Denitrification in hypersaline and coastal environments.

As the association of denitrification with global warming and nitrogen removal from ecosystems has gained attention in recent decades, numerous studies have examined denitrification rates and the distribution of denitrifiers across different environments. In this minireview, reported studies focused on coastal saline environments, including estuaries, mangroves, and hypersaline ecosystems, have been analysed to identify the relationship between denitrification and saline gradients. The analyses of the literature and databases stated the direct effect of salinity on the distribution patterns of denitrifiers. However, few works do not support this hypothesis thus making this topic controversial. The specific mechanisms by which salinity influences denitrifier distribution are not fully understood. Nevertheless, several physical and chemical environmental parameters, in addition to salinity, have been shown to play a role in structuring the denitrifying microbial communities. The prevalence of nirS or nirK denitrifiers in ecosystems is a subject of debate in this work. In general terms, in mesohaline environments, the predominant nitrite reductase is NirS type and, NirK is found predominantly in hypersaline environments. Moreover, the approaches used by different researchers are quite different, resulting in a huge amount of unrelated information, making it difficult to establish comparative analysis. The main techniques used to analyse the distribution of denitrifying populations along salt gradients have been also discussed.

Molecular Advances in Microbial Metabolism.

Climate change, global pollution due to plastics, greenhouse gasses, or heavy metals among other pollutants, as well as limited natural sources due to unsustainable lifestyles and consumption patterns, are revealing the need for more research to understand ecosystems, biodiversity, and global concerns from the microscale to the macroscale [...].

Analysis of the external signals driving the transcriptional regulation of the main genes involved in denitrification in Haloferax mediterranei.

Haloferax mediterranei is the model microorganism for the study of the nitrogen cycle in haloarchaea. This archaeon not only assimilate N-species such as nitrate, nitrite, or ammonia, but also it can perform denitrification under low oxygen conditions, using nitrate or nitrite as alternative electron acceptors. However, the information currently available on the regulation of this alternative respiration in this kind of microorganism is scarce. Therefore, in this research, the study of haloarchaeal denitrification using H. mediterranei has been addressed by analyzing the promoter regions of the four main genes of denitrification (narGH, nirK, nor, and nosZ) through bioinformatics, reporter gene assays under oxic and anoxic conditions and by site-directed mutagenesis of the promoter regions. The results have shown that these four promoter regions share a common semi-palindromic motif that plays a role in the control of the expression levels of nor and nosZ (and probably nirK) genes. Regarding the regulation of the genes under study, it has been concluded that nirK, nor, and nosZ genes share some expression patterns, and therefore their transcription could be under the control of the same regulator whereas nar operon expression displays differences, such as the activation by dimethyl sulfoxide with respect to the expression in the absence of an electron acceptor, which is almost null under anoxic conditions. Finally, the study with different electron acceptors demonstrated that this haloarchaea does not need complete anoxia to perform denitrification. Oxygen concentrations around 100 µM trigger the activation of the four promoters. However, a low oxygen concentration per se is not a strong signal to activate the promoters of the main genes involved in this pathway; high activation also requires the presence of nitrate or nitrite as final electron acceptors.

Hypersaline environments as natural sources of microbes with potential applications in biotechnology: The case of solar evaporation systems to produce salt in Alicante County (Spain).

Extremophilic microbes show a unique metabolism due to the adaptations they display to deal with extreme environmental parameters characterizing the extreme ecosystems that they inhabit (high salt concentration, high temperatures, and extreme pH values, high exposure to solar radiation etc.). Halophilic microorganisms characterised and isolated from saltmarshes, brines, salted ponds, salty lagoons etc. have recently attracted attention due to their potential biotechnological applications (as whole cells used for different purposes like wastewater treatments, or their biomolecules: enzymes, antibiotics, carotenoids, bioplastics). Alicante county (southeast of Spain) accounts for a significant number of salty environments like coastal or inland salty ponds from where sodium chloride (NaCl)is obtained, marshes, salty lagoons, etc. The best system characterised so far from a microbiological point of view is "Salinas de Santa Pola", also termed "Salinas Bras del Port". However, there are many other salty environments to be explored, like the natural park of Torrevieja and la Mata lagoons, salty lagoon located in Calpe city or inland salted ponds like those located in the northwest of the county. This review summarises the most relevant biotechnological applications of halophilic microbes described up to now. In addition, special attention is focused on ecosystems such as the lagoons of Torrevieja or inland salt marshes as natural environments whose microbial biodiversity is worthy of being studied in search of new strains and species with the aim to analyze their potential biotechnological applications (pharmaceutical, food industry, biomedicine, etc.).

