Red Sea Brine Pool Research Articles

Derivation of a novel antimicrobial peptide from the Red Sea Brine Pools modified to enhance its anticancer activity against U2OS cells

Cancer associated drug resistance is a major cause for cancer aggravation, particularly as conventional therapies have presented limited efficiency, low specificity, resulting in long term deleterious side effects. Peptide based drugs have emerged as potential alternative cancer treatment tools due to their selectivity, ease of design and synthesis, safety profile, and low cost of manufacturing. In this study, we utilized the Red Sea metagenomics database, generated during AUC/KAUST Red Sea microbiome project, to derive a viable anticancer peptide (ACP). We generated a set of peptide hits from our library that shared similar composition to ACPs. A peptide with a homeodomain was selected, modified to improve its anticancer properties, verified to maintain high anticancer properties, and processed for further in-silico prediction of structure and function. The peptide’s anticancer properties were then assessed in vitro on osteosarcoma U2OS cells, through cytotoxicity assay (MTT assay), scratch-wound healing assay, apoptosis/necrosis detection assay (Annexin/PI assay), RNA expression analysis of Caspase 3, KI67 and Survivin, and protein expression of PARP1. L929 mouse fibroblasts were also assessed for cytotoxicity treatment. In addition, the antimicrobial activity of the peptide was also examined on E coli and S. aureus, as sample representative species of the human bacterial microbiome, by examining viability, disk diffusion, morphological assessment, and hemolytic analysis. We observed a dose dependent cytotoxic response from peptide treatment of U2OS, with a higher tolerance in L929s. Wound closure was debilitated in cells exposed to the peptide, while annexin fluorescent imaging suggested peptide treatment caused apoptosis as a major mode of cell death. Caspase 3 gene expression was not altered, while KI67 and Survivin were both downregulated in peptide treated cells. Additionally, PARP-1 protein analysis showed a decrease in expression with peptide exposure. The peptide exhibited minimal antimicrobial activity on critical human microbiome species E. coli and S. aureus, with a low inhibition rate, maintenance of structural morphology and minimal hemolytic impact. These findings suggest our novel peptide displayed preliminary ACP properties against U2OS cells, through limited specificity, while triggering apoptosis as a primary mode of cell death and while having minimal impact on the microbiological species E. coli and S. aureus.

Sedimentary porewaters record regional tectonic and climate events that perturbed a deep-sea brine pool in the Gulf of Aqaba, Red Sea

Brine pools in deep-sea environments provide unique perspectives into planetary and geological processes, extremophile microbial communities, and sedimentary records. The NEOM Brine Pool Complex was the first deep-sea brine pool system found in the Gulf of Aqaba, representing a significant extension of the geographical range and depositional setting of Red Sea brine pools. Here, we use a combination of brine pool samples collected via cast using a conductivity, temperature, depth instrument (CTD), as well as interstitial porewaters extracted from a sediment core collected in the NEOM Brine Pool to characterize the chemical composition and subsurface evolution of the brine. New results indicate that the NEOM brines and the subsurface porewaters may originate from different sources. Elemental concentrations suggest the brines in the NEOM pool are likely derived from dissolution of sub-seabed evaporites. In contrast, the sedimentary porewaters appear to have been influenced by periodic turbidite flows, generated either by earthquakes, submarine landslides, or flash floods, in which normal marine waters from the overlying Red Sea became entrained, periodically disturbing the chemistry of the brine pool. Thus, sediment porewaters beneath brine pools may record transient and dynamic changes in these deep marine depositional environments, reflecting the interplay between climate, tectonics, and sedimentation patterns along a rapidly urbanizing coastline. In concert, new results from NEOM extend the range and chemical constraints on Red Sea Brine Pools and highlight the dynamic interplay between Red Sea Deep water, dissolving evaporites, turbidites, and subsurface fluids that produce these unique depositional environments which host microbial life at the edge of habitability. In concert with sedimentological indicators, the chemistry of porewaters beneath deep-sea brine pools may present detailed records of natural hazards arising from interactions between the atmosphere, lithosphere, hydrosphere, and anthroposphere.

