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The Archaea domain consists of a heterogeneous group of microorganisms with unique physiological properties that occupy a wide variety of niches in nature. Haloferax mediterranei is an extremely halophilic archaeon classified in the Phylum Euryarchaeota, which requires a high concentration of inorganic salts for optimal growth. In haloarchaea, transcription factors play a fundamental role in an adequate adaptation to environmental and nutritional changes, preserving the survival and integrity of the organism. To deepen knowledge of the Lrp/AsnC transcriptional regulator family, a lrp gene (HFX_RS01210) from this family has been studied. Site-directed mutagenesis has allowed us to identify the TATA-box and two potential sites of the transcriptional factor (TF) to its own promoter and autoregulate itself. Several approaches were carried out to elucidate whether this transcriptional regulator is involved in stresses due to heavy metals and limited nitrogen conditions. Characterization of the lrp deletion mutant and the Lrp overexpressed strain, suggests that the level of lrp expression depends on the nitrogen source and the presence of cobalt. The most striking results were obtained in the presence of nitrate as a nitrogen source due to the inability of the deletion mutant to grow. All these results confirm that Lrp is a powerful candidate for a regulatory role in the stress response, particularly under N-limiting conditions and the presence of cobalt. This work was supported by Programa Propio para el Fomento de la I + D + I del Vicerrectorado de Investigación y Transferencia de Conocimiento” of the University of Alicante (VIGRO-016).

Archaea are microorganisms with great ability to colonize some of the most inhospitable environments in nature, managing to survive in places with extreme characteristics for most microorganisms. Its proteins and enzymes are stable and can act under extreme conditions in which other proteins and enzymes would degrade. These attributes make them ideal candidates for use in a wide range of biotechnological applications. This review describes the most important applications, both current and potential, that archaea present in Biotechnology, classifying them according to the sector to which the application is directed. It also analyzes the advantages and disadvantages of its use.

Glutamine synthetase is an essential enzyme in ammonium assimilation and glutamine biosynthesis. The Haloferax mediterranei genome has two other glnA-type genes (glnA2 and glnA3) in addition to the glutamine synthetase gene glnA. To determine whether the glnA2 and glnA3 genes can replace glnA in nitrogen metabolism, we generated deletion mutants of glnA. The glnA deletion mutants could not be generated in a medium without glutamine, and thus, glnA is an essential gene in H. mediterranei. The glnA deletion mutant was achieved by adding 40 mM glutamine to the selective medium. This conditional HM26-ΔglnA mutant was characterised with different approaches in the presence of distinct nitrogen sources and nitrogen starvation. Transcriptomic analysis was performed to compare the expression profiles of the strains HM26-ΔglnA and HM26 under different growth conditions. The glnA deletion did not affect the expression of glnA2, glnA3 and nitrogen assimilation genes under nitrogen starvation. Moreover, the results showed that glnA, glnA2 and glnA3 were not expressed under the same conditions. These results indicated that glnA is an essential gene for H. mediterranei and, therefore, glnA2 and glnA3 cannot replace glnA in the conditions analysed. This work was funded by MICINN Grant Number BIO2013-42921P (to MJB), Generalitat Valenciana Grant Number ACIF/2018/200 (to GP) and Universidad de Alicante (VIGROB-016).

Halophilic archaea represent a promising natural source of carotenoids. However, little information is available about these archaeal metabolites and their biological effects. In the present work, carotenoids of strains Haloferax sp. ME16, Halogeometricum sp. ME3 and Haloarcula sp. BT9, isolated from Algerian salt lakes, were produced, extracted and identified by high‐performance liquid chromatography–diode array detector and liquid chromatography–mass spectrometry. Analytical results revealed a variation in the composition depending on the strain with a predominance of bacterioruberin. The evaluation of antioxidant capacity using ABTS [(2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] and DPPH (2,2-diphenyl-1-picrylhydrazyl) assays showed that these extracts have a strong antioxidant potential, in particular those of Haloferax sp. ME16 which displayed antioxidant power significantly higher than that of ascorbic acid used as standard. Antibacterial activity of carotenoid extracts against four human‐pathogenic strains and four fish‐pathogenic strains was evaluated by agar disk diffusion method. The results showed a good antibacterial activity. These findings suggest that the C50 carotenoids from the studied strains offer promising prospects for biotechnological applications. This work was supported by the Algerian Ministry of Higher Education and Scientific Research, FEDER Programa-Andalucia 2014–2020 (UHU-1257518) and the group (VIGROB-016) Universidad de Alicante.

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. This research was funded by Universidad de Alicante, VIGROB-016.

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. This work was supported by the Spanish Ministry of Economy and Competitiveness [grant BIO2013-42921-P]; Programa Propio para el fomento de la I+D+I del Vicerrectorado de Investigación y Transferencia de Conocimiento” of the University of Alicante (VIGRO-016) and the Generalitat Valenciana (Spain) [ACIF/2018/200].

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.

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.

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. This research was funded by Universidad de Alicante, VIGROB-016.

