title: Recovery of plasmid pEMB1, whose toxin-antitoxin system stabilizes an ampicillin resistance-conferring β-lactamase gene in Escherichia coli, from natural environments.
abstract: Non-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 into Escherichia coli TOP10, 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, the orf5 and orf6 genes were suggested to encode a CopG family transcriptional regulator and a plasmid stabilization system, respectively. Functional characterization of these genes using a knockout orf5 plasmid (pEMB1ΔparD) and the cloning and expression of orf6 (pET21bparE) indicated that these genes were antitoxin (parD) and toxin (parE) genes. Plasmid stability tests using pEMB1 and pEMB1ΔparDE in E. coli revealed that the orf5 and orf6 genes 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.
authors.affiliation: Department of Life Science, Chung-Ang University, Seoul, Republic of Korea.
authors.forename: Hyo Jung
authors.initials: HJ
authors.lastname: Lee
authors.affiliation: Department of Life Science, Chung-Ang University, Seoul, Republic of Korea.
authors.forename: Hyun Mi
authors.initials: HM
authors.lastname: Jin
authors.affiliation: Department of Life Science, Chung-Ang University, Seoul, Republic of Korea.
authors.forename: Moon Su
authors.initials: MS
authors.lastname: Park
authors.affiliation: Division of Environmental Science and Ecological Engineering, Korea University, Seoul, Republic of Korea.
authors.forename: Woojun
authors.initials: W
authors.lastname: Park
authors.affiliation: Department of Microbiology, Cornell University, Ithaca, New York, USA.
authors.forename: Eugene L
authors.initials: EL
authors.lastname: Madsen
authors.affiliation: Department of Life Science, Chung-Ang University, Seoul, Republic of Korea cojeon@cau.ac.kr.
authors.forename: Che Ok
authors.initials: CO
authors.lastname: Jeon
mesh_terms: D000668:Ampicillin Resistance; D001427:Bacterial Toxins / Q000235:genetics; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification*; D055786:Gene Knockout Techniques; D042822:Genomic Instability; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010957:Plasmids; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA; D012722:Sewage / Q000382:microbiology*; D012988:Soil Microbiology; D001618:beta-Lactamases / Q000235:genetics*
chemical_list: D001427:Bacterial Toxins; D004269:DNA, Bacterial; D012722:Sewage; D001618:beta-Lactamases