Biosensors: Development and Environmental Testing Investigators:

1. Biosensors: Development and Environmental Testing Investigators: Anne J. Anderson, Charles D. Miller and Joan E. McLean Utah State University Logan, Utah 84322-5305 RD-83090701-0

2. Metals of interest Copper:maximum drinking water level standard 1.3 mg/L aquatic chronic exposure limit 30.3 µg/L Human tolerance relatively high Used for microbial pest control Some lower organisms acute toxicity (phytoplankton, zooplankton, amphibians) Cadmium:maximum drinking water standard 0.005 mg/L aquatic chronic exposure limit 2.7µg/L Higher animal toxicity: accumulates in kidneys and liver, produces bone and blood problems Carcinogenic (Group B1) Minor use as pesticide

3. Cu is a trace element: required for certain enzymatic and carrier proteins Cd no known cellular use—toxic- may disrupt Ca and Zn metabolism Both cause oxidative stress Both act on –SH proteins Cell concentrations regulated by chelation, sorption, uptake, and efflux mechanisms MX

4. Biosensors • Final goals: • An array of promoter fusions that respond differentially and specifically • with light output upon exposure to toxic metals • 2) A gene chip array to detect transcript abundance from cells responding to • toxic metals. Pattern of gene activation would specify the metal.

5. Test organisms: P. putida strains Cells of both pseudomonads are killed by concentrations of Cu starting between 1 and 10 mg/L

6. P. putida KT2440 1000000000 100000000 10000000 1000000 100000 1 h 1 h # cells per 1 ml 4 h 4 h 10000 1000 100 10 1 0.001 0.01 0.1 1 10 100 mg/L of Cd2+ P. putida Corvallis 100000000 10000000 1000000 100000 10000 # cells per 1 ml 1000 100 10 1 0.001 0.01 0.1 1 10 100 mg/L of Cd2+ The pseudomonad cells show little change in culturability with Cd even at doses of 100 mg/L. Both wild type isolates behave similarly showing resistance to Cd

7. Generation of Luciferase Biosensor Metal stimulus Wild type lux A/B promoter ORF Transcript Transcript Light Protein product Normal cell Biosensor

8. P. putida Corvallis mutants-Cu [in .01M CaCl2] 30 25 20 Cat A*10 Lux Bfr 15 FeSOD 10 5 0 0 0.01 0.1 1 10 mg/L of Cu2+

9. P. putida Corvallis mutants-Cd [in .01M KNO ] 3 14 12 10 Cat A*10 8 Lux Bfr 6 FeSOD 4 2 0 0 1 10 50 100 mg/L of Cd2+ Corvallis mutants-Cd P. putida Cadmium detection [in .01M Ca(NO3)2] 14 Cat A*10 12 Bfr 10 FeSOD 8 Lux 6 4 2 0 0 1 10 50 100 mg/L Cd2+ FeSOD mutant is most sensitive Ca inhibits the Cd response

10. Response of KT2440 mutants to Cu 3.5 3 2.5 2 10 mg/L Relative change in lux (fold) 1 mg/L 1.5 1 0.5 0 68 18 45 47 41 4 43 66 70 69 7 31 46 21 3 30 Mutant strain

11. Response of KT2440 mutants to Cd 16 14 12 10 100 mg/L 10 mg/L 8 Relative change in lux (fold) 1 mg/L 6 4 2 0 54 18 31 30 43 4 68 46 45 69 7 70 21 41 3 47 45 Mutant strain

12. pI 4 pI 7 pI 4 pI7

13. Spot B: increased three fold Corresponded to lipoamide dehydrogenase in TCA cycle

14. pI 4 pI 7 4-15% Mr (kDa) Cd responses 97.0 66.0 45.0 30.0 20.1 14.4 Peptides of increased Intensity. KT2440 0.01M Ca(NO3)2 100 ppm Cd

15. Conclusions • The two approaches for biosensor development are feasible • 2) luxAB::Insertional mutants that detect Cu and Cd differentially have • been identified • Gene loci await determination • 3) Peptides that increase upon Cu exposure are detected and one has been • correlated with a specific function RD-83090701-0