title: Characterization of novel antibiotic resistance genes identified by functional metagenomics on soil samples.
abstract: The soil microbial community is highly complex and contains a high density of antibiotic-producing bacteria, making it a likely source of diverse antibiotic resistance determinants. We used functional metagenomics to search for antibiotic resistance genes in libraries generated from three different soil samples, containing 3.6 Gb of DNA in total. We identified 11 new antibiotic resistance genes: 3 conferring resistance to ampicillin, 2 to gentamicin, 2 to chloramphenicol and 4 to trimethoprim. One of the clones identified was a new trimethoprim resistance gene encoding a 26.8 kDa protein closely resembling unassigned reductases of the dihydrofolate reductase group. This protein, Tm8-3, conferred trimethoprim resistance in Escherichia coli and Sinorhizobium meliloti (γ- and α-proteobacteria respectively). We demonstrated that this gene encoded an enzyme with dihydrofolate reductase activity, with kinetic constants similar to other type I and II dihydrofolate reductases (K(m) of 8.9 µM for NADPH and 3.7 µM for dihydrofolate and IC(50) of 20 µM for trimethoprim). This is the first description of a new type of reductase conferring resistance to trimethoprim. Our results indicate that soil bacteria display a high level of genetic diversity and are a reservoir of antibiotic resistance genes, supporting the use of this approach for the discovery of novel enzymes with unexpected activities unpredictable from their amino acid sequences.
authors.affiliation: Genetic Ecology Group, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Calle Profesor Albareda 1, 18008 Granada, Spain.
authors.forename: Gloria
authors.initials: G
authors.lastname: Torres-Cortés
authors.forename: Vicenta
authors.initials: V
authors.lastname: Millán
authors.forename: Hugo C
authors.initials: HC
authors.lastname: Ramírez-Saad
authors.forename: Rafael
authors.initials: R
authors.lastname: Nisa-Martínez
authors.forename: Nicolás
authors.initials: N
authors.lastname: Toro
authors.forename: Francisco
authors.initials: F
authors.lastname: Martínez-Abarca
mesh_terms: D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects; D015723:Gene Library; D005798:Genes, Bacterial; D056186:Metagenomics; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010802:Phylogeny; D016962:Sinorhizobium meliloti / Q000187:drug effects; D012987:Soil / Q000032:analysis; D012988:Soil Microbiology; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D014295:Trimethoprim / Q000494:pharmacology; D014296:Trimethoprim Resistance / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012987:Soil; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase