National Science Foundation, Research Experience for Undergraduates, Utah State University August 5, 2010 serenalynn05@g

1. Pseudomonas putida KT2440 Bacterial Chemotaxis Behavior Analysis via Video Microscopy with Sodium Benzoate and Succinic Acid as Chemoattractants Serena Bennett National Science Foundation, Research Experience for Undergraduates, Utah State University August 5, 2010 serenalynn05@gmail.com

2. Outline • Results/Data • Growth Curve Parameter • Velocity • Turn Angle • X vs. Y Displacement • Project Contribution • Conclusion • Future Work • Acknowledgements • References • Questions Problem Pseudomonas Putida KT2440 Key Words Objective Equipment Chemoattractants Growth Curve Phases

3. Problem: Soil Pollution • Due to pesticides, oil spills, landfill leaching etc; soil is contaminated with toxic chemicals: • 2,4-Dichlorophenoxyacetic acid • Most widely used herbicide in the world • Major component in Agent Orange • Toluene • Naturally occurring in crude oil • Byproduct of gasoline production • Napthalene • Nitrobenzene • Water Contamination • Farmland Contamination • Environmental Damage • Health Risks Toxic Chemicals In Soil Effects

4. Key Words: Chemotaxis:The observable reaction of an organism moving toward or away from the concentration of a certain chemical. Chemoattractant: A certain chemical that the organism of interest is attracted to. Video Microscopy: Microscopic analysis of nano sized samples to capture real time videos: 15 frames/second

5. Pseudomonas putida KT2440 Potential as Soil Bioremediator • Motile due to flagella • Non-pathogenic: Safe to be released into the environment • Chemotactic towards soil pollutants • Naphthalene • Toluene • Certain aromatic compounds • 4-hydroxybenzoate • 2,4-Dichlorophenoxyacetic acid • Degrades certain toxic chemicals http://genome.jgi-psf.org/psepu/psepu.home.html

6. Taken by Serena Bennett with IX71 inverted microscope at 60X magnification at 15 frames/sec.

7. Objective • Observe P. Putida characteristic reactions/indicators from exposure to two separate chemoattractants: • Succinic Acid (Succinate) • Sodium Benzoate • Compare • Growth Curve Parameters • Velocity (Individual bacterium) • Turn Angle (Individual bacterium) • Displacement (Individual bacterium)

8. Equipment • Spectrophotometer • Measures absorbance (optical density) • Proportional to concentration • IX71 Inverted Video Microscope • (Olympus, Center Valley, PA, USA) • Digital CCD camera (Olympus DP30BW • ImagePro v. 6.1 • (Media Cybernetics, Bethesda, MD, USA) • Image Analysis • OriginPro v. 7.5 • (OriginLab Northampton, MA, USA) • PPKT2440 • Lysogeny Broth http://www.olympusamerica.com/seg_section/product.asp?product=1023&p=72

9. Chemoattracants • 15mM • 88.5mg in 50ml LB • Autoclave • 25mM • 180mg in 50ml LB • Autoclave • Concentrations taken from Nisar Ahmed Kanhar experiment, Utah State University. Succinic Acid Sodium Benzoate

10. Growth Curve Phases • Early Logarithmic • Mid Logarithmic • Late Logarithmic • Early Stationary General Phases Phases Compared in KT2440 Project

11. Growth Curve Comparison • OriginPro • Sigmoid Fit • Modified Gompertz Equations 1) 2) 3) 4) OriginPro fitted growth curve of PPKT2440 with no chemoattractant.

12. Growth Curve Parameters OriginPro growth curve of PPKT2440 with 25mM Benzoate OriginPro growth curve of PPKT2440 with 15mM Succinate

13. Growth Curve Parameters • A=a-asymptote-highest point in curve • k=rate coefficient • Xc= Center point of inflection • µm=Maximum specific growth rate • λ = Lag Phase

14. Velocity Comparison

15. Velocity Box Plots Mid Logarithmic Phase Early Logarithmic Phase Late Logarithmic Phase Early Stationary Phase

16. Turn Angle Comparison Smaller Turn Angle=Less Direction Change

17. Turn Angle Frequency PPKT2440 without chemoattractant favors small angles (under 90°). Notice bimodal distribution in above succinate experiment.