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. This study was funded by the Spanish Ministry of Economy and Competitiveness and FEDER (grant BIO2013-42921-P); the Generalitat Valenciana (Spain) (grant ACIF/2018/200); and the “Programa Propio para el Fomento de la I + D + i del Vicerrectorado de Investigación y Transferencia de Conocimiento” of the University of Alicante (Spain).

This work was funded by research grant from the MINECO Spain (CTM2013-43147-R) and Generalitat Valenciana (ACIF 2016/077).

A set of 110 extremely halophilic archaeal strains were isolated from seven distinct saline habitats located in different regions of Algeria. The physicochemical characterization of the samples showed that these habitats were thalassohaline. The carotenoid production from isolated strains varied from 0.1 to 3.68 µg/ml. Based on their physiological characteristics and pigment production, 43 strains were selected and identified by means of phenotypic tests and 16S ribosomal RNA gene sequencing. Phylogenetic analysis indicated that the isolates corresponded to the class Halobacteria and were closely related to genera Halorubrum, Haloarcula, Haloferax, Natrinema, Halogeometricum, Haloterrigena , and Halopiger . Carotenoids of the highest producer, strain Halorubrum sp. BS2 were identified using high‐performance liquid chromatography–diode array detector and liquid chromatography–mass spectrometry. Bacterioruberin and bisanhydrobacterioruberin were the predominant carotenoids. The scavenging activity of these carotenoids reached 99% at a concentration of 18 μg/ml, which was much higher than that of ascorbic acid used as a reference compound. These carotenoids also exhibited significant antibacterial activities against four human‐pathogenic strains and four fish‐pathogenic strains. Variations in salinity, agitation rate, temperature, and light intensity were found to influence growth and carotenoid production of Halorubrum sp. BS2. Our results suggest that halophilic archaea represent a potential source for carotenoids, which are characterized by high antioxidant and antibacterial activities. This study was supported by the Algerian Ministry of Higher Education and Scientific Research, European Research Grant (INTERREG VA POCTEP‐055 ALGARED_PLUS_5E) and the group (VIGROB‐016) Universidad de Alicante.

Background: Haloferax mediterranei is a denitrifying haloarchaeon using nitrate as a respiratory electron acceptor under anaerobic conditions in a reaction catalysed by pNarGH. Other ions such as bromate, perchlorate and chlorate can also be reduced. Methods: Hfx. mediterranei cells were grown anaerobically with nitrate as electron acceptor and chlorate reductase activity measured in whole cells and purified nitrate reductase. Results: No genes encoding (per)chlorate reductases have been detected either in the Hfx. mediterranei genome or in other haloarchaea. However, a gene encoding a chlorite dismutase that is predicted to be exported across the cytoplasmic membrane has been identified in Hfx. mediterranei genome. Cells did not grow anaerobically in presence of chlorate as the unique electron acceptor. However, cells anaerobically grown with nitrate and then transferred to chlorate-containing growth medium can grow a few generations. Chlorate reduction by the whole cells, as well as by pure pNarGH, has been characterised. No clear chlorite dismutase activity could be detected. Conclusions: Hfx. mediterranei pNarGH has its active site on the outer-face of the cytoplasmic membrane and reacts with chlorate and perchlorate. Biochemical characterisation of this enzymatic activity suggests that Hfx. mediterranei or its pure pNarGH could be of great interest for waste water treatments or to better understand biological chlorate reduction in early Earth or Martian environments. General significance: Some archaea species reduce (per)chlorate. However, results here presented as well as those recently reported by Liebensteiner and co-workers [1] suggest that complete perchlorate reduction in archaea follows different rules in terms of biological reactions. This work was funded by research grant from the MINECO Spain (CTM2013-43147-R), MEC Spain (BIO2008-00082), Generalitat Valenciana (GV/2011/038) and University of Alicante (GRE09-25).

Small RNAs have been studied in detail in domains Bacteria and Eukarya but, in the case of the domain Archaea, the knowledge is scarce and the physiological function of these small RNAs (sRNAs) is still uncertain. To extend the knowledge of sRNAs in the domain Archaea and their possible role in the regulation of the nitrogen assimilation metabolism in haloarchaea, Haloferax mediterranei has been used as a model microorganism. The bioinformatic approach has allowed for the prediction of 295 putative sRNAs genes in the genome of H. mediterranei, 88 of which have been verified by means of RNA-Sequencing (RNA-Seq). The secondary structure of these sRNAs and their possible targets have been identified. Curiously, some of them present as possible target genes relating to nitrogen assimilation, such as glutamate dehydrogenase and the nitrogen regulatory PII protein. Analysis of RNA-Seq data has also revealed differences in the expression pattern of 16 sRNAs according to the nitrogen source. Consequently, RNomic and bioinformatic approaches used in this work have allowed for the identification of new sRNAs in H. mediterranei, some of which show different expression patterns depending on the nitrogen source. This suggests that these sRNAs could be involved in the regulation of nitrogen assimilation and can constitute an important gene regulatory network. This work was supported by grant BIO2013-42921-P from the Spanish Ministry of Economy and Competitiveness. Funds from “Programa Propio para el fomento de la I+D+i del Vicerrectorado de Investigación y Transferencia de Conocimiento” de la Universidad de Alicante are also acknowledged.