Characterization of a novel peptide mined from the Red Sea brine pools and modified to enhance its anticancer activity

Drug resistance is a major cause of the inefficacy of conventional cancer therapies, and often accompanied by severe side effects. Thus, there is an urgent need to develop novel drugs with low cytotoxicity, high selectivity and minimal acquired chemical resistance. Peptide-based drugs (less than 0.5 kDa) have emerged as a potential approach to address these issues due to their high specificity and potent anticancer activity. In this study, we developed a support vector machine model (SVM) to detect the potential anticancer properties of novel peptides by scanning the American University in Cairo (AUC) Red Sea metagenomics library. We identified a novel 37-mer antimicrobial peptide through SVM pipeline analysis and characterized its anticancer potential through in silico cross-examination. The peptide sequence was further modified to enhance its anticancer activity, analyzed for gene ontology, and subsequently synthesized. To evaluate the anticancer properties of the modified 37-mer peptide, we assessed its effect on the viability and morphology of SNU449, HepG2, SKOV3, and HeLa cells, using an MTT assay. Additionally, we evaluated the migration capabilities of SNU449 and SKOV3 cells using a scratch-wound healing assay. The targeted selectivity of the modified peptide was examined by evaluating its hemolytic activity on human erythrocytes. Treatment with the peptide significantly reduced cell viability and had a critical impact on the morphology of hepatocellular carcinoma (SNU449 and HepG2), and ovarian cancer (SKOV3) cells, with a marginal effect on cervical cancer cell lines (HeLa). The viability of a human fibroblast cell line (1Br-hTERT) was also significantly reduced by peptide treatment, as were the proliferation and migration abilities of SNU449 and SKOV3 cells. The annexin V assay revealed programmed cell death (apoptosis) as one of the potential cellular death pathways in SNU449 cells upon peptide treatment. Finally, the peptide exhibited antimicrobial effects on both gram-positive and gram-negative bacterial strains. The findings presented here suggest the potential of our novel peptide as a potent anticancer and antimicrobial agent.

Metagenomic profiling of antibiotic resistance genes in Red Sea brine pools.

Antibiotic resistance (AR) is an alarming global health concern, causing an annual death rate of more than 35,000 deaths in the US. AR is a natural phenomenon, reported in several pristine environments. In this study, we report AR in pristine Red Sea deep brine pools. Antimicrobial resistance genes (ARGs) were detected for several drug classes with tetracycline and macrolide resistance being the most abundant. As expected, ARGs abundance increased in accordance with the level of human impact with pristine Red Sea samples having the lowest mean ARG level followed by estuary samples, while activated sludge samples showed a significantly higher ARG level. ARG hierarchical clustering grouped drug classes for which resistance was detected in Atlantis II Deep brine pool independent of the rest of the samples. ARG abundance was significantly lower in the Discovery Deep brine pool. A correlation between integrons and ARGs abundance in brine pristine samples could be detected, while insertion sequences and plasmids showed a correlation with ARGs abundance in human-impacted samples not seen in brine pristine samples. This suggests different roles of distinct mobile genetic elements (MGEs) in ARG distribution in pristine versus human-impacted sites. Additionally, we showed the presence of mobile antibiotic resistance genes in the Atlantis II brine pool as evidenced by the co-existence of integrases and plasmid replication proteins on the same contigs harboring predicted multidrug-resistant efflux pumps. This study addresses the role of non-pathogenic environmental bacteria as a silent reservoir for ARGs, and the possible horizontal gene transfer mechanism mediating ARG acquisition.

