

In this work, the biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) in Haloferax mediterranei was enhanced by continuous feeding of volatile fatty acids. Using this strategy, polymer production was doubled to around 5 g L–1 PHBV when compared to pulse-fed fed-batch fermentations. Polymer productivity and yield increased up to 12.8 mg L–1 h–1 and 0.63 g g–1 respectively when the carbon concentration in the fermentation media was kept constant at 0.25 molar. This biopolymer production was achieved in less than half the time when compared to pulse feeding, effectively quadrupling the overall PHA productivity. Control over co-polymer composition was achieved and maintained at around 40 mol% 3-hydroxyvalerate (3HV). A correlation between substrate consumption and cell growth was observed, providing a crucial tool for feeding rate selection in future fermentations. The higher productivity and yield obtained with the novel feeding strategy will be key to future industrial scale PHA production.

Classification and Identification of Archaea Using Single-Cell Raman Ejection and Artificial Intelligence: Implications for Investigating Uncultivated Microorganisms.

Archaea can produce special cellular components such as polyhydroxyalkanoates, carotenoids, rhodopsin, and ether lipids, which have valuable applications in medicine and green energy production. Most of the archaeal species are uncultivated, posing challenges to investigating their biomarker components and biochemical properties. In this study, we applied Raman spectroscopy to examine the biological characteristics of nine archaeal isolates, including halophilic archaea (Haloferax larsenii, Haloarcula argentinensis, Haloferax mediterranei, Halomicrobium mukohataei, Halomicrobium salinus, Halorussus sp., Natrinema gari), thermophilic archaea (Sulfolobus acidocaldarius), and marine group I (MGI) archaea (Nitrosopumilus maritimus). Linear discriminant analysis of the Raman spectra allowed visualization of significant separations among the nine archaeal isolates. Machine-learning classification models based on support vector machine achieved accuracies of 88-100% when classifying the nine archaeal species. The predicted results were validated by DNA sequencing analysis of cells isolated from the mixture by Raman-activated cell sorting. Raman spectra of uncultured archaea (MGII) were also obtained based on Raman spectroscopy and fluorescence in situ hybridization. The results combining multiple Raman-based techniques indicated that MGII may have the ability to produce lipids distinct from other archaeal species. Our study provides a valuable approach for investigating and classifying archaea, especially uncultured species, at the single-cell level.

Kinetics of DPPH\u2022 scavenging by bacterioruberin from Haloferax alexandrinus GUSF-1 (KF796625)

This is the first account of the kinetics of free radical scavenging by bacterioruberin obtained from cells of Haloferax alexandrinus GUSF-1(KF796625), grown at optimum conditions of 25% NaCl, pH 7, 42 °C, 150 rpm in NaCl Tryptone yeast extract medium and light. Bacterioruberin separated from methanolic extract displayed characteristics absorption peaks at 368, 386, 463, 492 and 525 nm and gave an m/z value of 740.4 (C50H76O4) in Liquid Chromatography-Mass Spectroscopy validating its purity. Bacterioruberin (13 µM) decolorized and decayed 0.2 mM 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH•) monitored at 517 nm and reached a steady state within 30 min. An EC50 of 6.50 µM ± 0.27 (4.81 µg/mL ± 0.2) was deduced for the 0.2 mM DPPH•-bacterioruberin reaction using the GraphPad Prism 9 statistical software and employing the right-angled triangle technique. The study also revealed a comprehensive information of the total kinetic activity of bacterioruberin with DPPH•: the antioxidant activity index was 16.38 ± 0.67; time needed to reach the steady state with the added EC50—30 min; the antiradical power 30.77 ± 1.27 and the antiradical efficiency of 54.7 × 10–3 ± 2.24, thus reflecting the strong antioxidant nature of bacterioruberin. Scavenging of DPPH• by bacterioruberin was a pseudo-first-order reaction with a rate constant k2 of 2.76 × 10–5 ± 0.001 µM−1 s−1 calculated at t = 0 or initial time and t = 30 min. The knowledge of the kinetics of bacterioruberin to scavenge DPPH• adds to its effective application as an antioxidant in medicinal use, pharmaceutical products and others. Additionally, the use of simple conventional method of DPPH• free radical scavenging, monitored using an easily available laboratory spectrophotometer, will certainly help in the effective use of any antioxidant compound.