18. Normalized X vs. Y Displacement-KT2440 Control Mid Logarithmic Phase Late Logarithmic Phase

19. Normalized X vs. Y Displacement-KT2440 with 15mM Succinate Mid Logarithmic Phase Late Logarithmic Phase

20. Normalized X vs. Y Displacement-KT2440 with 25mM Benzoate Mid Logarithmic Phase Late Logarithmic Phase

21. Accumulated Distance vs. Frame Number Without Chemoattractant With 15mM Succinate With 25mM Benzoate

22. Project Contribution Previous Research • Collective analysis • Swarm Plates • Individual bacterium analysis • Video Microscopy • Growth Curve Parameter Comparison

23. Conclusion: • Decreased velocity • Altered turn angle distribution-bimodal • Decrease in overall distance traveled • Extended lag phase • Lengthens distance traveled in mid log phase • Higher average direction change than without chemoattractant • Highest maximum absorbance 15mM Succinate 25mM Benzoate

24. Future Work • Find new sensitive indicators • More frames/sec in video analysis • Reactions to other chemoattractants • Research specific pseudomonas genes • Mutate genes to create custom bioremediator

25. Acknowledgments: • Funds from National Science Foundation • Research Experience for Undergraduates • Dr. YangQuan Chen • Program Director • Dr. Anhong Zhou • Molecular and Cellular Sensing and Imaging Laboratory • Dr. Charlie Miller • P. Putida cells • Mike Davis • Technical skills

26. References: • Harwood, Caroline S., Kathy Fosnaugh, and Marilyn Dispensa. "Flagellation of Pseudomonas putida and Analysis of Its Motile Behavior." Journal of Bacteriology. 171.7 (1989): 4063-4066. • Parales, Rebecca E., and John D Haddock. "Biocatalytic degradation of pollutants." Elsevier. 15. (2004): 374-379. • Reva, Oleg N. , Christian Weinel, MiryamWeinel, and KertsinBohm. "Functional Genomics of Stress Response in Pseudomonas putida KT2440." Journal of Bacteriology. 188.11 (2006): 4079-4092. • Ford, Roseanne M., and Ronald W. Harvey. "Role of chemotaxis in the transport of bacteria through saturated porous media." Elsevier. 30. (2007): 1608-1617. • Kim, Hye-Eun, Maiko Shitashiro, Akio Kurodo, Noboru Takiguchi, and Junichi Kato. “Ethylene Chemotaxis in Pseudomonas aeruginosaand Other Pseudomonas Species.” Microbes and Environments.22.2 (2007): 186-189. • Hawkins, Andrew C, and Caroline S. Harwood. "Chemotaxis of Ralstoniaeutropha JMP134(pJP4) to the Herbicide 2,4-Dichlorophenoxyacetate." Applied and Environmental Microbiology. 68(2), (2002): 968-972. • Parales, Rebecca E., Ditty, Jayna L., & Harwood, Carolin S. Toluene-degrading bacteria are chemotactic towards the environmental pollutants benzene, toluene, and trichloroethylene. Applied and Environmental Microbiology. 66(9), (2000): 4098-4104. • Zwietering, M. H., I. Jongenburger, F. M. Rombouts, and K. Van'tRiet. "Modeling of the Bacterial Growth Curve." Environmental Microbiology. 56.6 (1990): 1875-1881. • Kanhar, Nisar Ahmed, Charles Miller, and Anhong Zhou. "Chemotactic analysis of Pseudomonas putida KT2440 and Escherichia coli TOP10 towards environmental pollutants including methyl parathion." Manuscript, Utah State University. • Duffy, Kevin J., and Roseanne M. Ford. "Turn Angle and Run Time Distributions Characterize Swimming Behavior for Pseudomonas putida." Journal of Bacteriology. 179.4 (1997): 1428-1430.

27. Questions?