Discovery of the deep-sea NEOM Brine Pools in the Gulf of Aqaba, Red Sea

Deep-sea brine pools represent hypersaline environments famed for their extremophile microbes. With anoxia entirely excluding bioturbating megafauna, brine pools are also conducive to the pristine preservation of sedimentary sequences. Here we use bathymetric and geophysical observations to locate a complex of brine pools in the Gulf of Aqaba consisting of one 10,000 m2 pool and three minor pools of less than 10 m2. We further conduct sediment coring and direct sampling of the brine to confirm the sedimentary and environmental characteristics of these pools. We find that the main pool preserves a stratigraphy which spans at least 1200 years and contains a combination of turbidites, likely resulting from flashfloods and local seismicity, and tsunamigenic terrestrial sediment. The NEOM Brine Pools, as we name them, extend the known geographical range of Red Sea brine pools, and represent a unique preservational environment for the sedimentary signals of regional climatic and tectonic events.

Novel Enzymes From the Red Sea Brine Pools: Current State and Potential.

The Red Sea is a marine environment with unique chemical characteristics and physical topographies. Among the various habitats offered by the Red Sea, the deep-sea brine pools are the most extreme in terms of salinity, temperature and metal contents. Nonetheless, the brine pools host rich polyextremophilic bacterial and archaeal communities. These microbial communities are promising sources for various classes of enzymes adapted to harsh environments – extremozymes. Extremozymes are emerging as novel biocatalysts for biotechnological applications due to their ability to perform catalytic reactions under harsh biophysical conditions, such as those used in many industrial processes. In this review, we provide an overview of the extremozymes from different Red Sea brine pools and discuss the overall biotechnological potential of the Red Sea proteome.

Mineral systems and their putative link with mantle plumes

Abstract In this contribution, I discuss the putative link of mantle plumes with selected categories of mineral systems. Continental rifting and break-ups can be induced by the upwelling of mantle plumes, also resulting in the generation of a wide range of mineral deposits. These include magma-associated ores, anorogenic igneous events responsible for iron oxide–copper–gold (IOCG) deposits, carbonatites and hydrothermal-induced mineralization, as well as hydrocarbons, salt domes, petroleum and gas, and several mineral systems in continental passive margins. Amongst the magma-associated mineral systems, the Ni–Cu–platinum group element (PGE), Fe–Ti–V and Cr deposits are the economically most important, such as those of the Bushveld Igneous Complex in South Africa. Anorogenic magmas are generally alkaline and associated with IOCG mineral systems, as exemplified by the giant Olympic Dam and similar deposits in South America. Carbonatites are considered as a distal effect of hotspot mantle plumes, as shown by Mount Weld in Australia, which may be related to the Bushveld Superplume. Plume-related thermal anomalies are the principal factor for the inception of hydrothermal circulation and the genesis of a wide range of hydrothermal mineral systems in rift-related tectonic settings. These include large-scale sedimentary-rock-hosted metalliferous ores, such as sedimentary exhalative (SEDEX) deposits. A modern example of is provided by the Red Sea brine pools. Some key examples are presented in this paper.

Crystal Structure and Active Site Engineering of a Halophilic γ-Carbonic Anhydrase.

Environments previously thought to be uninhabitable offer a tremendous wealth of unexplored microorganisms and enzymes. In this paper, we present the discovery and characterization of a novel γ-carbonic anhydrase (γ-CA) from the polyextreme Red Sea brine pool Discovery Deep (2141 m depth, 44.8°C, 26.2% salt) by single-cell genome sequencing. The extensive analysis of the selected gene helps demonstrate the potential of this culture-independent method. The enzyme was expressed in the bioengineered haloarchaeon Halobacterium sp. NRC-1 and characterized by X-ray crystallography and mutagenesis. The 2.6 Å crystal structure of the protein shows a trimeric arrangement. Within the γ-CA, several possible structural determinants responsible for the enzyme’s salt stability could be highlighted. Moreover, the amino acid composition on the protein surface and the intra- and intermolecular interactions within the protein differ significantly from those of its close homologs. To gain further insights into the catalytic residues of the γ-CA enzyme, we created a library of variants around the active site residues and successfully improved the enzyme activity by 17-fold. As several γ-CAs have been reported without measurable activity, this provides further clues as to critical residues. Our study reveals insights into the halophilic γ-CA activity and its unique adaptations. The study of the polyextremophilic carbonic anhydrase provides a basis for outlining insights into strategies for salt adaptation, yielding enzymes with industrially valuable properties, and the underlying mechanisms of protein evolution.