The lanthipeptide biosynthetic clusters of the domain Archaea

Research on Archaea’s secondary metabolites is still lagging behind that of Bacteria and Eukarya. Our goal was to contribute to this knowledge gap by analyzing the lanthipeptide's clusters in Archaea. As previously proposed, Archaea encodes only class II synthetases (LanMs), which we found to be confined to the class Halobacteria (also known as haloarchaea). In total, we analyzed the phylogeny and the domains of 42 LanMs. Four types were identified, and the majority of them belong to the CCG group due to their cyclization domain, which includes LanMs of Cyanobacteria. Putative cognate peptides were predicted for most of LanMs and are a very diverse group of molecules that share a Kx(Y/F)(D/E)xx(F/Y) motif in their leader peptides. According to their homology, some of them were categorized into subfamilies, including Halolancins, Haladacins, Haloferaxcins and Halobiforcins. Many LanM genes were associated with mobile genetic elements, and their vicinities mainly encode ABC and MFS transporters, tailoring enzymes and uncharacterized proteins.Our results suggest that the biosynthesis of lanthipeptides in haloarchaea can entail distinct enzymology that must lead to the production of peptides with novel structures and unpredicted biological and ecological roles. Finally, an Haloferax mediterranei knockout, lacking its three lanM genes, was generated, and it was concluded that its antimicrobial activity is not primarily related to the production of lanthipeptides.

Evaluating haloarchaeal culture media for ultrahigh-molecular-weight polyhydroxyalkanoate biosynthesis by Haloferax mediterranei.

A series of culture media for haloarchaea were evaluated to optimize the production of ultrahigh-molecular-weight (UHMW) poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by Haloferax mediterranei. Cells of H. mediterranei grew (> 1g/L of dry cell weight) and accumulated PHBV upon flask cultivation in 10 medium types with neutral pH and NaCl concentration > 100g/L. Molecular weight and compositional analysis revealed that the number-average molecular weight (Mn) of PHBV produced with six selected types of media ranged from 0.8 to 3.5 × 106g/mol and the 3-hydroxyvalerate (3HV) composition ranged from 8 to 36mol%. Cultivation in two NBRC media, 1214 and 1380, resulted in the production of PHBV with an Mn of more than 3.0 × 106g/mol and a weight-average molecular weight of more than 5.0 × 106g/mol, indicating the production of UHMW-PHBV. These culture media contained small amount of complex nutrients like yeast extract and casamino acids, suggesting that H. mediterranei likely produced UHMW-PHBV on poor nutrient condition. Haloferax mediterranei grown in NBRC medium 1380 produced PHBV with the highest 3HV composition. A solvent-cast film of UHMW-PHBV with 26.4mol% 3HV produced from 1-L flask cultivation with NBRC medium 1380 was found to be flexible and semi-transparent. Thermal analysis of the UHMW-PHBV cast film revealed melting and glass-transition temperatures of 90.5°C and - 2.7°C, respectively. KEY POINTS: • Haloarchaeal culture media were evaluated to produce UHMW-PHBV by H. mediterranei. • UHMW-PHBV with varied molecular weight was produced dependent on culture media. • Semi-transparent film could be made from UHMW-PHBV with 26.4mol% 3HV.

Optimising PHBV biopolymer production in haloarchaea via CRISPRi-mediated redirection of carbon flux

The haloarchaeon Haloferax mediterranei is a potential strain for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production, yet the production yield and cost are the major obstacles hindering the use of this archaeal strain. Leveraging the endogenous type I-B CRISPR-Cas system in H. mediterranei, we develop a CRISPR-based interference (CRISPRi) approach that allows to regulate the metabolic pathways related to PHBV synthesis, thereby enhancing PHBV production. Our CRISPRi approach can downregulate the gene expression in a range of 25% to 98% depending upon the target region. Importantly, plasmid-mediated CRISPRi downregulation on the citrate synthase genes (citZ and gltA) improves the PHBV accumulation by 76.4% (from 1.78 to 3.14 g/L). When crRNA cassette integrated into chromosome, this further shortens the PHBV fermentation period and enhances PHA productivity by 165%. Our transcriptome analysis shows that repression of citrate synthase genes redirects metabolic flux from the central metabolic pathways to PHBV synthesis pathway. These findings demonstrate that the CRISPRi-based gene regulation is a transformative toolkit for fine-tuning the endogenous metabolic pathways in the archaeal system, which can be applied to not only the biopolymer production but also many other applications.