Distribution of Denitrification among Haloarchaea: A Comprehensive Study.

Microorganisms from the Halobacteria class, also known as haloarchaea, inhabit a wide range of ecosystems of which the main characteristic is the presence of high salt concentration. These environments together with their microbial communities are not well characterized, but some of the common features that they share are high sun radiation and low availability of oxygen. To overcome these stressful conditions, and more particularly to deal with oxygen limitation, some microorganisms drive alternative respiratory pathways such as denitrification. In this paper, denitrification in haloarchaea has been studied from a phylogenetic point of view. It has been demonstrated that the presence of denitrification enzymes is a quite common characteristic in Halobacteria class, being nitrite reductase and nitric oxide reductase the enzymes with higher co-occurrence, maybe due to their possible role not only in denitrification, but also in detoxification. Moreover, copper-nitrite reductase (NirK) is the only class of respiratory nitrite reductase detected in these microorganisms up to date. The distribution of this alternative respiratory pathway and their enzymes among the families of haloarchaea has also been discussed and related with the environment in which they constitute the major populations. Complete denitrification phenotype is more common in some families like Haloarculaceae and Haloferacaceae, whilst less common in families such as Natrialbaceae and Halorubraceae.

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.

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

Haloferaxmediterranei 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.

Haloarchaea as Cell Factories to Produce Bioplastics.

Plastic pollution is a worldwide concern causing the death of animals (mainly aquatic fauna) and environmental deterioration. Plastic recycling is, in most cases, difficult or even impossible. For this reason, new research lines are emerging to identify highly biodegradable bioplastics or plastic formulations that are more environmentally friendly than current ones. In this context, microbes, capable of synthesizing bioplastics, were revealed to be good models to design strategies in which microorganisms can be used as cell factories. Recently, special interest has been paid to haloarchaea due to the capability of some species to produce significant concentrations of polyhydroxyalkanoate (PHA), polyhydroxybutyrate (PHB), and polyhydroxyvalerate (PHV) when growing under a specific nutritional status. The growth of those microorganisms at the pilot or industrial scale offers several advantages compared to that of other microbes that are bioplastic producers. This review summarizes the state of the art of bioplastic production and the most recent findings regarding the production of bioplastics by halophilic microorganisms with special emphasis on haloarchaea. Some protocols to produce/analyze bioplastics are highlighted here to shed light on the potential use of haloarchaea at the industrial scale to produce valuable products, thus minimizing environmental pollution by plastics made from petroleum.

Exploring the Molecular Machinery of Denitrification in Haloferax mediterranei Through Proteomics.

Many proteins and enzymes involved in denitrification in haloarchaea can be inferred to be located between the cytoplasmic membrane and the S-layer, based on the presence of a Tat signal sequence and the orientation of the active site that some of these enzymes have. The membrane fraction of the haloarchaeon Haloferax mediterranei (R-4), grown under anaerobic conditions in the presence of nitrate, was solubilized to identify the respiratory proteins associated or anchored to it. Using Triton X-100, CHAPS, and n-Octyl-ß-d-glucopyranoside at different concentrations we found the best conditions for isolating membrane proteins in micelles, in which enzymatic activity and stability were maintained. Then, they were subjected to purification using two chromatographic steps followed by the analysis of the eluents by NANO-ESI Chip-HPLC-MS/MS. The results showed that the four main enzymes of denitrification (nitrate, nitrite, nitric oxide, and nitrous oxide reductases) in H. mediterranei were identified and they were co-purified thanks to the micelles made with Triton X-100 (20% w/v for membrane solubilisation and 0.2% w/v in the buffers used during purification). In addition, several accessory proteins involved in electron transfer processes during anaerobic respiration as well as proteins supporting ATP synthesis, redox balancing and oxygen sensing were detected. This is the first characterization of anaerobic membrane proteome of haloarchaea under denitrifying conditions using liquid chromatography-mass spectrometry. It provides new information for a better understanding of the anaerobic respiration in haloarchaea.

Haloferax mediterranei, an Archaeal Model for Denitrification in Saline Systems, Characterized Through Integrated Physiological and Transcriptional Analyses.