Biotechnological processes and, in general, industrial processes require in many cases the use of catalyzers able to work at extreme conditions in terms of temperature, ionic strength, pressure, and extreme pH values, or even in the presence of toxic or heavy metals. Several approaches have been explored so far involving the use of mesophilic enzymes isolated mainly from bacteria and plants. However, these enzymes are not stable under the extreme conditions previously mentioned, and consequently, they are not active. For these reasons, extremophilic enzymes have been revealed as promising catalyzers to improve several important features sustaining the reactions that are the key processes in biotechnology. Thus, these enzymes are capable of catalyzing their respective reactions in nonaqueous environments, in water–solvent mixtures, at extremely high pressures, at acidic and alkaline pH, at temperatures up to 140°C, or near the freezing point of water. These enzymes, also called extremozymes, are produced by extremophiles, which are microorganisms able to grow and live in environments of extreme temperature (–2°C to 15°C for cold environments, 60°C–120°C in hot environments), ionic strength (2–5 M NaCl), or pH ( 9.0). These organisms mainly belong to the Bacteria and Archaea domains. Besides, extremozymes can be enhanced through genetic engineering to obtain biocatalysts more in line with the needs of each moment. This chapter summarizes the current knowledge about extremozymes and their potential uses in biotechnology, paying special attention to those isolated from members of the Archaea domain.

Several amylolytic activities have been isolated from a controlled growing media containing starch and nitrate or ammonium acetate as a carbon and energy source, excreted by the halophilic archaeon Haloferax mediterranei. These enzymes produced in nitrate-containing medium were different from those produced by the organism when cultured in ammonium acetate-containing medium. Haloferax mediterranei was able to grow optimally in both the media but not in a medium with ammonium chloride and starch as exclusive source of nitrogen and carbon, respectively. Growth was significantly lower when nitrate was replaced by ammonium, although there was significant amylolytic activity in the medium. At least six different activities were obtained in the nitrate-containing medium, but only five for the ammonium containing one. These enzymes displayed a different affinity for starch as a chromatographic matrix, when eluted with maltose in a range from 0.02 M to 0.2 M, and differed in their kinetic parameters for starch as a substrate. The average medium length of the products obtained from cracking starch was different for each amylolytic activity, ranging from glucose to larger polysaccharides. Moreover, they exhibited different molecular masses, from 15 to 80 kDa. On the other hand, all of them behaved as typical halophilic enzymes, requiring high salt concentrations from 2M to 4M NaCl for stability and activity. Also, it exhibited an optimal pH ranged from 7 to 8 and showed certain thermophilic behaviour, with maximal activity within 50°C to 60°C. The study of the presence and behaviour of this set of starch degrading enzymes will allow for a better understanding of how halophilic organism obtain the adequate carbohydrates to be incorporated and optimally used. This work was supported by project BIO2013-42921-P from the Spanish Ministry of Economy and Competitiveness.

Microorganisms, including Bacteria and Archaea, play a key role in denitrification, which is the major mechanism by which fixed nitrogen returns to the atmosphere from soil and water. While the enzymology of denitrification is well understood in Bacteria, the details of the last two reactions in this pathway, which catalyse the reduction of nitric oxide (NO) via nitrous oxide (N

Gene homologs of GlnK PII regulators and AmtB-type ammonium transporters are often paired on prokaryotic genomes, suggesting these proteins share an ancient functional relationship. Here, we demonstrate for the first time in Archaea that GlnK associates with AmtB in membrane fractions after ammonium shock, thus, providing a further insight into GlnK-AmtB as an ancient nitrogen sensor pair. For this work, Haloferax mediterranei was advanced for study through the generation of a pyrE2-based counterselection system that was used for targeted gene deletion and expression of Flag-tagged proteins from their native promoters. AmtB1-Flag was detected in membrane fractions of cells grown on nitrate and was found to coimmunoprecipitate with GlnK after ammonium shock. Thus, in analogy to bacteria, the archaeal GlnK PII may block the AmtB1 ammonium transporter under nitrogen-rich conditions. In addition to this regulated protein–protein interaction, the archaeal amtB-glnK gene pairs were found to be highly regulated by nitrogen availability with transcript levels high under conditions of nitrogen limitation and low during nitrogen excess. While transcript levels of glnK-amtB are similarly regulated by nitrogen availability in bacteria, transcriptional regulators of the bacterial glnK promoter including activation by the two-component signal transduction proteins NtrC (GlnG, NRI) and NtrB (GlnL, NRII) and sigma factor σN (σ54) are not conserved in archaea suggesting a novel mechanism of transcriptional control. This work was supported by project BIO2008-00082 from the Spanish Ministry of Science and Innovation MICINN awarded to MBP, which includes funding from the European Union (“FEDER”). During this work, L. P.-R. was supported by a fellowship (AP2007-02932) from the Spanish Ministry of Education. This work was also funded by project NIH R01 GM057498 from the National Institute of General Medical Science awarded to J. M.-F. and by project DOE DE-FG02-05ER15650 from the Chemical Sciences, Geosciences, and Biosciences Division of the Office of Basic Energy Sciences (BES) in the U.S. Department of Energy (DOE) awarded to J. M.-F.