Novel Glutamate-Putrescine Ligase Activity in Haloferax mediterranei: A New Function for glnA-2 Gene.

The genome of the halophilic archaea Haloferax mediterranei contains three ORFs that show homology with glutamine synthetase (GS) (glnA-1, glnA-2, and glnA-3). Previous studies have focused on the role of GlnA-1, suggesting that proteins GlnA-2 and GlnA-3 could play a different role to that of GS. Glutamine synthetase (EC 6.3.1.2) belongs to the class of ligases, including 20 subclasses of other different enzymes, such as aspartate–ammonia ligase (EC 6.3.1.1), glutamate–ethylamine ligase (EC 6.3.1.6), and glutamate–putrescine ligase (EC 6.3.1.11). The reaction catalyzed by glutamate–putrescine ligase is comparable to the reaction catalyzed by glutamine synthetase (GS). Both enzymes can bind a glutamate molecule to an amino group: ammonium (GS) or putrescine (glutamate–putrescine ligase). In addition, they present the characteristic catalytic domain of GS, showing significant similarities in their structure. Although these proteins are annotated as GS, the bioinformatics and experimental results obtained in this work indicate that the GlnA-2 protein (HFX_1688) is a glutamate–putrescine ligase, involved in polyamine catabolism. The most significant results are those related to glutamate–putrescine ligase’s activity and the analysis of the transcriptional and translational expression of the glnA-2 gene in the presence of different nitrogen sources. This work confirms a new metabolic pathway in the Archaea domain which extends the knowledge regarding the utilization of alternative nitrogen sources in this domain.

Biomineralization of Fe3+ to Nanosized γFe2O3 by Haloferax alexandrinus GUSF-1

Hypersaline environment is a habitat with extreme osmotic conditions along with low Aw, serving as a home to extremely halophilic and halotolerant bacteria. The hypersaline environments, such as solar salterns located along the rivers, are exposed to fluxes of iron from iron ore transportation and other industrial wastes. The solar salterns often serve as a sink for metal intoxicants. Studies on archaea interaction with metal ions indicate the formation of minerals such as goethite, hematite, rhodochrosite, etc. However, studies exploring haloarchaeal candidates interacting with metals such as Fe3+ in a hypersaline growth condition are scarce. This study unveils for the first time formation of γFe2O3 from Fe3+ by the haloarchaeon, Haloferax sp. GUSF-1 thus implying the significance of the culture synthesizing minerals in hypersaline sediments. γFe2O3 is formed from Fe3+ by the haloarchaeon Haloferax sp. GUSF-1 (GenBank accession no.GU-1KF796625), under microaerophilic growth on sodium acetate. A 50 mg L−1 of Fe2+ and 30.6 mg L−1 of Fe3+ was detected inside the cells. Simultaneously, a brown-colored crystalline material deposited in the culture broth through an iron reductase inhibited by Zn2+ ions. The XRD of the deposit exhibited d values of 2.96, 2.514, 2.086, 1.6, and 1.45, while SEM-EDX displayed cubic and irregularly shaped minute particles with peaks for Fe at 0.6, 6.4, and 6.6 keV, respectively. TEM profiles revealed polycrystalline particles of 12–23 nm in size. Further, the SAED concentric pattern of light scattering with well-defined diffraction spots was consistent and matched with maghemite's crystal structure (γFe2O3). The FTIR spectrum revealed a peak at 1450 cm−1 indicating iron oxyhydroxide formation as an intermediate having γ-FeOOH stretching bond vibrations. Conclusively, this study opens the possibility of the haloarchaea isolated from solar salterns for its exploitation in nanobiotechnology.

In Silico Analysis of the Enzymes Involved in Haloarchaeal Denitrification.

During the last century, anthropogenic activities such as fertilization have led to an increase in pollution in many ecosystems by nitrogen compounds. Consequently, researchers aim to reduce nitrogen pollutants following different strategies. Some haloarchaea, owing to their denitrifier metabolism, have been proposed as good model organisms for the removal of not only nitrate, nitrite, and ammonium, but also (per)chlorates and bromate in brines and saline wastewater. Bacterial denitrification has been extensively described at the physiological, biochemical, and genetic levels. However, their haloarchaea counterparts remain poorly described. In previous work the model structure of nitric oxide reductase was analysed. In this study, a bioinformatic analysis of the sequences and the structural models of the nitrate, nitrite and nitrous oxide reductases has been described for the first time in the haloarchaeon model Haloferax mediterranei. The main residues involved in the catalytic mechanism and in the coordination of the metal centres have been explored to shed light on their structural characterization and classification. These results set the basis for understanding the molecular mechanism for haloarchaeal denitrification, necessary for the use and optimization of these microorganisms in bioremediation of saline environments among other potential applications including bioremediation of industrial waters.

