The eastern Mediterranean pre-MSC brine pool as an analogue for future subtropical hydroclimate

<p>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 (TEX<sub>86</sub>-) and/or alkenone unsaturation ratio (U<sup>K′</sup><sub>37</sub>) based SSTs and oxygen isotopes (δ<sup>18</sup>O) 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.</p>

Insights into Red Sea Brine Pool Specialized Metabolism Gene Clusters Encoding Potential Metabolites for Biotechnological Applications and Extremophile Survival

The recent rise in antibiotic and chemotherapeutic resistance necessitates the search for novel drugs. Potential therapeutics can be produced by specialized metabolism gene clusters (SMGCs). We mined for SMGCs in metagenomic samples from Atlantis II Deep, Discovery Deep and Kebrit Deep Red Sea brine pools. Shotgun sequence assembly and secondary metabolite analysis shell (antiSMASH) screening unraveled 2751 Red Sea brine SMGCs, pertaining to 28 classes. Predicted categorization of the SMGC products included those (1) commonly abundant in microbes (saccharides, fatty acids, aryl polyenes, acyl-homoserine lactones), (2) with antibacterial and/or anticancer effects (terpenes, ribosomal peptides, non-ribosomal peptides, polyketides, phosphonates) and (3) with miscellaneous roles conferring adaptation to the environment/special structure/unknown function (polyunsaturated fatty acids, ectoine, ladderane, others). Saccharide (80.49%) and putative (7.46%) SMGCs were the most abundant. Selected Red Sea brine pool sites had distinct SMGC profiles, e.g., for bacteriocins and ectoine. Top promising candidates, SMs with pharmaceutical applications, were addressed. Prolific SM-producing phyla (Proteobacteria, Actinobacteria, Cyanobacteria), were ubiquitously detected. Sites harboring the largest numbers of bacterial and archaeal phyla, had the most SMGCs. Our results suggest that the Red Sea brine niche constitutes a rich biological mine, with the predicted SMs aiding extremophile survival and adaptation.

Thermal Stability of a Mercuric Reductase from the Red Sea Atlantis II Hot Brine Environment as Analyzed by Site-Directed Mutagenesis

ABSTRACTThe 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.IMPORTANCEThe 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.