|AN, RAN - University Of Kentucky|
|HANDELSMAN, JO - Yale University|
|MOE, LUKE - University Of Kentucky|
Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/10/2015
Publication Date: 3/17/2015
Citation: Allen, H.K., An, R., Handelsman, J., Moe, L. 2015. A response regulator from a soil metagenome enhances resistance to the beta-lactam antibiotic carbenicillin in Escherichia coli. PLoS One. 10(3):e0120094. DOI: 10.1371/journal.pone.0120094.
Interpretive Summary: Whether and what kinds of antibiotic resistance genes reside in the environment are relevant research questions in order to understand all possible ways that a bacterium could become resistant to antibiotics. This is important because once pathogenic bacteria become resistant to antibiotics, they become harder to treat. We studied antibiotic resistance in Alaskan soil because it is an environment far from human activities, and so we could investigate antibiotic resistance that naturally occurs in soil bacterial communities. We found a gene that makes Escherichia coli (E. coli) resistant to a certain beta-lactam antibiotic called carbenicillin, and it is different from known mechanisms of resistance to this type of antibiotics. Turning on this Alaskan-soil gene in E. coli indirectly makes E. coli resistant to carbenicillin. The Alaskan-soil gene does not interact directly with carbenicillin; rather, it tells E. coli to make more of the pumps that it already has to kick carbenicillin out of the cell. It also does not change the resistance to other antibiotics that we tested. This research is interesting because it presents an unexpected mechanism of antibiotic resistance. This study contributes useful information to scientists, veterinarians, and medical professionals interested in the full potential of antibiotic resistance genes from the environment.
Technical Abstract: Environmental reservoirs of antibiotic resistance genes are thought to harbor as-yet-unknown mechanisms of antibiotic resistance. Here we report on an unconventional mode by which a metagenomic response regulator confers resistance to the beta-lactam antibiotic carbenicillin in Escherichia coli. A recombinant metagenomic clone (Blr16) harboring a 5,169 bp deoxyribonucleic acid (DNA) insert was selected from a metagenomic library previously constructed from a remote Alaskan soil. The Blr16 recombinant clone conferred specific resistance to carbenicillin, with limited increases in resistance conferred to other tested antibiotics, including other beta-lactams (penicillins and cephalosporins), rifampin, ciprofloxacin, erythromycin, chloramphenicol, nalidixic acid, fusidic acid, and gentamicin. Resistance was more pronounced at 24 Celsius than at 37 Celsius. Zone-of-inhibition assays showed that the mechanism of carbenicillin resistance was not due to antibiotic inactivation. The DNA insert did not encode any genes known to confer antibiotic resistance, but did have two putative open reading frames (ORFs) that were annotated as a metallopeptidase and a two-component response regulator. Transposon mutagenesis and subcloning of the two open reading frames followed by minimum inhibitory concentration assays showed that the response regulator gene was necessary and sufficient to confer the phenotype. Quantitative reverse transcriptase PCR showed that the response regulator suppresses expression of the ompF porin gene, independently of the small RNA regulator micF, and enhances exression of the acrD, mdtA, and mdtB efflux pump genes. This work demonstrates that antibiotic resistance can be achieved by the modulation of gene regulation by heterologous DNA. Functional analyses such as these continue to be important for making discoveries in antibiotic resistance gene ecology.