Search

A quantitative analysis of coccoliths is presented in 18 core-top samples ranging between 26° N and 21° N and covering two major deep brine pools in the northern part of the Red Sea. Non-brine sites are characterized by rich coccoliths that may reach up to 3.31 × 109 coccoliths/g made by 22 species, whereas brine sites of Shaban and Kebrit Deeps with additional two non-brine sites are characterized by a decline in coccoliths/g (3.25 × 108 coccoliths/g), Shannon diversity, CaCO3 (%), and high TOC (%). Carbonate dissolution, inferred by qualitative observation and quantitative indices, was only observed at one brine site GeoB7828 in Kebrit Deep. This suggests that the decline in coccolith assemblages may not entirely be attributed to carbonate dissolution. The major decline, however, is probably related to the suspension of fecal pellets and marine aggregates containing delicate coccolith shields within a nepheloid layer and subsequently grazed by zooplankters in which reduced the numbers of coccolith that reached the bottom of the brine sites, or alternatively a deep-sea flow current that carried and remobilized some suspended particles outside the brine pool. Latitudinal fluctuations of eutrophic/oligotrophic coccoliths suggest profound trophic changes in the photic zone in the northern part of the Red Sea. C. braarudii, a valuable nutrient-indicator species is here reported for the first time, along with G. oceanica, H. carteri as well as biogenic opal dominating the assemblage between 26° N and 24° N, suggesting elevated nutrient conditions and supporting recent high chl-a records, whereas areas between 21° N and 23° N lie under oligotrophic conditions due to the presence of U. sibogae, U. tenuis, R. clavigera, F. profunda, and S. pulchra.

The previously uncharted Afifi brine pool was discovered in the eastern shelf of the southern Red Sea. It is the shallowest brine basin yet reported in the Red Sea (depth range: 353.0 to 400.5 m). It presents a highly saline (228 g/L), thalassohaline, cold (23.3 °C), anoxic brine, inhabited by the bacterial classes KB1, Bacteroidia and Clostridia and the archaeal classes Methanobacteria and Deep Sea Euryarcheota Group. Functional assignments deduced from the taxonomy indicate methanogenesis and sulfur respiration to be important metabolic processes in this environment. The Afifi brine was remarkably enriched in dissolved inorganic carbon due to microbial respiration and in dissolved nitrogen, derived from anammox processes and denitrification, according to high δN values (+6.88‰, AIR). The Afifi brine show a linear increase in δO and δD relative to seawater that differs from the others Red Sea brine pools, indicating a non-hydrothermal origin, compatible with enrichment in evaporitic environments. Afifi brine was probably formed by venting of fossil connate waters from the evaporitic sediments beneath the seafloor, with a possible contribution from the dehydration of gypsum to anhydrite. Such origin is unique among the known Red Sea brine pools., This research was funded with King Abdullah University of Science and Technology funding through baseline funding and Red Sea Research Center competitive fund to C.M.D. and D.D. We thank J.C. Santamarina, M. Terzariol, K. Pigeon, A. Granados and J.M. Arrieta, and the crew of R/V Thuwal for their help.

The Fengcheng Formation is a nonmarine, carbonate-dominated succession that formed under arid climatic conditions in a hydrologically closed basin. Two transects and two seismic profiles were examined, the characteristics and environmental significance of different lithofacies were studied, and a model of depositional environment divisions was proposed. The sedimentary model involved an alkaline lake in which the depositional environments consisted of a shallow saline lake margin, slope, saline lake center, and steep lake margin from northeast to southwest. The perennial central salty lake was located in the southwestern part of the study area, whereas there were widespread, low-gradient lake margins in the northeast, east, southeast, and southern parts of the study area. Lake-level fluctuations had a major influence on the shallow saline lake system and complicated the depositional environments in these areas. The deposits are derived from bedrock reworking, volcanic eruptions, and authigenic minerals that precipitated from brine during the hypersaline phase. Fine-grained terrigenous clastic sediments, volcanic ashes and dusts, and authigenic minerals mixed in the depocenter (concentration center of the brine pool), which was covered by high-salinity brines, and the depositional environment was anoxic as a result of salinity-based brine stratification. A thick sodium carbonate succession occurred in the depocenter of the ancient Mahu lake, where bedded sodium carbonate alternated with fine-grained, organic-rich tuff or tuffaceous hydrocarbon source rocks. Microorganisms bloomed in the alkaline, high-salinity brine, and the organic matter was well preserved, which is similar to those modern alkaline saline lakes in eastern Africa and western North America. Thus, the Permian Fengcheng Formation contains source rocks that formed in an alkaline saline lake.