Haloferax mediterranei (R4) belongs to the group of halophilic archaea, one of the predominant microbial populations in hypersaline environments. In these ecosystems, the low availability of oxygen pushes the microbial inhabitants toward anaerobic pathways and the presence of N-oxyanions favor denitrification. In a recent study comparing three Haloferax species carrying dissimilatory N-oxide reductases, H. mediterranei showed promise as a future model for archaeal denitrification. This work further explores the respiratory physiology of this haloarchaeon when challenged with ranges of nitrite and nitrate concentrations and at neutral or sub-neutral pH during the transition to anoxia. Moreover, to begin to understand the transcriptional regulation of N-oxide reductases, detailed gas kinetics was combined with gene expression analyses at high resolution. The results show that H. mediterranei has an expression pattern similar to that observed in the bacterial Domain, well-coordinated at low concentrations of N-oxyanions. However, it could only sustain a few generations of exponential anaerobic growth, apparently requiring micro-oxic conditions for de novo synthesis of denitrification enzymes. This is the first integrated study within this field of knowledge in haloarchaea and Archaea in general, and it sheds lights on denitrification in salty environments.

Deciphering Pathways for Carotenogenesis in Haloarchaea.

Bacterioruberin and its derivatives have been described as the major carotenoids produced by haloarchaea (halophilic microbes belonging to the Archaea domain). Recently, different works have revealed that some haloarchaea synthetize other carotenoids at very low concentrations, like lycopene, lycopersene, cis- and trans-phytoene, cis- and trans-phytofluene, neo-ß-carotene, and neo-α-carotene. However, there is still controversy about the nature of the pathways for carotenogenesis in haloarchaea. During the last decade, the number of haloarchaeal genomes fully sequenced and assembled has increased significantly. Although some of these genomes are not fully annotated, and many others are drafts, this information provides a new approach to exploring the capability of haloarchaea to produce carotenoids. This work conducts a deeply bioinformatic analysis to establish a hypothetical metabolic map connecting all the potential pathways involved in carotenogenesis in haloarchaea. Special interest has been focused on the synthesis of bacterioruberin in members of the Haloferax genus. The main finding is that in almost all the genus analyzed, a functioning alternative mevalonic acid (MVA) pathway provides isopentenyl pyrophosphate (IPP) in haloarchaea. Then, the main branch to synthesized carotenoids proceeds up to lycopene from which ß-carotene or bacterioruberin (and its precursors: monoanhydrobacterioriberin, bisanhydrobacterioruberin, dihydrobisanhydrobacteriuberin, isopentenyldehydrorhodopsin, and dihydroisopenthenyldehydrorhodopsin) can be made.

DMSO Reductase Family: Phylogenetics and Applications of Extremophiles.

Dimethyl sulfoxide reductases (DMSO) are molybdoenzymes widespread in all domains of life. They catalyse not only redox reactions, but also hydroxylation/hydration and oxygen transfer processes. Although literature on DMSO is abundant, the biological significance of these enzymes in anaerobic respiration and the molecular mechanisms beyond the expression of genes coding for them are still scarce. In this review, a deep revision of the literature reported on DMSO as well as the use of bioinformatics tools and free software has been developed in order to highlight the relevance of DMSO reductases on anaerobic processes connected to different biogeochemical cycles. Special emphasis has been addressed to DMSO from extremophilic organisms and their role in nitrogen cycle. Besides, an updated overview of phylogeny of DMSOs as well as potential applications of some DMSO reductases on bioremediation approaches are also described.

Denitrifying haloarchaea within the genus Haloferax display divergent respiratory phenotypes, with implications for their release of nitrogenous gases.

Haloarchaea are extremophiles, generally thriving at high temperatures and salt concentrations, thus, with limited access to oxygen. As a strategy to maintain a respiratory metabolism, many halophilic archaea are capable of denitrification. Among them are members of the genus Haloferax, which are abundant in saline/hypersaline environments. Three reported haloarchaeal denitrifiers, Haloferax mediterranei, Haloferax denitrificans and Haloferax volcanii, were characterized with respect to their denitrification phenotype. A semi-automatic incubation system was used to monitor the depletion of electron acceptors and accumulation of gaseous intermediates in batch cultures under a range of conditions. Out of the species tested, only H. mediterranei was able to consistently reduce all available N-oxyanions to N2 , while the other two released significant amounts of NO and N2 O, which affect tropospheric and stratospheric chemistries respectively. The prevalence and magnitude of hypersaline ecosystems are on the rise due to climate change and anthropogenic activity. Thus, the biology of halophilic denitrifiers is inherently interesting, due to their contribution to the global nitrogen cycle, and potential application in bioremediation. This work is the first detailed physiological study of denitrification in haloarchaea, and as such a seed for our understanding of the drivers of nitrogen turnover in hypersaline systems.

Denitrifying haloarchaea: sources and sinks of nitrogenous gases.