In this work we report for the first time a post-translational modification of PII homologues from the Archaea Domain. Haloferax mediterranei is the first haloarchaea whose PII proteins have been studied, it possesses two of them (GlnK1 and GlnK2), both encoded adjacent to a gene for the ammonia transporter Amt. An approach based on 2DE, anti-GlnK immunoblot and peptide mass fingerprint (MALDI-TOF-MS) of the reactive spots showed that GlnK proteins in H. mediterranei are post-translationally uridylylated. A third spot with lower pI suggests the existence of a non-descript post-translational modification in this protein family. This work was supported by project BIO2008-00082 from the Spanish Ministry of Science and Innovation (MICINN), which includes funding from the European Union (“FEDER”). L.P.R. is supported by a fellowship (AP2007-02932) from the Ministry of Education.

GlnK proteins belong to the PII superfamily of signal transduction proteins and are involved in the regulation of nitrogen metabolism. These proteins are normally encoded in an operon together with the structural gene for the ammonium transporter AmtB. Haloferax mediterranei possesses two genes encoding for GlnK, specifically, glnK1 and glnK2. The present study marks the first investigation of PII proteins in haloarchaea, and provides evidence for the direct interaction between glutamine synthetase and both GlnK1 and GlnK2. Complex formation between glutamine synthetase and the two GlnK proteins is demonstrated with pure recombinant protein samples using in vitro activity assays, gel filtration chromatography and western blotting. This protein–protein interaction increases glutamine synthetase activity in the presence of 2-oxoglutarate. Separate experiments that were carried out with GlnK1 and GlnK2 produced equivalent results. This work was supported by project BIO2008-00082 from the Spanish Ministry of Science and Innovation (MICINN), which includes funding from the European Union (“FEDER”). LPR is supported by a fellowship (AP2007-02932) from the Ministry of Education.

Stegophorus macronectes (Johnston & Mawson, 1942) is a gastrointestinal parasite found in Antarctic seabirds. The original description of the species, which was based only on females, is poor and fragmented with some unclear diagnostic characters. This study provides new morphometric and molecular data on this previously poorly described parasite. Nuclear rDNA sequences (18S, 5.8S, 28S and internal transcribed spacer (ITS) regions) were isolated from S. macronectes specimens collected from the chinstrap penguin Pygoscelis antarctica Forster on Deception Island, Antarctica. Using 18S rDNA sequences, phylogenetic analyses (maximum likelihood, maximum parsimony and Bayesian inference) of the order Spirurida were performed to determine the phylogenetic location of this species. Primer pairs of the ITS regions were designed for genus-level identification of specimens, regardless of their cycle, as an alternative to coprological methods. The utility of this molecular method for identification of morphologically altered specimens is also discussed., This study was funded by the Spanish Ministry of Economy and Competiveness and the European Regional Development Fund Projects CGL2004-01348, POL2006-05175, CGL2007-60369 and CTM2011-24427. V.V. was supported by a PhD grant from the Spanish Council of Scientific Research (CSIC) and the European Social Fund (JAEPre08-01053).M.J.P.was supported by a PhDgrant from the Spanish Ministry of Science and Innovation (BES2005-8465).

A cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) was successfully isolated and characterized from the halophilic archaeon Haloferax mediterranei. The enzyme is a monomer with a molecular mass of 77 kDa and optimum activity at 55°C, pH 7.5 and 1.5 M NaCl. The enzyme displayed many activities related to the degradation and transformation of starch. Cyclization was found to be the predominant activity, yielding a mixture of cyclodextrins, mainly α-CD, followed by hydrolysis and to a lesser extent coupling and disproportionation activities. Gene encoding H. mediterranei CGTase was cloned and heterologously overexpressed. Sequence analysis revealed an open reading frame of 2142 bp that encodes a protein of 713 amino acids. The amino acid sequence displayed high homology with those belonging to the α-amylase family. The CGTase is secreted to the extracellular medium by the Tat pathway. Upstream of the CGTase gene, four maltose ABC transporter genes have been sequenced (malE, malF, malG, malK). The expression of the CGTase gene yielded a fully active CGTase with similar kinetic behavior to the wild-type enzyme. The H. mediterranei CGTase is the first halophilic archaeal CGTase characterized, sequenced and expressed.

Haloferax mediterranei is an extreme halophilic micro-organism belonging to the Archaea domain that was isolated from the Santa Pola solar salterns (Alicante, Spain) in 1983. The biochemistry of the proteins involved in nitrogen metabolism is being studied, but the knowledge of their regulation is very scarce at present. The PII superfamily is constituted by major regulators of nitrogen metabolism, which are widespread in prokaryotic and eukaryotic organisms. These trimeric proteins (12 kDa per subunit) have in Escherichia coli long been known to regulate GS (glutamine synthetase) activity via its adenylyltransferase/adenylyl-removing enzyme and, more recently, to be able to interact directly with this enzyme in methanogenic archaea. We have tested the possible role of PII proteins in the regulation of ammonium assimilation in our model organism and the results clearly indicate that the direct influence of GS by PII proteins can also take place in halophilic archaea, starting with the comprehension of nitrogen regulation in those organisms.