Nitrous oxide (N2O) is a climate-relevant atmospheric trace gas. It is produced as an intermediate of the nitrogen cycle. The open and coastal oceans are major sources of atmospheric N2O. However, its oceanic distribution is still largely unknown. Here we present the first measurements of the water column distribution of N2O in the Gulf of Aqaba and the Red Sea. Samples for N2O depth profiles were collected at the time-series site Station A in the northern Gulf of Aqaba (June and September 2003, and February 2004) and at several stations in the central Red Sea (October 2014, January and August 2016). Additionally, we measured N2O concentrations in brine pool samples collected in the northern and central Red Sea (January 2005 and August 2016). In the Gulf of Aqaba, N2O surface concentrations ranged from 6 to 8 nmol L−1 (97–111% saturation) and were close to the equilibrium with the overlying atmosphere. A pronounced temporal variability of the N2O water column distribution was observed. We suggest that this variability is a reflection of the interplay between N2O production by nitrification and its consumption by N2 fixation in the layers below 150 m during summer. N2O surface concentrations and saturations in the central Red Sea basin ranged from 2 to 9 nmol L−1 (43–155% saturation). A pronounced temporal variability with significant supersaturation in October 2014 and undersaturation in January and August 2016 was observed in the surface layer. In October 2014, N2O in the water column seemed to result from production via nitrification. Low N2O water column concentrations in January and August 2016 indicated a significant removal of N2O. We speculate that either in-situ consumption or remote loss processes of N2O such as denitrification in coastal regions were responsible for this difference. Strong meso- and submesoscale processes might have transported the coastal signals across the Red Sea. In addition, enhanced N2O concentrations of up to 39 nmol L−1 were found at the seawater-brine pool interfaces which point to an N2O production via nitrification and/or denitrification at low O2 concentrations. Our results indicate that the Red Sea and the Gulf of Aqaba are unique natural laboratories for the study of N2O production and consumption pathways under extreme conditions in one of the warmest and most saline region of the global oceans.

In this paper, a review is offered on lacustrine sites in Australia where both dolomite and magnesite are found to precipitate today (but not through diagenesis) and have been doing so during the Holocene. Information thus far obtained in the Coorong region of SE South Australia is provided and this follows with a discussion of the published literature from the volcanic plains of Western Victoria where modern dolomite and magnesite are also precipitating in saline playa lakes today. The work presented here clearly identifies that the volcanic rises (and topographic highs) in Western Victoria, such as Warrion Hill, play a vital role in groundwater recharge first, with subsequent flow paths towards the low plains in the region where saline playas are located. In the topographically-high recharge areas, the waters are predominantly rich in Ca and, as water progressively flows towards the playas, Ca is lost through calcite precipitation in scorias and soils, and is also presumably exchanged for K, with a considerable enrichment of the groundwaters with respect to Mg/Ca and K/Ca. This cationic exchange is thought to occur on clays in the subsurface, which are the by-product of weathering of the local Quaternary basalts. Eventually, the lake waters become supersaturated with respect to Mg-rich minerals and both dolomite and magnesite precipitate naturally in the lake waters to finally settle on the lake floors. It is more than likely that a brine pool occurs below each lake and it is from this that precipitation occurs once that pool waters reaches the lake surface. High levels of bicarbonate are found in the groundwaters principally as a result of carbonic acid supplied through rain and also from hydrolysis of silicate minerals that originally occurred in the basalts. The high concentrations of bicarbonate contribute to the precipitation of the carbonate minerals. Chemical analyses of the groundwaters and the lakes are tabled and help understand the chemical changes along the groundwater flow paths. In addition, examination of the δ18O and δ13C of the Western Victorian lake sediments, by analogy with those of Coorong lakes, indicates that although the carbonates in the Coorong lakes are precipitated as a result of sulphate bacterial reduction, this appears not to be the case for the Western Victorian lakes. Due to the high turbidity of the Western Victorian dolomite-precipitating lakes as well because of their very high salinities (often ≫50TDS), there are no macrophytes, and little if any algal growth.