Discovery of Afifi, the shallowest and southernmost brine pool reported in the Red Sea

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 δ15N values (+6.88‰, AIR). The Afifi brine show a linear increase in δ18O 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.

Enrichment of Marinobacter sp. and Halophilic Homoacetogens at the Biocathode of Microbial Electrosynthesis System Inoculated With Red Sea Brine Pool.

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.

Mining the deep Red-Sea brine pool microbial community for anticancer therapeutics

BackgroundMicrobial 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.MethodsA 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.ResultsIsolates 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.ConclusionIn 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.

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.

Antibacterial and anticancer activities of orphan biosynthetic gene clusters from Atlantis II Red Sea brine pool

BackgroundCancer and infectious diseases are problematic because of continuous emergence of drug resistance. One way to address this enormous global health threat is bioprospecting the unlikeliest environments, such as extreme marine niches, which have tremendous biodiversity that is barely explored. One such environment is the Red Sea brine pool, Atlantis II Deep (ATII). Here, we functionally screened a fosmid library of metagenomic DNA isolated from the ATII lower convective layer (LCL) for antibacterial and anticancer activities.ResultsSelected clones, 14-7E and 10-2G, displayed antibacterial effects on the marine strain Bacillus sp. Cc6. Moreover, whole cell lysates from 14-7E and 10-2G exhibited decreased cell viability against MCF-7 (39.1% ± 6.6, 42% ± 8.1 at 50% v/v) and U2OS cells (35.7% ± 1.9, 79.9% ± 5.9 at 50% v/v), respectively. By sequencing the insert DNA from 14-7E and 10-2G, we identified two putative orphan biosynthetic gene clusters. Both clusters harbored putative ATP-binding cassette (ABC) transporter permeases and S-adenosylmethionine-related genes. Interestingly, the biosynthetic gene cluster identified on 14-7E is of archaeal origin and harbors a putative transcription factor. Several identified genes may be responsible for the observed antibacterial and anticancer activities. The 14-7E biosynthetic gene cluster may be encoding enzymes producing a specialized metabolite (effect of detected genes involved in C–C bond formation and glycosylation). The bioactivity may also be due to predicted subtilases encoded by this cluster. The 10-2G cluster harbored putative glycosyltransferase and non-ribosomal peptide synthase genes; thus the observed activity of this clone could be caused by a bioactive peptide.ConclusionsThe ATII LCL prokaryotic metagenome hosts putative orphan biosynthetic gene clusters that confer antibiotic and anticancer effects. Further biochemical studies should characterize the detected bioactive components, and the potential use of 14-7E metabolite for antibiosis and 10-2G metabolite as a selective anti-breast cancer drug.

A polyextremophilic alcohol dehydrogenase from the Atlantis II Deep Red Sea brine pool.

Enzymes originating from hostile environments offer exceptional stability under industrial conditions and are therefore highly in demand. Using single‐cell genome data, we identified the alcohol dehydrogenase (ADH) gene, adh/a1a, from the Atlantis II Deep Red Sea brine pool. ADH/A1a is highly active at elevated temperatures and high salt concentrations (optima at 70 °C and 4 m KCl) and withstands organic solvents. The polyextremophilic ADH/A1a exhibits a broad substrate scope including aliphatic and aromatic alcohols and is able to reduce cinnamyl‐methyl‐ketone and raspberry ketone in the reverse reaction, making it a possible candidate for the production of chiral compounds. Here, we report the affiliation of ADH/A1a to a rare enzyme family of microbial cinnamyl alcohol dehydrogenases and explain unique structural features for halo‐ and thermoadaptation.

