title: Functional Metagenome Mining of Soil for a Novel Gentamicin Resistance Gene.
abstract: Extensive use of antibiotics over recent decades has led to bacterial resistance against antibiotics, including gentamicin, one of the most effective aminoglycosides. The emergence of resistance is problematic for hospitals, since gentamicin is an important broad-spectrum antibiotic for the control of bacterial pathogens in the clinic. Previous study to identify gentamicin resistance genes from environmental samples have been conducted using culture-dependent screening methods. To overcome these limitations, we employed a metagenome-based culture-independent protocol to identify gentamicin resistance genes. Through functional screening of metagenome libraries derived from soil samples, a fosmid clone was selected as it conferred strong gentamicin resistance. To identify a specific functioning gene conferring gentamicin resistance from a selected fosmid clone (35-40 kb), a shot-gun library was constructed and four shot-gun clones (2-3 kb) were selected. Further characterization of these clones revealed that they contained sequences similar to that of the RNA ligase, T4 rnlA that is known as a toxin gene. The overexpression of the rnlA-like gene in Escherichia coli increased gentamicin resistance, indicating that this toxin gene modulates this trait. The results of our metagenome library analysis suggest that the rnlA-like gene may represent a new class of gentamicin resistance genes in pathogenic bacteria. In addition, we demonstrate that the soil metagenome can provide an important resource for the identification of antibiotic resistance genes, which are valuable molecular targets in efforts to overcome antibiotic resistance.
authors.affiliation: Molecular Phytobacteriology Laboratory, KRIBB, Daejeon 34113, Republic of Korea.|Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 34141, Republic of Korea.
authors.forename: Hyunjoo
authors.initials: H
authors.lastname: Im
authors.affiliation: Microbial Resource Center, KRIBB, Jeongeup 56212, Republic of Korea.|Department of Bioinformatics, University of Science and Technology (UST), Daejeon 34141, Republic of Korea.
authors.forename: Kyung Mo
authors.initials: KM
authors.lastname: Kim
authors.affiliation: Microbial Resource Center, KRIBB, Jeongeup 56212, Republic of Korea.|Department of Bioinformatics, University of Science and Technology (UST), Daejeon 34141, Republic of Korea.
authors.forename: Sang-Heon
authors.initials: SH
authors.lastname: Lee
authors.affiliation: Molecular Phytobacteriology Laboratory, KRIBB, Daejeon 34113, Republic of Korea.|Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 34141, Republic of Korea.
authors.forename: Choong-Min
authors.initials: CM
authors.lastname: Ryu
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001419:Bacteria / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification; D016680:Genome, Bacterial; D005839:Gentamicins / Q000494:pharmacology*; D056186:Metagenomics; D012988:Soil Microbiology
chemical_list: D000900:Anti-Bacterial Agents; D005839:Gentamicins
keywords: Antibiotic-resistance; Gentamicin; Metagenome; RnlA; Shot-gun library