During the Late Miocene the Mediterranean Sea experienced severe disruption of its connectivity to the Atlantic Ocean, highlighted by a rapid sea-level drop, culminating to the Messinian Salinity Crisis (MSC; 5.97-5.33 Ma). Such a paleoceanographic change, triggered by the cumulative effect of climate and tectonics, caused high-amplitude fluctuations in the hydrology of the entire basin, and further influenced the geological history of the Mediterranean Sea. Although a consistent pattern of the paleoclimate has started to emerge, we currently lack a continuous sea surface salinity (SSS) record linking the timing of sea surface temperature (SST) variations, sea-level fluctuations, and the overall environmental change, particularly for the pre-evaporitic period. Initial viewpoints of a linear and gradual salinity increase prior to the onset of the MSC have been recently revised and replaced by highly variable salinity-related patterns representative of the stepwise restriction of the Mediterranean Sea. Here we use the combined Tetra Ether (TEX86-) and/or alkenone unsaturation ratio (UK′37) based SSTs and oxygen isotopes (δ18O) from the cyclic marl-sapropel sedimentary succession of Agios Myron section (north-central Crete, Greece) to assess hydroclimate changes during that time, and we finally present the first record of SSS in the eastern Mediterranean Sea for the earliest Messinian (7.2–6.5 Ma). The relatively stable marine conditions after the Tortonian/Messinian boundary, expressed through a cool and fresh upper water column, significantly changed at ∼6.9 Ma, when an important reversal in the heart of the Messinian cooling trend supplemented by a coherent hypersaline water column took place. The observed SST and SSS patterns provide context for a two-fold evolution of this event (centered at 6.9–6.8 and 6.72 Ma), which finally led to the onset of a brine pool into the eastern Mediterranean basin. The transitional character of the following time interval (6.7–6.5 Ma) marks another important step in the basin restriction with a wider range of salinity fluctuations from highly saline to diluted conditions and enhanced water column stratification prior to the deposition of evaporites. Overall, this evolution supports the concept of a stepwise restriction of the Mediterranean Sea associated with substantial hydroclimate variability and increasing environmental (thermal and salinity) stress, and further confirms its position as a preferred laboratory for developing new conceptual models in paleoceanography, allowing the investigation and scale assessment of a phenomenon with high chances of representing a future analogue scenario.

The Arroyo Rojo VMS deposit is the most studied polymetallic ore of Tierra del Fuego, Argentina. The first mentions of its existence began in the 80s by Minera Aguilar, which has discovered a small nunatak containing a lens of polymetallic massive sulfides, since then various authors have studied the geology and mineralization of the site. Several anomalies of copper, lead and zinc were also identified in the region during regional prospection in the 70s. The deposit is within the Lemaire formation, a rhyolitic low-Jurassic marine volcano-sedimentary complex formed during the fragmentation of Gondwana. The mineralization consists of a massive sulfide lens that varies from 1.5 to 4 m in thickness in surface and two more massive sulfide lenses at depth with a thickness that varies between 3 and 18.6 m. The ore is composed mostly of pyrite and sphalerite, minor galena, chalcopyrite and pyrrhotite. Some of the original textures like framboidal, colloidal and euhedral type can be still recognized among other deformation and recrystallization textures. The hydrothermal alteration consists of a roughly concentric zonation with an inner silica and clorithic zone and an external sericitic zone. Even though previous works have classified the Arroyo Rojo deposit as Kuroko type, more recent studies have shown evidence that associates this deposit with a brine pool deposition.

Two extremely halophilic archaeal strains, designated SB29T and SB3T, were isolated from the brine-seawater interface of Discovery Deep in the Red Sea. Cells of both strains were pleomorphic (irregular polyhedrals, ovals, and rods) and stained Gram-negative, colonies were pigmented pink. The sequence similarity of the 16S rRNA gene of strain SB29T with that of its most closely related validly described species (Hfx. sulfurifontis DSM 16227T) and that of strain SB3T with its closest validly described relative (Hfx. denitrificans ATCC 35960T) was 98.1% and 98.6%, respectively. The incomplete draft genomes of SB29T and SB3T are 3,871,125 bp and 3,904,985 bp in size, respectively, and their DNA G + C contents are 60.75% and 65.64%, respectively. The highest ANI values between the genomes of SB29T and SB3T and the most closely related genomes in GenBank were determined as 82.6% (Hfx. sulfurifontis ATCC BAA-897T, GenBank accession no. GCA_000337835.1) and 92.6% (Haloferax denitrificans ATCC 35960T, GenBank accession no. GCA_000337795.1), respectively. These data indicate that the two new isolates cannot be classified into any recognized species of the genus Haloferax, and, therefore, two novel species of the genus Haloferax are proposed: Haloferax profundi sp. nov. (type strain SB29T = JCM 19567T = CGMCC 1.14960T) and Haloferax marisrubri sp. nov. (type strain SB3T = JCM 19566T = CGMCC 1.14958T).