Structural analysis of glucose dehydrogenase from Haloferax mediterranei revealed that the adenosine 2′-phosphate of NADP + was stabilized by the side chains of Arg207 and Arg208. To investigate the structural determinants for coenzyme specificity, several mutants involving residues Gly206, Arg207 and Arg208 were engineered and kinetically characterized. The single mutants G206D and R207I were less efficient with NADP + than the wild type, and the double and triple mutants G206D/R207I and G206D/R207I/R208N showed no activity with NADP + . In the single mutant G206D, the relation k cat / K NA D + was 1.6 times higher than in the wild type, resulting in an enzyme that preferred NAD + over NADP + . The single mutation was sufficient to modify coenzyme specificity, whereas other dehydrogenases usually required more than one or two mutations to change coenzyme specificity. However, the highest reaction rates were reached with the double mutant G206D/R207I and with coenzyme NAD + , where the k cat was 1.6 times higher than the k cat of the wild-type enzyme with NADP + . However, catalytic efficiency with NAD + was lower, as the K m value for coenzyme was 77 times higher than the wild type with NADP + .

Despite being the subject of intensive investigations, many aspects of the mechanism of the zinc-dependent medium chain alcohol dehydrogenase (MDR) superfamily remain contentious. We have determined the high-resolution structures of a series of binary and ternary complexes of glucose dehydrogenase, an MDR enzyme from Haloferax mediterranei . In stark contrast to the textbook MDR mechanism in which the zinc ion is proposed to remain stationary and attached to a common set of protein ligands, analysis of these structures reveals that in each complex, there are dramatic differences in the nature of the zinc ligation. These changes arise as a direct consequence of linked movements of the zinc ion, a zinc-bound bound water molecule, and the substrate during progression through the reaction. These results provide evidence for the molecular basis of proton traffic during catalysis, a structural explanation for pentacoordinate zinc ion intermediates, a unifying view for the observed patterns of metal ligation in the MDR family, and highlight the importance of dynamic fluctuations at the metal center in changing the electrostatic potential in the active site, thereby influencing the proton traffic and hydride transfer events.

Halophilic archaea belong to the third domain of life, which live and survive in a highly salty environment. Nitrate assimilation is one of the main processes of the N-cycle, allowing the use of NO3−, NO2− and/or NH4+ as N source for growth. This pathway in general termed “Assimilatory nitrate pathway or assimilatory nitrate reduction” includes not only assimilatory nitrate reduction but also the assimilation of nitrite and ammonium. In the assimilatory nitrate reduction, NO3− is finally reduced to NH4+ by two sequential reactions catalysed by a ferredoxin-dependent nitrate reductase (Nas; EC 1.6.6.2) and a ferredoxin-dependent nitrite reductase (Nir; EC 1.7.7.1). The glutamine synthetase/glutamate synthase pathway (GS-GOGAT; EC 6.3.1.2, EC 1.4.7.1, respectively) or l-glutamate dehydrogenase (GDH; EC 1.4.1.2) are responsible for incorporating NH4+ into carbon skeletons. This chapter reviews current knowledge on nitrogen metabolism in haloarchaea with emphasis on assimilatory nitrate reduction, proteins involved and its regulation.

A number of species of Haloferax genus (halophilic archaea) are able to grow microaerobically or even anaerobically using different alternative electron acceptors such as fumarate, nitrate, chlorate, dimethyl sulphoxide, sulphide and/or trimethylamine. This metabolic capability is also shown by other species of the Halobacteriaceae and Haloferacaceae families (Archaea domain) and it has been mainly tested by physiological studies where cell growth is observed under anaerobic conditions in the presence of the mentioned compounds. This work summarises the main reported features on anaerobic metabolism in the Haloferax, one of the better described haloarchaeal genus with significant potential uses in biotechnology and bioremediation. Special attention has been paid to denitrification, also called nitrate respiration. This pathway has been studied so far from Haloferax mediterranei and Haloferax denitrificans mainly from biochemical point of view (purification and characterisation of the enzymes catalysing the two first reactions). However, gene expression and gene regulation is far from known at the time of writing this chapter.