Anti-Pseudomonas aeruginosa biofilm activity of tellurium nanorods biosynthesized by cell lysate of Haloferax alexandrinus GUSF-1(KF796625).

Pseudomonas aeruginosa, an opportunistic human pathogen, is a major health concern as it grows as a biofilm and evades the host's immune defenses. Formation of biofilms on catheter and endotracheal tubes demands the development of biofilm-preventive (anti-biofilm) approaches and evaluation of nanomaterials as alternatives to antibiotics. The present study reports the successful biosynthesis of tellurium nanorods using cell lysate of Haloferax alexandrinus GUSF-1 (KF796625). The black particulate matter had absorption bands at 0.5 and 3.6keV suggestive of elemental tellurium; showed x-ray diffraction peaks at 2θ values 24.50°, 28.74°, 38.99°, 43.13°, 50.23° and displayed a crystallite size of 36.99nm. The black nanorods of tellurium were an average size of 40nm × 7nm, as observed in transmission electron microscopy. To our knowledge, the use of cell lysate of Haloferax alexandrinus GUSF-1 (KF796625) as a green route for the biosynthesis of tellurium nanorods with a Pseudomonas aeruginosa biofilm inhibiting capacity is novel to haloarchaea. At 50µgmL-1, these tellurium nanorods exhibited 75.03% in-vitro reduction of biofilms of Pseudomonas aeruginosa ATCC 9027, comparable to that of ciprofloxacin, which is used in treatment of Pseudomonas infections. Further, the observed ability of these nanoparticles to inhibit the formation of Pseudomonas biofilms is worthy of future research perusal.

Analysis of Haloferax mediterranei Lrp Transcriptional Regulator.

Haloferax mediterranei is an extremely halophilic archaeon, able to live in hypersaline environments with versatile nutritional requirements, whose study represents an excellent basis in the field of biotechnology. The transcriptional machinery in Archaea combines the eukaryotic basal apparatus and the bacterial regulation mechanisms. However, little is known about molecular mechanisms of gene expression regulation compared with Bacteria, particularly in Haloarchaea. The genome of Hfx. mediterranei contains a gene, lrp (HFX_RS01210), which encodes a transcriptional factor belonging to Lrp/AsnC family. It is located downstream of the glutamine synthetase gene (HFX_RS01205), an enzyme involved in ammonium assimilation and amino acid metabolism. To study this transcriptional factor more deeply, the lrp gene has been homologously overexpressed and purified under native conditions by two chromatographic steps, namely nickel affinity and gel filtration chromatography, showing that Lrp behaves asa tetrameric protein of approximately 67 kDa. Its promoter region has been characterized under different growth conditions using bgaH as a reporter gene. The amount of Lrp protein was also analyzed by Western blotting in different nitrogen sources and under various stress conditions. To sum up, regarding its involvement in the nitrogen cycle, it has been shown that its expression profile does not change in response to the nitrogen sources tested. Differences in its expression pattern have been observed under different stress conditions, such as in the presence of hydrogen peroxide or heavy metals. According to these results, the Lrp seems to be involved in a general response against stress factors, acting as a first-line transcriptional regulator.

Analysis of Polyhydroxyalkanoates Granules in Haloferax mediterranei by Double-Fluorescence Staining with Nile Red and SYBR Green by Confocal Fluorescence Microscopy.

Haloferax mediterranei is a haloarchaeon of high interest in biotechnology because it produces and mobilizes intracellular polyhydroxyalkanoate (PHA) granules during growth under stress conditions (limitation of phosphorous in the culture media), among other interesting metabolites (enzymes, carotenoids, etc.). The capability of PHA production by microbes can be monitored with the use of staining-based methods. However, the staining of haloarchaea cells is a challenging task; firstly, due to the high ionic strength of the medium, which is inappropriate for most of dyes, and secondly, due to the low permeability of the haloarchaea S-layer to macromolecules. In this work, Haloferax mediterranei is used as a halophilic archaeon model to describe an optimized protocol for the visualization and analysis of intracellular PHA granules in living cells. The method is based on double-fluorescence staining using Nile red and SYBR Green by confocal fluorescence microscopy. Thanks to this method, the capability of PHA production by new haloarchaea isolates could be easily monitored.