Subsea hypersaline anoxic brine pools are among the most extreme habitable environments on Earth that offer clues to life on other planets. Brine is toxic to macrofauna as remotely operated vehicles commonly observe dead and preserved remains in brine pools. While brine pools are often assumed to be stable stratified systems, we show that underwater landslides can cause significant disturbances. Moreover, landslides create large-amplitude waves upon impact with the brine pool, similar to tsunami waves. We focus on the Orca Basin brine pool in the deepwater Gulf of Mexico, which contains numerous landslide deposits and blocks that originated from scarps several hundred meters above the brine pool. The impact of massive fast-moving landslides generated waves with amplitude on the order of 100 s of meters, which rival the largest known ocean waves. Brine waves can negatively affect biological communities and potentially overspill to spread hypersaline brine into surrounding basins.

In this work, the potential of harvesting energy from ocean salt basins is discussed. These deep ocean depressions which host thick salts deposits whose salinities could be up to 5-10 times higher than normal seawater open an interesting -and so far, unexplored possibility for ocean energy. It is known that upon mixing two solutions of different concentrations spontaneous energy can be released, and then, if the upper waters surrounding the basin are deliberately brought and mixed with the hypersaline brine pool, osmotic energy can be obtained. Although the extractable osmotic energy density is certainly low in comparison with other ocean energy sources, nevertheless, ocean basins can be several kilometers broad and then a vast amount of energy is stored in those basins, which, in addition, could be steadily extracted without interruption avoiding the intermittence problem besetting traditional renewable energies. The economical viability for an ocean brine pool power station could be in any case higher or at least similar than that from Ocean Thermal Energy Conversion (OTEC). Additional R&D is required in order to arrive at a reliable practical design and to explore this yet untapped new source of ocean energy.

The incorporation of metabolic carbon (Cm) into shells of mollusks has been used as an indicator of animal condition and availability of food resources in estuarine and freshwater settings. This study examines Cm in Bathymodiolus childressi, a marine cold seep mussel dependent on methanotrophic symbionts. As seeps develop, mature, and go quiescent, methane supply will vary and affect the amount of metabolic carbon deposited into the growing shell. B. childressi (n = 136) were live-collected from two seep sites over a 17 year period in the Northern Gulf of Mexico to investigate whether changes in Cm were detectable between sites and across years. Significant differences in Cm were observed between mussel populations at Brine Pool (15.4 ± 0.4%) and Bush Hill (10.3 ± 0.3%). Cm also changed significantly within each site across year (Bush Hill 1991: 12.2 ± 0.5%, 1992: 17.3 ± 0.8%) and decadal time scales (Brine Pool 1989: 15.5 ± 0.7%, 2006: 19.5 ± 0.7%). These findings agree with previous studies that found mussel condition was higher at Brine Pool and correlate well with a trophic mixing model that indicated significantly higher methane source utilization at the Brine Pool (65 ± 1.1%) than at Bush Hill (49 ± 1.6%). Further development of this method should allow for assessment of Cm in shell assemblages as an indicator of historical resource availability at both active and former cold seep sites.

