Novel Enzymes From the Red Sea Brine Pools: Current State and Potential
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ABSTRACT:
SUBMITTER: Renn D
PROVIDER: S-EPMC8578733 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:In the Red Sea, two neighboring deep-sea brine pools, Atlantis II and Discovery, have been studied extensively, and the results have shown that the temperature and concentrations of metal and methane in Atlantis II have increased over the past decades. Therefore, we investigated changes in the microbial community and metabolic pathways. Here, we compared the metagenomes of the two pools to each other and to those of deep-sea water samples. Archaea were generally absent in the Atlantis II metagenome; Bacteria in the metagenome were typically heterotrophic and depended on aromatic compounds and other extracellular organic carbon compounds as indicated by enrichment of the related metabolic pathways. In contrast, autotrophic Archaea capable of CO2 fixation and methane oxidation were identified in Discovery but not in Atlantis II. Our results suggest that hydrothermal conditions and metal precipitation in the Atlantis II pool have resulted in elimination of the autotrophic community and methanogens.
| S-EPMC3638166 | biostudies-literature
Project description: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 (Tm = 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 Tm = 43.3°C. In conclusion, we have discovered two novel AR enzymes with potential application as thermophilic selection markers.
| S-EPMC5270753 | biostudies-literature
Project description: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.
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Project description:The candidate Division MSBL1 (Mediterranean Sea Brine Lakes 1) comprises a monophyletic group of uncultured archaea found in different hypersaline environments. Previous studies propose methanogenesis as the main metabolism. Here, we describe a metabolic reconstruction of MSBL1 based on 32 single-cell amplified genomes from Brine Pools of the Red Sea (Atlantis II, Discovery, Nereus, Erba and Kebrit). Phylogeny based on rRNA genes as well as conserved single copy genes delineates the group as a putative novel lineage of archaea. Our analysis shows that MSBL1 may ferment glucose via the Embden-Meyerhof-Parnas pathway. However, in the absence of organic carbon, carbon dioxide may be fixed via the ribulose bisphosphate carboxylase, Wood-Ljungdahl pathway or reductive TCA cycle. Therefore, based on the occurrence of genes for glycolysis, absence of the core genes found in genomes of all sequenced methanogens and the phylogenetic position, we hypothesize that the MSBL1 are not methanogens, but probably sugar-fermenting organisms capable of autotrophic growth. Such a mixotrophic lifestyle would confer survival advantage (or possibly provide a unique narrow niche) when glucose and other fermentable sugars are not available.
| S-EPMC4725937 | biostudies-literature
Project description: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.
| S-EPMC10943910 | biostudies-literature
Project description:The seafloor is a unique environment, which allows insights into how geochemical processes affect the diversity of biological life. Among its diverse ecosystems are deep-sea brine pools - water bodies characterized by a unique combination of extreme conditions. The 'polyextremophiles' that constitute the microbial assemblage of these deep hot brines have not been comprehensively studied. We report a comparative taxonomic analysis of the prokaryotic communities of the sediments directly below the Red Sea brine pools, namely, Atlantis II, Discovery, Chain Deep, and an adjacent brine-influenced site. Analyses of sediment samples and high-throughput pyrosequencing of PCR-amplified environmental 16S ribosomal RNA genes (16S rDNA) revealed that one sulfur (S)-rich Atlantis II and one nitrogen (N)-rich Discovery Deep section contained distinct microbial populations that differed from those found in the other sediment samples examined. Proteobacteria, Actinobacteria, Cyanobacteria, Deferribacteres, and Euryarchaeota were the most abundant bacterial and archaeal phyla in both the S- and N-rich sections. Relative abundance-based hierarchical clustering of the 16S rDNA pyrotags assigned to major taxonomic groups allowed us to categorize the archaeal and bacterial communities into three major and distinct groups; group I was unique to the S-rich Atlantis II section (ATII-1), group II was characteristic for the N-rich Discovery sample (DD-1), and group III reflected the composition of the remaining sediments. Many of the groups detected in the S-rich Atlantis II section are likely to play a dominant role in the cycling of methane and sulfur due to their phylogenetic affiliations with bacteria and archaea involved in anaerobic methane oxidation and sulfate reduction.
| S-EPMC3423430 | biostudies-literature
Project description: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 ∼54% 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.
| S-EPMC4303633 | biostudies-literature
Project description:Red Sea brine pool Genome sequencing and assembly
| PRJNA514885 | ENA
Project description:The central rift of the Red Sea contains 25 brine pools with different physicochemical conditions, dictating the diversity and abundance of the microbial community. Three of these pools, the Atlantis II, Kebrit and Discovery Deeps, are uniquely characterized by a high concentration of hydrocarbons. The brine-seawater interface, described as an anoxic-oxic (brine-seawater) boundary, is characterized by a high methane concentration, thus favoring aerobic methane oxidation. The current study analyzed the aerobic free-living methane-oxidizing bacterial communities that potentially contribute to methane oxidation at the brine-seawater interfaces of the three aforementioned brine pools, using metagenomic pyrosequencing, 16S rRNA pyrotags and pmoA library constructs. The sequencing of 16S rRNA pyrotags revealed that these interfaces are characterized by high microbial community diversity. Signatures of aerobic methane-oxidizing bacteria were detected in the Atlantis II Interface (ATII-I) and the Kebrit Deep Upper (KB-U) and Lower (KB-L) brine-seawater interfaces. Through phylogenetic analysis of pmoA, we further demonstrated that the ATII-I aerobic methanotroph community is highly diverse. We propose four ATII-I pmoA clusters. Most importantly, cluster 2 groups with marine methane seep methanotrophs, and cluster 4 represent a unique lineage of an uncultured bacterium with divergent alkane monooxygenases. Moreover, non-metric multidimensional scaling (NMDS) based on the ordination of putative enzymes involved in methane metabolism showed that the Kebrit interface layers were distinct from the ATII-I and DD-I brine-seawater interfaces.
| S-EPMC4172156 | biostudies-literature
Project description: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.
| S-EPMC6544261 | biostudies-literature