Functional analysis of Lsm protein under multiple stress conditions in the extreme haloarchaeon Haloferax mediterranei

The Sm, like-Sm, and Hfq proteins belonging to the Sm superfamily of proteins are represented in all domains of life. These proteins are involved in several RNA metabolism pathways. The functions of bacterial Hfq and eukaryotic Sm proteins have been described, but knowledge about the in vivo functions of archaeal Sm proteins remains limited. This study aims to improve the understanding of Lsm proteins and their role using the haloarchaeon Haloferax mediterranei as a model microorganism. The Haloferax mediterranei genome contains one lsm gene that overlaps with the rpl37e gene. To determine the expression of lsm and rpl37e genes and the co-transcription of both, reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed under different standard and stress conditions. The results suggest that the expression of lsm and rpl37e is constitutive. Co-transcription occurs at sub-optimal salt concentrations and temperatures, depending on the growth phase. The halophilic Lsm protein contains two Sm motifs, Sm1 and Sm2, and the sequence encoding the Sm2 motif also constitutes the promoter of the rpl37e gene. To investigate their biological functions, the lsm deletion mutant and the Sm1 motif deletion mutant, where the Sm2 motif remained intact, were generated and characterised. Comparison of the lsm deletion mutant, Sm1 deletion mutant, and the parental strain HM26 under standard and stress growth conditions revealed growth differences. Finally, swarming assays in complex and defined media showed greater swarming capacity in the deletion mutants.

Towards the Elucidation of Assimilative nasABC Operon Transcriptional Regulation in Haloferax mediterranei.

The assimilatory pathway of the nitrogen cycle in the haloarchaeon Haloferax mediterranei has been well described and characterized in previous studies. However, the regulatory mechanisms involved in the gene expression of this pathway remain unknown in haloarchaea. This work focuses on elucidating the regulation at the transcriptional level of the assimilative nasABC operon (HFX_2002 to HFX_2004) through different approaches. Characterization of its promoter region using β-galactosidase as a reporter gene and site-directed mutagenesis has allowed us to identify possible candidate binding regions for a transcriptional factor. The identification of a potential transcriptional regulator related to nitrogen metabolism has become a real challenge due to the lack of information on haloarchaea. The investigation of protein–DNA binding by streptavidin bead pull-down analysis combined with mass spectrometry resulted in the in vitro identification of a transcriptional regulator belonging to the Lrp/AsnC family, which binds to the nasABC operon promoter (p.nasABC). To our knowledge, this study is the first report to suggest the AsnC transcriptional regulator as a powerful candidate to play a regulatory role in nasABC gene expression in Hfx. mediterranei and, in general, in the assimilatory nitrogen pathway.

Halolysin R4 of Haloferax mediterranei confers its host antagonistic and defensive activities.

Halolysins, which are subtilisin-like serine proteases of haloarchaea, are usually secreted into the extracellular matrix via the twin-arginine translocation pathway. A small number of activated molecules can greatly affect cell growth owing to their proteolytic activity. It is, however, unclear as to whether this proteolysis-based growth inhibition by halolysins conveys antagonistic or defensive effects against other resident abd potentially competitive microorganisms. Here, we report that halolysin R4 (HlyR4), encoded by the hlyR4 gene, is the key enzyme in the initial steps of extracellular protein utilization in Haloferax mediterranei HlyR4 shows significant antagonistic activity against other haloarchaeal strains. Deletion of hlyR4 completely halts the inhibition activity of Hfx. mediterranei towards other haloarchaea, while correspondingly, complementation of hlyR4 almost completely restores the inhibition activity. Furthermore, Hfx. mediterranei strains containing hlyR4 showed a certain amount of resistance to halocins and halolysins in milieu, and this function of hlyR4 is reproducible in Haloarcula hispanica The versatility of HlyR4 enables its host to outcompete other haloarchaea living in the same hypersaline environment. Intriguingly, unlike the growth phase-dependent halolysins SptA and Nep, it is likely that HlyR4 may be secreted independent of growth phase. This study provides a new peptide antibiotics candidate in haloarchaea, as well as new insight towards a better understanding of the ecological roles of halolysins.Importance: This study shows that halolysin R4 from Haloferax mediterranei provides its host antagonistic and defensive activities against other haloarchaea, which expands our knowledge on the traditional function of haloarchaeal extracellular proteases. Haloarchaeal extracellular serine proteases have been previously discussed as growth-phase-dependent proteins, whereas our study reports constitutive expression of halolysin R4. This work also clearly reveals a hidden diversity of extracellular proteases from haloarchaea. Studies on multifunctional halolysins reveal that they play an important ecological role in shaping microbial community composition and provide a new perspective towards understanding the intricate interactions between haloarchaeal cells in hypersaline environments. HlyR4 can lyse competing cells living in the same environment, and the cell debris may probably be utilized as nutrients, which may constitute an important part of nutrient cycling in extremely hypersaline environments.