Homoacetogens are efficient CO2 fixing bacteria using H2 as electron donor to produce acetate. These organisms can be enriched at the biocathode of microbial electrosynthesis (MES) for electricity-driven CO2 reduction to acetate. Studies exploring homoacetogens in MES are mainly conducted using pure or mix-culture anaerobic inocula from samples with standard environmental conditions. Extreme marine environments host unique microbial communities including homoacetogens that may have unique capabilities due to their adaptation to harsh environmental conditions. Anaerobic deep-sea brine pools are hypersaline and metalliferous environments and homoacetogens can be expected to live in these environments due to their remarkable metabolic flexibility and energy-efficient biosynthesis. However, brine pools have never been explored as inocula for the enrichment of homacetogens in MES. Here we used the saline water from a Red Sea brine pool as inoculum for the enrichment of halophilic homoacetogens at the biocathode (−1 V vs. Ag/AgCl) of MES. Volatile fatty acids, especially acetate, along with hydrogen gas were produced in MES systems operated at 25 and 10% salinity. Acetate concentration increased when MES was operated at a lower salinity ∼3.5%, representing typical seawater salinity. Amplicon sequencing and genome-centric metagenomics of matured cathodic biofilm showed dominance of the genus Marinobacter and phylum Firmicutes at all tested salinities. Seventeen high-quality draft metagenome-assembled genomes (MAGs) were extracted from the biocathode samples. The recovered MAGs accounted for 87 ± 4% of the quality filtered sequence reads. Genome analysis of the MAGs suggested CO2 fixation via Wood–Ljundahl pathway by members of the phylum Firmicutes and the fixed CO2 was possibly utilized by Marinobacter sp. for growth by consuming O2 escaping from the anode to the cathode for respiration. The enrichment of Marinobacter sp. with homoacetogens was only possible because of the specific cathodic environment in MES. These findings suggest that in organic carbon-limited saline environments, Marinobacter spp. can live in consortia with CO2 fixing bacteria such as homoacetogens, which can provide them with fixed carbon as a source of carbon and energy.

The lower convective layer (LCL) at Atlantis II brine pool of the Red sea represents one of the exceptional, unique ecosystems. Thioredoxin is a multi-functional antioxidant redox protein that has a crucial role in various vital cellular processes. In the current study, a functional metagenomics approach was used to isolate and characterize thioredoxin from the LCL of Atlantis II Deep brine pool (Trx-ATII). From the metagenomic DNA of the LCL, the thioredoxin gene was directly retrieved and sequenced. Sequence analysis showed that the gene belonged to thioredoxin-like superfamily with classical Trx motif (-CXXC-). Phylogenetic analysis revealed that Trx-ATII was closely related to Trx of Prochlorococcus marinus with a maximum identity of 86%. Successfully, Trx-ATII was cloned and expressed in E. coli, where the purified protein had M.wt of 16 kDa. Characterization studies revealed that Trx-ATII protein is halophilic; can tolerate up to 2.5 M NaCl and thermostable, where 90% of its activity was retained at 60 °C. Trx-ATII can reduce both DTNB and insulin disulfide- containing substrates. In conclusion, a unique thioredoxin protein was isolated from a harsh environment that can maintain its activity under extreme conditions of salinity and temperature as a promising redox protein for biotechnological applications.

Background Microbial species in the brine pools of the Red Sea and the brine pool-seawater interfaces are exposed to high temperature, high salinity, low oxygen levels and high concentrations of heavy metals. As adaptations to these harsh conditions require a large suite of secondary metabolites, these microbes have a huge potential as a source of novel anticancer molecules. Methods A total of 60 ethyl-acetate extracts of newly isolated strains from extreme environments of the Red-Sea were isolated and tested against several human cancer cell lines for potential cytotoxic and apoptotic activities. Results Isolates from the Erba brine-pool accounted for 50% of active bacterial extracts capable of inducing 30% or greater inhibition of cell growth. Among the 60 extracts screened, seven showed selectivity towards triple negative BT20 cells compared to normal fibroblasts. Conclusion In this study, we identified several extracts able to induce caspase-dependent apoptosis in various cancer cell lines. Further investigations and isolation of the active compounds of these Red Sea brine pool microbes may offer a chemotherapeutic potential for cancers with limited treatment options.

Summary The advent of metagenomics has greatly facilitated the discovery of enzymes with useful biochemical characteristics for industrial and biomedical applications, from environmental niches. In this study, we used sequence‐based metagenomics to identify two antibiotic resistance enzymes from the secluded, lower convective layer of Atlantis II Deep Red Sea brine pool (68°C, ~2200 m depth and 250‰ salinity). We assembled > 4 000 000 metagenomic reads, producing 43 555 contigs. Open reading frames (ORFs) called from these contigs were aligned to polypeptides from the Comprehensive Antibiotic Resistance Database using BLASTX. Two ORFs were selected for further analysis. The ORFs putatively coded for 3′‐aminoglycoside phosphotransferase [APH(3′)] and a class A beta‐lactamase (ABL). Both genes were cloned, expressed and characterized for activity and thermal stability. Both enzymes were active in vitro, while only APH(3′) was active in vivo. Interestingly, APH(3′) proved to be thermostable (T m = 61.7°C and ~40% residual activity after 30 min of incubation at 65°C). On the other hand, ABL was not as thermostable, with a T m = 43.3°C. In conclusion, we have discovered two novel AR enzymes with potential application as thermophilic selection markers.