Physiology, regulation and biochemical aspects of the nitrogen assimilation are well known in Prokarya or Eukarya but they are poorly described in Archaea domain. The haloarchaeon Haloferax mediterranei can use different nitrogen inorganic sources (NO 3 − , NO 2 − or NH 4 + ) for growth. Different approaches were considered to study the effect of NH 4 + on nitrogen assimilation in Hfx. mediterranei cells grown in KNO3 medium. The NH 4 + addition to KNO3 medium caused a decrease of assimilatory nitrate (Nas) and nitrite reductases (NiR) activities. Similar effects were observed when nitrate-growing cells were transferred to NH 4 + media. Both activities increased when NH 4 + was removed from culture, showing that the negative effect of NH 4 + on this pathway is reversible. These results suggest that ammonium causes the inhibition of the assimilatory nitrate pathway, while nitrate exerts a positive effect. This pattern has been confirmed by RT-PCR. In the presence of both NO 3 − and NH 4 + , NH 4 + was preferentially consumed, but NO 3 − uptake was not completely inhibited by NH 4 + at prolonged time scale. The addition of MSX to NH 4 + or NO 3 − cultures results in an increase of Nas and NiR activities, suggesting that NH 4 + assimilation, rather than NH 4 + per se, has a negative effect on assimilatory nitrate reduction in Hfx. mediterranei.

Haloferax mediterranei is a denitrifying halophilic archaeon, able to assimilate nitrate or nitrite in the presence of oxygen by the assimilatory nitrate pathway. It can also grow in the presence of high nitrate or nitrite concentrations under anoxic conditions, using both nitrogen species as electron acceptors. In this study, the ability of H. mediterranei to remove high nitrate and nitrite concentrations from culture media has been demonstrated. This suggests that this haloarchaeon could be applied in water bioremediation processes to repair damage caused by anthropogenic activities. This could be beneficial in regions such as Comunidad Valenciana or Murcia (Spain), where the water tables contain high nitrate and nitrite concentrations due to fertiliser addition, and high salt concentrations due to marine intrusions.

The production of pigments by halophilic archaea has been analysed during the last half a century. The main reasons that sustains this research are: (i) many haloarchaeal species possess high carotenoids production availability; (ii) downstream processes related to carotenoid isolation from haloarchaea is relatively quick, easy and cheap; (iii) carotenoids production by haloarchaea can be improved by genetic modification or even by modifying several cultivation aspects such as nutrition, growth pH, temperature, etc.; (iv) carotenoids are needed to support plant and animal life and human well-being; and (v) carotenoids are compounds highly demanded by pharmaceutical, cosmetic and food markets. Several studies about carotenoid production by haloarchaea have been reported so far, most of them focused on pigments isolation or carotenoids production under different culture conditions. However, the understanding of carotenoid metabolism, regulation, and roles of carotenoid derivatives in this group of extreme microorganisms remains mostly unrevealed. The uses of those haloarchaeal pigments have also been poorly explored. This work summarises what has been described so far about carotenoids production by haloarchaea and their potential uses in biotechnology and biomedicine. In particular, new scientific evidence of improved carotenoid production by one of the better known haloarchaeon (Haloferax mediterranei) is also discussed., This work was funded by research grant from the MINECO Spain (CTM2013-43147-R).

1) to propose a new classification of the trace elements based on a study of the recently reported research; 2) to offer detailed and actualized information about trace elements.the analysis of the research results recently reported reveals that the advances of the molecular analysis techniques point out the importance of certain trace elements in human health. A detailed analysis of the catalytic function related to several elements not considered essential o probably essentials up to now is also offered. To perform the integral analysis of the enzymes containing trace elements informatics tools have been used. Actualized information about physiological role, kinetics, metabolism, dietetic sources and factors promoting trace elements scarcity or toxicity is also presented.Oligotherapy uses catalytic active trace elements with therapeutic proposals. The new trace element classification here presented will be of high interest for different professional sectors: doctors and other professions related to medicine; nutritionist, pharmaceutics, etc. Using this new classification and approaches, new therapeutic strategies could be designed to mitigate symptomatology related to several pathologies, particularly carential and metabolic diseases.Objetivos: 1) Proponer una nueva clasificación de los oligoelementos fundamentada en el estudio detallado de las investigaciones más recientes sobre los mismos; 2) ofrecer información detallada y actualizada sobre todos los oligoelementos. Resultados: el análisis de todos los resultados de investigación consultados pone de manifiesto que los avances en las técnicas de análisis molecular permiten dilucidar la importancia que presentan ciertos oligoelementos para la salud humana. Se ofrece un análisis detallado de la función catalítica que podrían tener determinados elementos no considerados hasta ahora como esenciales o posiblemente esenciales, gracias al uso de plataformas informáticas que permiten el análisis integrado de datos sobre enzimas. Asimismo se presenta información integrada y actualizada del papel fisiológico, cinéticas y metabolismo, fuentes dietéticas y factores que propician la carencia o la toxicidad de cada uno de los oligoelementos. Conclusiones: La Oligoterapia plantea el uso de oligoelementos catalíticamente activos con fines terapéuticos. La nueva clasificación de oligoelementos planteada en este trabajo será de interés para diversos sectores profesionales: médicos y demás personal sanitario, nutricionistas, farmacéuticos, etc. Así podrán diseñarse nuevas estrategias terapéuticas que permitan paliar la sintomatología de diversas patologías, en particular las enfermedades carenciales y metabólicas.

