Amplified detection of transcriptional and translational inhibitors in bioluminescent Escherichia coli K-12.
The excessive prescription of antimicrobial agents and their use as animal growth promoters lead to the spread of resistance among pathogenic bacteria. Consequently, unnecessary use should be minimized, and new chemicals with novel mechanisms of action are needed. The authors have developed a fast method to measure the activity of antibiotics by means of a genetically engineered strain of Escherichia coli K-12. The system is based on the full-length bacterial luciferase operon coupled to the tetracycline-inducible tetA promoter in the reporter plasmid pTetLux1. Sublethal doses of tetracycline are used to start the luciferase synthesis in cultures that were previously incubated with the antibiotic under investigation. After a variable time frame-from 1 to 4 h, depending on the antimicrobial mode of action-the level of light emission from treated cultures is compared to the level obtained in control cultures. The gap in bioluminescence outlines the antibiotic interference in bacterial metabolism. Throughout this study, freeze-dried sensor cells were used to avoid repeated cultures from day to day. The authors show the results of 10 model antibiotics, representing different molecular structures and mechanisms of action. The results show that no actively dividing cells are needed for sensitive responses, especially when transcriptional and translational inhibitors, directly interfering with the luciferase production, are tested. The assay can be easily automated for high-throughput screening purposes of pharmaceutical industry.
Galluzzi
Lorenzo
L
University of Turku, Department of Biotechnology, Finland.
Karp
Matti
M
D001331:Automation; D020650:Combinatorial Chemistry Techniques; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism*; D017930:Genes, Reporter; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008027:Light; D008156:Luciferases / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D013997:Time Factors; D014158:Transcription, Genetic
D008156:Luciferases