Bathymodiolus childressi is a foundation species at methane seeps on the upper- continental slope of the Gulf of Mexico. Other species of the genus are known to gain the advan- tage of variable food availability through trophic plasticity, by hosting dual microbial symbionts while retaining their own particle feeding ability. B. childressi, however, hosts only a single methanotrophic symbiont, and the possibility of trophic plasticity has not been fully examined in this species. Feeding strategies of archival specimens from 2 geochemically contrasting seeps from the Gulf of Mexico (Bush Hill and Brine Pool NR-1) were characterized using 4-source mix- ing analysis of δ 13 C, δ 15 N and δ 34 S values. Bush Hill mussels used a single thermogenic methane pool and derived N and S from different sources. Brine Pool mussels used 2 separate methane pools; the primary one being biogenic and the secondary possibly a mix of biogenic and thermo- genic. Utilization of particulate material was less common at Brine Pool than at Bush Hill. Bush Hill appears to offer lower levels of methane-based resources with particulate material having a greater, and sometimes dominant role in nutrition. Spatial patterns within the seeps were found but were not reflective of simple gradients. Some temporal changes occurred at both yearly scales and between samples, which were collected 17 yr apart. The 4-source mixing model used extrap- olations of mussel isotope values and limited environment characterization to infer likely trophic sources. The actual sources, however, remain unidentified. Future research across a wider range of seeps as well as experimental studies should be used to test the validity of the model.

The deep-sea anoxic brines of the Red Sea comprise unique, complex and extreme habitats. These environments are too harsh for metazoans, while the brine–seawater interface harbors dense microbial populations. We investigated the adjacent pelagic fauna at two brine pools using net tows, video records from a remotely operated vehicle and submerged echosounders. Waters just above the brine pool of Atlantis II Deep (2000 m depth) appeared depleted of macrofauna. In contrast, the fauna appeared to be enriched at the Kebrit Deep brine–seawater interface (1466 m).

The highly extreme conditions of the lower convective layer in the Atlantis II (ATII) Deep brine pool of the Red Sea make it an ideal environment for the search for novel enzymes that can function under extreme conditions. In the current study, we isolated a novel sequence of a thioredoxin reductase (TrxR) enzyme from the metagenomic dataset established from the microbial community that resides in the lower convective layer of Atlantis II. The gene was cloned, expressed and characterized for redox activity, halophilicity, and thermal stability. The isolated thioredoxin reductase (ATII-TrxR) was found to belong to the high-molecular-weight class of thioredoxin reductases. A search for conserved domains revealed the presence of an extra domain (Crp) in the enzyme sequence. Characterization studies of ATII-TrxR revealed that the enzyme was halophilic (maintained activity at 4 M NaCl), thermophilic (optimum temperature was 65°C) and thermostable (60% of its activity was retained at 70°C). Additionally, the enzyme utilized NADH in addition to NADPH as an electron donor. In conclusion, a novel thermostable and halophilic thioredoxin reductase has been isolated with a unique sequence that adapts to the harsh conditions of the brine pools making this protein a good candidate for biological research and industrial applications.