The structure of glucose dehydrogenase from the extreme halophile Haloferax mediterranei has been solved at 1.6-Å resolution under crystallization conditions which closely mimic the “ in vivo ” intracellular environment. The decoration of the enzyme’s surface with acidic residues is only partially neutralized by bound potassium counterions, which also appear to play a role in substrate binding. The surface shows the expected reduction in hydrophobic character, surprisingly not from changes associated with the loss of exposed hydrophobic residues but rather arising from a loss of lysines consistent with the genome wide-reduction of this residue in extreme halophiles. The structure reveals a highly ordered, multilayered solvation shell that can be seen to be organized into one dominant network covering much of the exposed surface accessible area to an extent not seen in almost any other protein structure solved. This finding is consistent with the requirement of the enzyme to form a protective shell in a dehydrating environment.

Haloferax volcanii Ds-threo-isocitrate dehydrogenase (ICDH) was highly expressed in bacteria as inclusion bodies. The recombinant enzyme was refolded, purified and characterized, and was found to be NADP-dependent like the wild-type protein. Sequence alignment of several isocitrate dehydrogenases from evolutionarily divergent organisms including H. volcanii revealed that the amino acid residues involved in coenzyme specificity are highly conserved. Our objective was to switch the coenzyme specificity of halophilic ICDH by altering these conserved amino acids. We were able to switch coenzyme specificity from NADP+ to NAD+ by changing five amino acids by site-directed mutagenesis (Arg291, Lys343, Tyr344, Val350 and Tyr390). The five mutants of ICDH were overexpressed in Escherichia coli as inclusion bodies and each recombinant ICDH protein was refolded and purified, and its kinetic parameters were determined. Coenzyme specificity did not switch until all five amino acids were substituted.

The Haloferax mediterranei nar operon has been sequenced and its regulation has been characterized at transcriptional level. The nar operon encodes seven open reading frames(ORFs) (ORF1 narB, narC, ORF4, narG, narH, ORF7 and narJ). ORF1, ORF4 and ORF7 are open reading frames with no assigned function, however the rest of them encoded different proteins. narB codes for a 219-amino-acid-residue iron Rieske protein. narC encodes a protein of 486 amino acid residues identified by databases searches as cytochrome-b (narC). The narG gene encodes a protein with 983 amino acid residues and is identified as a respiratory nitrate reductase catalytic subunit (narG). NarH protein has been identified as an electron transfer respiratory nitrate reductase subunit (narH). The last ORF encodes a chaperonin-like protein (narJ) of 242 amino acid residues. The respiratory nitrate reductase was purified 21-fold from H. mediterranei membranes. Based on SDS-PAGE and gel-filtration chromatography under native conditions, the enzyme complex consists of two subunits of 112 and 61 kDa. The optimum temperature for activity was 70 degrees C at 3.4 M NaCl and the stability did not show a direct dependence on salt concentration. Respiratory nitrate reductase showed maximum activity at pH 7.9 and pH 8.2 when assays were carried out at 40 and 60 degrees C, respectively. The absorption spectrum indicated that Nar contains Fe-S clusters. Reverse transcriptase (RT-PCR) shows that regulation of nar genes occurs at transcriptional level induced by oxygen-limiting conditions and the presence of nitrate.

Reverse micelles were used as a cytoplasmic model to study the kinetics of an extreme halophilic enzyme such as the recombinant glucose dehydrogenase from the Archaeon Haloferax mediterranei. This enzyme was solubilized in reverse micelles of hexadecyltrimethylammoniumbromide in cyclohexane, with 1-butanol as co-surfactant. Glucose dehydrogenase retained its catalytic properties in this organic medium, showing good stability at low water content, even at low salt concentration (125 mM NaCl). The dependence of the enzymatic activity on the molar water surfactant ratio (w0=[H2O]/[surfactant]) increased with rising water content. Surprisingly, the activity of this extreme halophilic enzyme did not depend on the salt concentration in reverse micelles. The kinetic of the enzymatic oxidation of β-D-glucose to D-glucono-1,5-lactone using NADP+ as coenzyme for the glucose dehydrogenase from Haloferax mediterranei was also studied in the reverse micellar system.

Haloferax mediterranei is a halophilic archaeon that can grow in aerobic conditions with nitrate as sole nitrogen source. The electron donor in the aerobic nitrate reduction to ammonium was a ferredoxin. This ferredoxin has been purified and characterised. Air-oxidized H. mediterranei ferredoxin has a UV-visible absorption spectra typical of 2Fe-type ferredoxins with an A420/A280 of 0.21. The nuclear magnetic resonance (NMR) spectra of the ferredoxin showed similarity to those of ferredoxins from plant and bacteria, containing a [2Fe-2S] cluster. The physiological function of ferredoxin might be to serve as an electron donor for nitrate reduction to ammonium by assimilatory nitrate (EC 1.6.6.2) and nitrite reductases (EC 1.7.7.1). The apparent molecular weight (Mr) of the ferredoxin was estimated to be 21 kDa on SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

The extreme conditions under which haloarchaea survive make them good bioremediation agents in water treatment processes and in saline and hypersaline environments contaminated with toxic compounds such as nitrate, nitrite and ammonia, chlorine compounds such as perchlorate and chlorate, heavy metals, and aromatic compounds. New advances in the understanding of haloarchaea metabolism, biochemistry, and molecular biology suggest that general biochemical pathways related to nitrogen (Nitrogen cycle), metals (iron, mercury), hydrocarbons, or phenols can be used for bioremediation proposals. The main goal of the chapter is to present a review about the main characteristics of the archaeal species and their possible uses for bioremediation processes paying special attention to the Halobacteriaceae family. Several examples about the role of these microorganisms in salty brines or soils with high concentrations of nitrogenous compounds, heavy metals, aliphatic or aromatic hydrocarbons, or oxyanions are also discussed. This work was funded by a research grant from MINECO, Spain (CTM2013-43147-R).

