Improved Sensitivity of the in vivo lux Reporter System in Measuring Reactive Oxygen Species during Antibiotic Treatment of Escherichia coli

Reactive oxygen species (ROS) in Escherichia coli can be produced through aerobic respiration as well as through environmental stressors and has been shown to cause direct damage to various cell components. Measurements of internal ROS production have previously been performed through the use of a lux operon with luxCDABE genes under the control of the katG promoter. This study tested the sensitivity and effectiveness of two split lux systems ( luxAB and luxAB + luxCDE ) for measurement of internal ROS enhanced through the external substrate, nonanal, and induced through the antibiotics, tobramycin and norfloxacin, compared to the sensitivity of the full luxCDABE reporter system. The split lux system was found to have an enhanced luminescence response to antibiotic treatment, where luxAB and luxAB + luxCDE produced 2.8 times and 1.5 times higher signal than the full luxCDABE system, respectively. The response ratio of the split system containing only luxAB was the highest at 5.5 times and 30 times larger than the full luxCDABE and split luxAB + luxCDE systems, respectively. Through calculations of EC and the detection limit, the split luxAB + luxCDE system was shown to be the most sensitive in 200 detecting ROS. Variation within the data suggests that other factors may contribute to the effectiveness and sensitivity of the lux systems, possibly competition between external and internal substrates in the luxAB + luxCDE system. Overall it was determined that the split lux reporter systems were more sensitive to ROS than the full luxCDABE system, and also produced a stronger luminescence signal.