1. Abstract A biosensor that utilizes bacterial stress responses to oxidative toxins, such as the glutathione-gated potassium efflux (GGKE) system found in Gram negative heterotrophic bacteria, can be used to detect the presence of toxins in environmental water systems. Our team has previously reported a direct link between GGKE and activated sludge biomass deflocculation in response to oxidative chemicals, showing that GGKE can be used for upset early warning in biological wastewater treatment (1, 2). The bacterial GGKE system also involves the sacrificial antioxidant compound glutathione, which is similarly implicated in the detoxification of reactive oxygen species in many eukaryotic cells. In this study, we have developed a bacterial GGKE-based biosensor prototype that consists of two main components: the biological element (Pseudomonas aeruginosa cells immobilized in a polymer matrix) and the sensing unit that measures extracellular potassium. We have shown that the microscale immobilization of P. aeruginosa in alginate can be achieved. The dose-response curve for bacterial GGKE in response to N-ethylmaleimide (NEM), a model oxidative chemical, correlates very well with mitochondrial damage to a mixed rat brain cell culture exposed to the same concentrations of NEM. Overall, our proof-of-concept study indicates that robust bacterial-based biosensors may potentially be used to predict public health threats in a variety of systems. Predicting the public health impact of oxidative toxins using a bacterial glutathione-gated potassium efflux stress response biosensor Kaoru Ikuma 1, Ines D. S. Henriques 1, Beverly A. Rzigalinski 4, Brian J. Love 2, and Nancy G. Love 1,3* Departments of 1 Civil & Environmental Engineering, 2 Materials Sciences and Engineering, and 3 Biological Sciences, Virginia Tech, Blacksburg, VA 24061 4 Edward Via Virginia College of Osteopathic Medicine, Blacksburg, VA 24060 * Corresponding Author Acknowledgements Funding for this project was provided by the EPA Midwest Hazardous Substances Research Center and the National Institution of Standards and Technology. The authors would like to thank Jody Smiley, Julie Petruska, Chengya Liang, and Neeraj Singh for their support and assistance in the laboratories. Research Significance Oxidative stress resulting from reactive oxygen species (ROS) buildup in brain tissues is a common precursor to neurodegenerative disorders such as Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease (3, 4). Although further work is necessary, this study indicates that bacterial-based biosensors may potentially be used to predict public health threats in a variety of engineered and natural systems. Rat brain cell mitochondrial damage assay (MTT assay): Microscale immobilization of P. aeruginosa cells were accomplished in alginate beads with an average diameter of 200  m Cells were stressed immediately after immobilization but viability recovered over time 3 hours after immobilization 10 days after immobilization Figure 2. LIVE/DEAD ® staining of immobilized cells. Green dots indicate live cells, red dots indicate dead cells. Figure 1. Comparison of alginate bead sizes to a dime. Microscopic image of alginate microbeads are also shown at 200x resolution. Biosensor model Immobilized bacterial cells K+K+ K+K+ K+K+ K+K+ Upstream K + sensing unit (ion-selective electrode/ ISE) Downstream K + sensing unit (ISE) Conclusions  Microscale immobilization of P. aeruginosa cells was achieved in alginate using an octanol-emulsifier mixture  Immobilized cells were viable for at least 14 days  Bacterial GGKE response to NEM is dose-dependent  Mitochondrial damage to rat brain cells can be used as a measure of oxidative damage and is a dose- dependent response  Bacterial GGKE response corresponds to rat brain cell mitochondrial damage upon exposure to NEM Bacterial GGKE response corresponds well to rat brain cell mitochondrial damage upon exposure to similar concentrations of NEM Figure 3. Dose-dependent responses of bacterial GGKE and mitochondrial damage to mixed cultures of rat brain cells upon exposure to NEM. Bacterial GGKE measured as K + effluxed per cell, rat brain cell mitochondrial damage measured as the decrease in formazan production in comparison to the unshocked cells. Error bars indicate the standard deviations from at least three separate experiments. References 1. Bott, C. B., and N. G. Love. 2004. Implicating the Glutathione-Gated Potassium Efflux System as a Cause of Electrophile-Induced Activated-Sludge Deflocculation. App. Environ. Microbiol. 70:001-010. 2. Bott, C. B., and N. G. Love. 2002. Investigating a Mechanistic Cause for Activated-Sludge Deflocculation in Response to Shock Loads of Toxic Electrophilic Chemicals. Water Environ. Res. 74:306-315. 3. Dringen, R., and J. Hirrlinger. 2003. Glutathione Pathways in the Brain. Biol. Chem. 384:505- 516. 4. Fonnum, F., and E. A. Lock. 2004. The contributions of excitotoxicity, glutathione depletion and DNA repair in chemically induced injury to neurones: exemplified with toxic effects on cerebellar granule cells. J. Neurochem. 88:513-531. Future work GSH (reduced glutathione) GS - /GSX (may be conjugated to electrophiles) GSSG (oxidized glutathione) Hypothesis: The intracellular glutathione is oxidized or conjugated in a dose-dependent manner in response to NEM exposure, and the response corresponds to the bacterial GGKE and rat brain cell mitochondrial damage dose-response curves. The hypothesis will be tested by measuring intracellular glutathione (reduced, oxidized, and conjugated) by high-performance liquid chromatography (HPLC) in collaboration with Dr. Heileen Hsu-Kim at Duke University. Methods Stirred at 1200 rpm CaCl 2 -octanol solutions added Stirred at 1200 rpm to cure 5% alginate gel and bacteria mixture dropped into emulsifier-containing octanol Octanol washed off by centrifugation Bacterial strain used: Pseudomonas aeruginosa environmental isolate Microscale bacterial cell immobilization in alginate: Long-term viability assays of immobilized cells: Late log phase cells (400x) Each batch exposed to different concentration of toxin (NEM/Cu) Samples taken over time (t = 0, 3, 20, 40, 60 min) Analyzed for extracellular K + using atomic absorption spectrophotometry Bacterial GGKE experiment: Mixed rat brain cells matured for 12 days (100x) Cells exposed to different doses of NEM/Cu MTT assay MTT yellow solution Formazan blue crystals (absorbance measured at 570nm) Mitochondrial dehydrogenase microbeads Autoclaved primary wastewater effluent + yeast extract Shake at room temperature Take samples at 0, 1, 3, 5, 8, 11, 14 days after immobilization Dissolve microbeads with high salt solution (high Na + concentration) Perform fluorescent viability assays on the microscope (LIVE/DEAD ® staining) K+K+ GS- Oxidative chemical stressor Bacterial GGKE responseEukaryotic cell damage cascade ROS ↑ Antioxidants e.g. GSH APOPTOSIS Exposure to oxidative chemicals results in various stress responses including GGKE in Gram-negative bacterial cells and mitochondrial damage in eukaryotic cells GSH = reduced glutathione Rat brain cell mitochondrial damage assay (MTT assay): Dime 2 cm Alginate bead 2 mm Alginate microbeads 200  m