Enrichment of extremophilic exoelectrogens in microbial electrolysis cells using Red Sea brine pools as inocula

Applying microbial electrochemical technologies for the treatment of highly saline or thermophilic solutions is challenging due to the lack of proper inocula to enrich for efficient exoelectrogens. Brine pools from three different locations (Valdivia, Atlantis II and Kebrit) in the Red Sea were investigated as potential inocula sources for enriching exoelectrogens in microbial electrolysis cells (MECs) under thermophilic (70°C) and hypersaline (25% salinity) conditions. Of these, only the Valdivia brine pool produced high and consistent current 6.8±2.1A/m2-anode in MECs operated at a set anode potential of +0.2V vs. Ag/AgCl (+0.405V vs. standard hydrogen electrode). These results show that exoelectrogens are present in these extreme environments and can be used to startup MEC under thermophilic and hypersaline conditions. Bacteroides was enriched on the anode of the Valdivia MEC, but it was not detected in the open circuit voltage reactor seeded with the Valdivia brine pool.

Novel thermostable antibiotic resistance enzymes from the Atlantis II Deep Red Sea brine pool.

SummaryThe 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.

Insertion sequences enrichment in extreme Red sea brine pool vent.

Mobile genetic elements are major agents of genome diversification and evolution. Limited studies addressed their characteristics, including abundance, and role in extreme habitats. One of the rare natural habitats exposed to multiple-extreme conditions, including high temperature, salinity and concentration of heavy metals, are the Red Sea brine pools. We assessed the abundance and distribution of different mobile genetic elements in four Red Sea brine pools including the world's largest known multiple-extreme deep-sea environment, the Red Sea Atlantis II Deep. We report a gradient in the abundance of mobile genetic elements, dramatically increasing in the harshest environment of the pool. Additionally, we identified a strong association between the abundance of insertion sequences and extreme conditions, being highest in the harshest and deepest layer of the Red Sea Atlantis II Deep. Our comparative analyses of mobile genetic elements in secluded, extreme and relatively non-extreme environments, suggest that insertion sequences predominantly contribute to polyextremophiles genome plasticity.

Red Sea Atlantis II brine pool nitrilase with unique thermostability profile and heavy metal tolerance

BackgroundNitrilases, which hydrolyze nitriles in a one-step reaction into carboxylic acids and ammonia, gained increasing attention because of the abundance of nitrile compounds in nature and their use in fine chemicals and pharmaceutics. Extreme environments are potential habitats for the isolation and characterization of extremozymes including nitrilases with unique resistant properties. The Red Sea brine pools are characterized by multitude of extreme conditions. The Lower Convective Layer (LCL) of the Atlantis II Deep Brine Pool in the Red Sea is characterized by elevated temperature (68 °C), high salt concentrations (250 ‰), anoxic conditions and high heavy metal concentrations.ResultsWe identified and isolated a nitrilase from the Atlantis II Deep Brine Pool in the Red Sea LCL. The isolated 338 amino-acid nitrilase (NitraS-ATII) is part of a highly conserved operon in different bacterial phyla with indiscernible function. The enzyme was cloned, expressed and purified. Characterization of the purified NitraS-ATII revealed its selectivity towards dinitriles, which suggests a possible industrial application in the synthesis of cyanocarboxylic acids. Moreover, NitraS-ATII showed higher thermal stability compared to a closely related nitrilase, in addition to its observed tolerance towards high concentrations of selected heavy metals.ConclusionThis enzyme sheds light on evolution of microbes in the Atlantis II Deep LCL to adapt to the diverse extreme environment and can prove to be valuable in bioremediation processes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0244-2) contains supplementary material, which is available to authorized users.