The haloarchaeon Haloferax mediterranei is able to grow in the presence of different inorganic and organic nitrogen sources by means of the assimilatory pathway under aerobic conditions. In order to identify genes of potential importance in nitrogen metabolism and its regulation in the halophilic microorganism, we have analysed its global gene expression in three culture media with different nitrogen sources: (a) cells were grown stationary and exponentially in ammonium, (b) cells were grown exponentially in nitrate, and (c) cells were shifted to nitrogen starvation conditions. The main differences in the transcriptional profiles have been identified between the cultures with ammonium as nitrogen source and the cultures with nitrate or nitrogen starvation, supporting previous results which indicate the absence of ammonium as the factor responsible for the expression of genes involved in nitrate assimilation pathway. The results have also permitted the identification of transcriptional regulators and changes in metabolic pathways related to the catabolism and anabolism of amino acids or nucleotides. The microarray data was validated by real-time quantitative PCR on 4 selected genes involved in nitrogen metabolism. This work represents the first transcriptional profiles study related to nitrogen assimilation metabolism in extreme halophilic microorganisms using microarray technology. This work was supported by the Spanish Ministry of Science and Innovation (MICINN, Grant BIO2008-00082), the Spanish Ministry of Economy and Competitivity (MINECO, Grant BIO2013-42921-P) and by project AEST/2013/062 from the Generalitat Valenciana (Spain).

The kinetic mechanism of NADP-glutamate dehydrogenase (EC 1.4.1.4) from the Archaeon Haloferax mediterranei was studied in 3 M KC1 and in glycerol. Haloferax mediterranei is a halophilic organism requiring 20-25% NaCl for optimal growth, so its enzymes are stabilised by high salt concentrations. We have replaced the salt by 20% (v/v) glycerol in order to analyse if the kinetic properties and kinetic mechanism of the enzyme change at lower salt concentrations in order to apply the enzyme in a bioreactor. The kinetic mechanism was studied by initial velocity measurements with, product and substrate analog inhibition. Product and substrates analog inhibition experiments indicate that (a), NADP+ is a competitive inhibitor versus NADPH indicating that NADP+ and NADPH both bind to free enzyme and (b), the binding order of substrates is NADPH, α-ketoglutarate and ammonium. The results suggest that the mechanism in the amination reaction, with and without salt, is the same: sequential ordered with A (NADPH) bindi...

An NAD-dependent D-2-hydroxyacid dehydrogenase (EC 1.1.1.) was isolated and characterized from the halophilic Archaeon Haloferax mediterranei. The enzyme is a dimer with a molecular mass of 101.4 +/- 3.3 kDa. It is strictly NAD-dependent and exhibits its highest activity in 4 M NaCl. The enzyme is characterized by a broad substrate specificity 2-ketoisocaproate and 2-ketobutyrate being the substrates with the higher Vmax/Km. When pyruvate and 2-ketobutyrate were the substrates the optimal pH was acidic (pH 5) meanwhile for 2-ketoisocaproate maximum activity was achieved at basic pH between 7.5 and 8.5. The optimum temperature was 52 degrees C and at 65 degrees C there was a pronounced activity decrease. This new enzyme can be used for the production of D-2-hydroxycarboxylic acid.

The kinetic mechanism and metal content of Haloferax mediterranei NAD(P)+-glucose dehydrogenase have been investigated. The kinetic mechanism has been determined by initial rate and inhibition studies. Initial velocity studies were performed with d -glucose as well as with the alternative substrate d -xylose, with NADP+ as coenzyme. The results show that the mechanism is sequential with respect to substrate addition. The product inhibition patterns agree with an ordered binding of NADP+ and d -glucose, followed by an ordered release of gluconolactone and NADPH. The activity of Hf. mediterranei glucose dehydrogenase was markedly dependent on the concentration of metal ions. Inactivation by metal chelators and reactivation by certain divalent ions indicated that glucose dehydrogenase from Hf. mediterranei contains tightly bound metal ions which are essential for activity. Metal analyses demonstrated that the enzyme binds 3.6±0.3 mol of Zn(II)/mol of protein, which corresponds to the binding of two atoms of Zn(II) per subunit. Alignment of the N-terminal sequence of glucose dehydrogenase from Hf. mediterranei with medium chain zinc-containing dehydrogenases reveals a clear similarity between them, suggesting that glucose dehydrogenase from Hf. mediterranei belongs to this family.