Haloarchaea thrive under saline and hypersaline conditions and often dominate microbial communities in saltmarshes, salted lakes/soils and some oceanic areas. Some of the predominant species show denitrifying capabilities, although it remains unclear whether they are complete or partial denitrifiers. As complete denitrifiers, they could play important roles buffering ecosystems in which nitrate and nitrite appear as contaminants. However, partial denitrifying haloarchaea could contribute to the emission of nitrogenous gasses, thus acting as drivers of climate change and ozone depletion. In this review, we summarise some recent results on denitrification in haloarchaea, discuss the environmental implications and outline possible applications in mitigation. Finally, we list questions to be addressed in the near future, facilitating increased understanding of the role of these organisms in N turnover in arid and hypersaline environments.

Analysis of multiple haloarchaeal genomes suggests that the quinone-dependent respiratory nitric oxide reductase is an important source of nitrous oxide in hypersaline environments.

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 (N2 O) to nitrogen (N2 ), are little studied in Archaea, and hardly at all in haloarchaea. This work describes an extensive interspecies analysis of both complete and draft haloarchaeal genomes aimed at identifying the genes that encode respiratory nitric oxide reductases (Nors). The study revealed that the only nor gene found in haloarchaea is one that encodes a single subunit quinone dependent Nor homologous to the qNor found in bacteria. This surprising discovery is considered in terms of our emerging understanding of haloarchaeal bioenergetics and NO management.

Effects of nitrogen sources on the nitrate assimilation in Haloferax mediterranei: growth kinetics and transcriptomic analysis.

The haloarchaeon Haloferax mediterranei is able to grow in a defined culture media not only in the presence of inorganic nitrogen salt but also with amino acid as the sole nitrogen source. Assimilatory nitrate and nitrite reductases, respectively, catalyze the first and second reactions. The genes involved in this process are nasA, which encodes nitrate reductase and is found within the operon nasABC, and nasD, which encodes nitrite reductase. These genes are subjected to transcriptional regulation, being repressed in the presence of ammonium and induced with either nitrate or nitrite. This type of regulation has also been described when the amino acids are used as nitrogen source in the minimal media. Furthermore, it has been observed that the microorganism growth depends on nitrogen source, obtaining the lowest growth rate in the presence of nitrate and aspartate. In this paper, we present the results of a comparative study of microorganism growth and transcriptomic analysis of the operon nasABC and gene nasD in different nitrogen sources. The results are the first ever produced in relation to amino acids as nitrogen sources within the Halobacteriaceae family.

Active site dynamics in the zinc-dependent medium chain alcohol dehydrogenase superfamily.

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.

Nitrogen metabolism in haloarchaea.

The nitrogen cycle (N-cycle), principally supported by prokaryotes, involves different redox reactions mainly focused on assimilatory purposes or respiratory processes for energy conservation. As the N-cycle has important environmental implications, this biogeochemical cycle has become a major research topic during the last few years. However, although N-cycle metabolic pathways have been studied extensively in Bacteria or Eukarya, relatively little is known in the Archaea. Halophilic Archaea are the predominant microorganisms in hot and hypersaline environments such as salted lakes, hot springs or salted ponds. Consequently, the denitrifying haloarchaea that sustain the nitrogen cycle under these conditions have emerged as an important target for research aimed at understanding microbial life in these extreme environments.The haloarchaeon Haloferax mediterranei was isolated 20 years ago from Santa Pola salted ponds (Alicante, Spain). It was described as a denitrifier and it is also able to grow using NO3(-), NO2(-) or NH4(+) as inorganic nitrogen sources. This review summarizes the advances that have been made in understanding the N-cycle in halophilic archaea using Hfx mediterranei as a haloarchaeal model. The results obtained show that this microorganism could be very attractive for bioremediation applications in those areas where high salt, nitrate and nitrite concentrations are found in ground waters and soils.

Analysis of protein solvent interactions in glucose dehydrogenase from the extreme halophile Haloferax mediterranei.

The structure of glucose dehydrogenase from the extreme halophile Haloferax mediterranei has been solved at 1.6-A 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.

Gene cloning, heterologous overexpression and optimized refolding of the NAD-glutamate dehydrogenase from Haloferax mediterranei.

The NAD-dependent glutamate dehydrogenase (GDH) gene from the halophilic archaeon Haloferax mediterranei has been cloned. The analysis of the nucleotide sequence revealed an open reading frame of 1323 bp that encodes a NAD-GDH. The amino acid sequence displayed high homology with those from other sources, especially the highly conserved residues involved in 2-oxoglutarate binding. The expression of this gene in Escherichia coli, the refolding and further characterization, yielded a fully active NAD-GDH with the same features than those found for the wild-type enzyme. This halophilic NAD-GDH showed a highly dependence on salts for both stability and activity, being essential for the refolding of the recombinant enzyme.