ABSTRACTNon-culture-based procedures were used to investigate plasmids showing ampicillin resistance properties in two different environments: remote mountain soil (Mt. Jeombong) and sludge (Tancheon wastewater treatment plant). Total DNA extracted from the environmental samples was directly transformed intoEscherichia coliTOP10, and a single and three different plasmids were obtained from the mountain soil and sludge samples, respectively. Interestingly, the restriction fragment length polymorphism pattern of the plasmid from the mountain soil sample, designated pEMB1, was identical to the pattern of one of the three plasmids from the sludge sample. Complete DNA sequencing of plasmid pEMB1 (8,744 bp) showed the presence of six open reading frames, including a β-lactamase gene. Using BLASTX, theorf5andorf6genes were suggested to encode a CopG family transcriptional regulator and a plasmid stabilization system, respectively. Functional characterization of these genes using a knockoutorf5plasmid (pEMB1ΔparD) and the cloning and expression oforf6(pET21bparE) indicated that these genes were antitoxin (parD) and toxin (parE) genes. Plasmid stability tests using pEMB1 and pEMB1ΔparDEinE. colirevealed that theorf5andorf6genes enhanced plasmid maintenance in the absence of ampicillin. Using a PCR-based survey, pEMB1-like plasmids were additionally detected in samples from other human-impacted sites (sludge samples) and two other remote mountain soil samples, suggesting that plasmids harboring a β-lactamase gene with a ParD-ParE toxin-antitoxin system occurs broadly in the environment. This study extends knowledge about the dissemination and persistence of antibiotic resistance genes in naturally occurring microbial populations.
Recovery of Plasmid pEMB1, Whose Toxin-Antitoxin System Stabilizes an Ampicillin Resistance-Conferring β-Lactamase Gene in Escherichia coli, from Natural Environments