Mining a database of single amplified genomes from Red Sea brine pool extremophiles-improving reliability of gene function prediction using a profile and pattern matching algorithm (PPMA).

Reliable functional annotation of genomic data is the key-step in the discovery of novel enzymes. Intrinsic sequencing data quality problems of single amplified genomes (SAGs) and poor homology of novel extremophile's genomes pose significant challenges for the attribution of functions to the coding sequences identified. The anoxic deep-sea brine pools of the Red Sea are a promising source of novel enzymes with unique evolutionary adaptation. Sequencing data from Red Sea brine pool cultures and SAGs are annotated and stored in the Integrated Data Warehouse of Microbial Genomes (INDIGO) data warehouse. Low sequence homology of annotated genes (no similarity for 35% of these genes) may translate into false positives when searching for specific functions. The Profile and Pattern Matching (PPM) strategy described here was developed to eliminate false positive annotations of enzyme function before progressing to labor-intensive hyper-saline gene expression and characterization. It utilizes InterPro-derived Gene Ontology (GO)-terms (which represent enzyme function profiles) and annotated relevant PROSITE IDs (which are linked to an amino acid consensus pattern). The PPM algorithm was tested on 15 protein families, which were selected based on scientific and commercial potential. An initial list of 2577 enzyme commission (E.C.) numbers was translated into 171 GO-terms and 49 consensus patterns. A subset of INDIGO-sequences consisting of 58 SAGs from six different taxons of bacteria and archaea were selected from six different brine pool environments. Those SAGs code for 74,516 genes, which were independently scanned for the GO-terms (profile filter) and PROSITE IDs (pattern filter). Following stringent reliability filtering, the non-redundant hits (106 profile hits and 147 pattern hits) are classified as reliable, if at least two relevant descriptors (GO-terms and/or consensus patterns) are present. Scripts for annotation, as well as for the PPM algorithm, are available through the INDIGO website.

Induction of apoptosis in cancer cell lines by the Red Sea brine pool bacterial extracts

BackgroundMarine microorganisms are considered to be an important source of bioactive molecules against various diseases and have great potential to increase the number of lead molecules in clinical trials. Progress in novel microbial culturing techniques as well as greater accessibility to unique oceanic habitats has placed the marine environment as a new frontier in the field of natural product drug discovery.MethodsA total of 24 microbial extracts from deep-sea brine pools in the Red Sea have been evaluated for their anticancer potential against three human cancer cell lines. Downstream analysis of these six most potent extracts was done using various biological assays, such as Caspase-3/7 activity, mitochondrial membrane potential (MMP), PARP-1 cleavage and expression of γH2Ax, Caspase-8 and -9 using western blotting.ResultsIn general, most of the microbial extracts were found to be cytotoxic against one or more cancer cell lines with cell line specific activities. Out of the 13 most active microbial extracts, six extracts were able to induce significantly higher apoptosis (>70%) in cancer cells. Mechanism level studies revealed that extracts from Chromohalobacter salexigens (P3-86A and P3-86B(2)) followed the sequence of events of apoptotic pathway involving MMP disruption, caspase-3/7 activity, caspase-8 cleavage, PARP-1 cleavage and Phosphatidylserine (PS) exposure, whereas another Chromohalobacter salexigens extract (K30) induced caspase-9 mediated apoptosis. The extracts from Halomonas meridiana (P3-37B), Chromohalobacter israelensis (K18) and Idiomarina loihiensis (P3-37C) were unable to induce any change in MMP in HeLa cancer cells, and thus suggested mitochondria-independent apoptosis induction. However, further detection of a PARP-1 cleavage product, and the observed changes in caspase-8 and -9 suggested the involvement of caspase-mediated apoptotic pathways.ConclusionAltogether, the study offers novel findings regarding the anticancer potential of several halophilic bacterial species inhabiting the Red Sea (at the depth of 1500–2500 m), which constitute valuable candidates for further isolation and characterization of bioactive molecules.