Stress-activated bioluminescent Escherichia coli sensors for antimicrobial agents detection.
A DNA cartridge encoding Photinus pyralis luciferase (luc), lacZ homology extensions and an excisable marker was constructed to facilitate the conversion of Escherichia coli lacZ fusions to luc fusions by lambda Red-mediated recombination. This tool was used to transform a cspA::lacZ strain into a luminescent biosensor for C-group translational inhibitors. Comparison of cspA::lacZ and cspA::luc cells showed native firefly luciferase to be a more rapid and sensitive reporter than beta-galactosidase for chloramphenicol detection. To evaluate the usefulness of a red-shifted variant of P. pyralis luciferase (LucR1) for biosensor development, a single copy translational fusion between the SOS-inducible sulA promoter and the lucR1 gene was inserted at the malP site of the E. coli chromosome. The sulA::lucR1 fusion allowed high signal detection of the quinolone ofloxacin to levels as low as 15% of the minimum inhibitory concentration and could be combined with a cspA::lacZ fusion to yield a biosensor suitable for the independent and dual detection of chloramphenicol and ofloxacin.
Shapiro
Elyse
E
Department of Chemical Engineering, University of Washington, Box 351750, Seattle, WA 98195, USA.
Baneyx
François
F
D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002701:Chloramphenicol / Q000032:analysis*; D057773:Cold Shock Proteins and Peptides; D004926:Escherichia coli / Q000187:drug effects* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics; D048888:Fireflies / Q000201:enzymology; D006360:Heat-Shock Proteins / Q000235:genetics; D007763:Lac Operon / Q000235:genetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D015242:Ofloxacin / Q000032:analysis*; D010957:Plasmids / Q000235:genetics
D057773:Cold Shock Proteins and Peptides; C498192:CspA protein, E coli; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D002701:Chloramphenicol; D015242:Ofloxacin; D008156:Luciferases