title: Novel thermostable antibiotic resistance enzymes from the Atlantis II Deep Red Sea brine pool.
abstract: 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.
authors.affiliation: Graduate Program of Biotechnology, The American University in Cairo, Cairo, Egypt.|Department of Biology and Biochemistry, University of Bath, Bath, UK.
authors.forename: Ali H A
authors.initials: AH
authors.lastname: Elbehery
authors.affiliation: Department of Biology and Biochemistry, University of Bath, Bath, UK.
authors.forename: David J
authors.initials: DJ
authors.lastname: Leak
authors.affiliation: Graduate Program of Biotechnology, The American University in Cairo, Cairo, Egypt.|Biology Department and YJ-Science and Technology Research Center, The American University in Cairo, Cairo, Egypt.
authors.forename: Rania
authors.initials: R
authors.lastname: Siam
mesh_terms: D003001:Cloning, Molecular; D019295:Computational Biology; D024881:Drug Resistance, Bacterial; D004795:Enzyme Stability; D015870:Gene Expression; D019015:Geologic Sediments; D019156:Indian Ocean; D019868:Kanamycin Kinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D054892:Metagenome; D056186:Metagenomics; D016366:Open Reading Frames; D012492:Salts; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D013696:Temperature; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D012492:Salts; C017082:brine; D019868:Kanamycin Kinase; D001618:beta-Lactamases