Cellular and Genomic Properties of Haloferax gibbonsii LR2-5, the Host of Euryarchaeal Virus HFTV1.

Hypersaline environments are the source of many viruses infecting different species of halophilic euryarchaea. Information on infection mechanisms of archaeal viruses is scarce, due to the lack of genetically accessible virus–host models. Recently, a new archaeal siphovirus, Haloferax tailed virus 1 (HFTV1), was isolated together with its host belonging to the genus Haloferax, but it is not infectious on the widely used model euryarcheon Haloferax volcanii. To gain more insight into the biology of HFTV1 host strain LR2-5, we studied characteristics that might play a role in its virus susceptibility: growth-dependent motility, surface layer, filamentous surface structures, and cell shape. Its genome sequence showed that LR2-5 is a new strain of Haloferax gibbonsii. LR2-5 lacks obvious viral defense systems, such as CRISPR-Cas, and the composition of its cell surface is different from Hfx. volcanii, which might explain the different viral host range. This work provides first deep insights into the relationship between the host of halovirus HFTV1 and other members of the genus Haloferax. Given the close relationship to the genetically accessible Hfx. volcanii, LR2-5 has high potential as a new model for virus–host studies in euryarchaea.

Haloferax mediterranei Cells as C50 Carotenoid Factories.

Haloarchaea produce C50 carotenoids such as bacterioruberin, which are of biotechnological in-terest. This study aimed to analyze the effect of different environmental and nutritional conditions on the cellular growth and dynamics of carotenoids accumulation in Haloferax mediterranei. The maximum production of carotenoids (40 µg·mL−1) was obtained during the stationary phase of growth, probably due to nutrient-limiting conditions (one-step culture). By seven days of culture, 1 mL culture produced 22.4 mg of dry weight biomass containing 0.18 % (w/w) of carotenoids. On the other hand, carbon-deficient cultures (low C/N ratio) were observed to be optimum for C50 bacterioruberin production by Hfx. mediterranei, but negatively affected the growth of cells. Thus, a two-steps process was evaluated for optimum carotenoids yield. In the first step, a nutri-ent-repleted culture medium enabled the haloarchaea to produce biomass, while in the second step, the biomass was incubated under osmotic stress and in a carbon-deficient medium. Under the conditions used, the obtained biomass contained 0.27% (w/w) of carotenoids after seven days, which accounts for 58.49 µg·mL−1 of carotenoids for a culture with turbidity 14.0.

The Survival of Haloferax mediterranei under Stressful Conditions.

Haloarchaea can survive and thrive under exposure to a wide range of extreme environmental factors, which represents a potential interest to biotechnology. Growth responses to different stressful conditions were examined in the haloarchaeon Haloferax mediterranei R4. It has been demonstrated that this halophilic archaeon is able to grow between 10 and 32.5% (w/v) of sea water, at 32–52 °C, although it is expected to grow in temperatures lower than 32 °C, and between 5.75 and 8.75 of pH. Moreover, it can also grow under high metal concentrations (nickel, lithium, cobalt, arsenic), which are toxic to most living beings, making it a promising candidate for future biotechnological purposes and industrial applications. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analysis quantified the intracellular ion concentrations of these four metals in Hfx. mediterranei, concluding that this haloarchaeon can accumulate Li+, Co2+, As5+, and Ni2+ within the cell. This paper is the first report on Hfx. mediterranei in which multiple stress conditions have been studied to explore the mechanism of stress resistance. It constitutes the most detailed study in Haloarchaea, and, as a consequence, new biotechnological and industrial applications have emerged.