The hypersaline Kebrit Deep brine pool in the Red Sea is characterized by high levels of toxic heavy metals. Here, we describe two structurally related mercuric reductases (MerAs) from this site which were expressed in Escherichia coli. Sequence similarities suggest that both genes are derived from proteobacteria, most likely the Betaproteobacteria or Gammaproteobacteria. We show that one of the enzymes (K35NH) is strongly inhibited by NaCl, while the other (K09H) is activated in a NaCl-dependent manner. We infer from this difference that the two forms might support the detoxification of mercury in bacterial microorganisms that employ the compatible solutes and salt-in strategies, respectively. Three-dimensional structure modeling shows that all amino acid substitutions unique to each type are located outside the domain responsible for formation of the active MerA homodimer, and the vast majority of these are found on the surface of the molecule. Moreover, K09H exhibits the predominance of acidic over hydrophobic side chains that is typical of halophilic salt-dependent proteins. These findings enhance our understanding of how selection pressures imposed by two environmental stressors have endowed MerA enzymes with catalytic properties that can potentially function in microorganisms that utilize distinct mechanisms for osmotic balance in hypersaline environments. IMPORTANCE Analysis of two structurally homologous but catalytically distinct mercuric reductases from the Kebrit Deep brine in the Red Sea sheds light on the adaptations that enable microorganisms to cope simultaneously with extreme salinity and toxic mercury compounds. One is strongly inhibited by high NaCl concentrations, while the other exhibits NaCl-dependent activation. Their different activity profiles imply that they may derive from bacterial microorganisms that utilize compatible solutes and salt-in strategies, respectively, to maintain osmotic balance. Three-dimensional modeling reveals that regions not involved in formation of the active homodimer are conserved between the two. However, in the NaCl-dependent form, distinct amino acid substitutions are found in areas that are critical for stability in high salt. The work provides insights into how two environmental stressors have shaped the structure of orthologous enzymes through selection and adaptation, enabling them to retain their catalytic function in what may be very different cellular contexts.

The demonstrated existence of vast brine pools in a number of places in the ocean basin around the world offers an interesting opportunity to the production of power from ocean, which has not been yet considered. The attractiveness of these vast deposits of brine lie in the fact that can be readily dissolved with the surrounding top waters extracting the spontaneous osmotic mixing energy released. In addition, osmotic engines can pumped out the brackish streams by buoyancy without the add of pressure exchangers (PEXs) as is required in current pressure retarded osmosis (PRO) technology and then simplifying significatively the overall process. Utilizing a simplified physical model, a first estimation for the density of power per unit of membrane area was calculated

FEBS Open Bio 9(2), 194-205 (2019). doi:10.1002/2211-5463.12557, Published by Elsevier on behalf of the Federation of European Biochemical Societies, Cambridge

The lower convective layer (LCL) of the Atlantis II brine pool of the Red Sea is a unique environment in terms of high salinity, temperature, and high concentrations of heavy metals. Mercuric reductase enzymes functional in such extreme conditions could be considered a potential tool in the environmental detoxification of mercurial poisoning and might alleviate ecological hazards in the mining industry. Here, we constructed a mercuric reductase library from Atlantis II, from which we identified genes encoding two thermostable mercuric reductase (MerA) isoforms: one is halophilic (designated ATII-LCL) while the other is not (designated ATII-LCL-NH). The ATII-LCL MerA has a short motif composed of four aspartic acids (4D414–417) and two characteristic signature boxes that played a crucial role in its thermal stability. To further understand the mechanism behind the thermostability of the two studied enzymes, we mutated the isoform ATII-LCL-NH and found that the substitution of 2 aspartic acids (2D) at positions 415 and 416 enhanced the thermal stability, while other mutations had the opposite effect. The 2D mutant showed superior thermal tolerance, as it retained 81% of its activity after 10 min of incubation at 70°C. A three-dimensional structure prediction revealed newly formed salt bridges and H bonds in the 2D mutant compared to the parent molecule. To the best of our knowledge, this study is the first to rationally design a mercuric reductase with enhanced thermal stability, which we propose to have a strong potential in the bioremediation of mercurial poisoning. IMPORTANCE The Red Sea is an attractive environment for bioprospecting. There are 25 brine-filled deeps in the Red Sea. The Atlantis II brine pool is the biggest and hottest of such hydrothermal ecosystems. We generated an environmental mercuric reductase library from the lowermost layer of the Atlantis II brine pool, in which we identified two variants of the mercuric reductase enzyme (MerA). One is the previously described halophilic and thermostable ATII-LCL MerA and the other is a nonhalophilic relatively less thermostable enzyme, designated ATII-LCL-NH MerA. We used the ATII-LCL-NH enzyme as a parent molecule to locate the amino acid residues involved in the noticeably higher thermotolerance of the homolog ATII-LCL MerA. Moreover, we designed a novel enzyme with superior thermal stability. This enzyme might have strong potential in the bioremediation of mercuric toxicity.