The bottom of the Red Sea harbors over 25 deep hypersaline anoxic basins that are geochemically distinct and characterized by vertical gradients of extreme physicochemical conditions. Because of strong changes in density, particulate and microbial debris get entrapped in the brine-seawater interface (BSI), resulting in increased dissolved organic carbon, reduced dissolved oxygen toward the brines and enhanced microbial activities in the BSI. These features coupled with the deep-sea prevalence of ammonia-oxidizing archaea (AOA) in the global ocean make the BSI a suitable environment for studying the osmotic adaptations and ecology of these important players in the marine nitrogen cycle. Using phylogenomic-based approaches, we show that the local archaeal community of five different BSI habitats (with up to 18.2% salinity) is composed mostly of a single, highly abundant Nitrosopumilus-like phylotype that is phylogenetically distinct from the bathypelagic thaumarchaea; ammonia-oxidizing bacteria were absent. The composite genome of this novel Nitrosopumilus-like subpopulation (RSA3) co-assembled from multiple single-cell amplified genomes (SAGs) from one such BSI habitat further revealed that it shares B54% of its predicted genomic inventory with sequenced Nitrosopumilus species. RSA3 also carries several, albeit variable gene sets that further illuminate the phylogenetic diversity and metabolic plasticity of this genus. Specifically, it encodes for a putative proline-glutamate 'switch' with a potential role in osmotolerance and indirect impact on carbon and energy flows. Metagenomic fragment recruitment analyses against the composite RSA3 genome, Nitrosopumilus maritimus, and SAGs of mesopelagic thaumarchaea also reiterate the divergence of the BSI genotypes from other AOA.

Oceanic deep hypersaline anoxic basins (DHABs) are characterized by drastic changes in physico-chemical conditions in the transition from overlaying seawater to brine body. Brine-seawater interfaces (BSIs) of several DHABs across the Mediterranean Sea have been shown to possess methanogenic and sulfate-reducing activities, yet no systematic studies have been conducted to address the potential functional diversity of methanogenic and sulfate-reducing communities in the Red Sea DHABs. Here, we evaluated the relative abundance of Bacteria and Archaea using quantitative PCR and conducted phylogenetic analyses of nearly full-length 16S rRNA genes as well as functional marker genes encoding the alpha subunits of methyl-coenzyme M reductase (mcrA) and dissimilatory sulfite reductase (dsrA). Bacteria predominated over Archaea in most locations, the majority of which being affiliated with Deltaproteobacteria, while Thaumarchaeota were the most prevalent Archaea in all sampled locations. The upper convective layers of Atlantis II Deep, which bear increasingly harsh environmental conditions, were dominated by members of the class Thermoplasmata (Marine Benthic Group E and Mediterranean Sea Brine Lakes Group 1). Our study revealed unique microbial compositions, the presence of niche-specific groups, and collectively a higher diversity of the sulfatereducing communities compared to the methanogenic communities in all five studied locations. Dear Dr. Jebbar, Please find attached our revised version of RESMIC-D-15-00081 for publication in Research in Microbiology. The only comment from the referee was on the measurements of CO 2 in our samples. As these were done by a commercial service provide by GEOMAR in Germany, we now refer to their website.We hope that you will find the manuscript now suitable for publication in Research in Microbiology. were dominated by members of the class Thermoplasmata (Marine Benthic Group E and 37Mediterranean Sea Brine Lakes Group 1). Our study revealed unique microbial compositions, the 38 presence of niche-specific groups, and collectively a higher diversity of the sulfate-reducing 39 communities compared to the methanogenic communities in all five studied locations. 40

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.

BackgroundHigh salinity and temperature combined with presence of heavy metals and low oxygen renders deep-sea anoxic brines of the Red Sea as one of the most extreme environments on Earth. The ability to adapt and survive in these extreme environments makes inhabiting bacteria interesting candidates for the search of novel bioactive molecules.MethodsTotal 20 i.e. lipophilic (chloroform) and hydrophilic (70% ethanol) extracts of marine bacteria isolated from brine-seawater interface of the Red Sea were tested for cytotoxic and apoptotic activity against three human cancer cell lines, i.e. HeLa (cervical carcinoma), MCF-7 (Breast Adenocarcinoma) and DU145 (Prostate carcinoma).ResultsAmong these, twelve extracts were found to be very active after 24 hours of treatment, which were further evaluated for their cytotoxic and apoptotic effects at 48 hr. The extracts from the isolates P1-37B and P3-37A (Halomonas) and P1-17B (Sulfitobacter) have been found to be the most potent against tested cancer cell lines.ConclusionOverall, bacterial isolates from the Red Sea displayed promising results and can be explored further to find novel drug-like molecules. The cell line specific activity of the extracts may be attributed to the presence of different polarity compounds or the cancer type i.e. biological differences in cell lines and different mechanisms of action of programmed cell death prevalent in different cancer cell lines.

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