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

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slide1

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

outline
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

problem soil pollution
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

key words
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

pseudomonas putida kt2440 potential as soil bioremediator
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

objective
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)
equipment
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

chemoattracants
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

growth curve phases
Growth Curve Phases
  • Early Logarithmic
  • Mid Logarithmic
  • Late Logarithmic
  • Early Stationary

General Phases

Phases Compared in KT2440 Project

growth curve comparison
Growth Curve Comparison
  • OriginPro
    • Sigmoid Fit
    • Modified Gompertz Equations

1)

2)

3)

4)

OriginPro fitted growth curve of PPKT2440 with no chemoattractant.

growth curve parameters
Growth Curve Parameters

OriginPro growth curve of PPKT2440 with 25mM Benzoate

OriginPro growth curve of PPKT2440 with 15mM Succinate

growth curve parameters13
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
velocity box plots
Velocity Box Plots

Mid Logarithmic Phase

Early Logarithmic Phase

Late Logarithmic Phase

Early Stationary Phase

turn angle comparison
Turn Angle Comparison

Smaller Turn Angle=Less Direction Change

turn angle frequency
Turn Angle Frequency

PPKT2440 without chemoattractant favors small angles (under 90°).

Notice bimodal distribution in above succinate experiment.

normalized x vs y displacement kt2440 control
Normalized X vs. Y Displacement-KT2440 Control

Mid Logarithmic Phase

Late Logarithmic Phase

normalized x vs y displacement kt2440 with 15mm succinate
Normalized X vs. Y Displacement-KT2440 with 15mM Succinate

Mid Logarithmic Phase

Late Logarithmic Phase

normalized x vs y displacement kt2440 with 25mm benzoate
Normalized X vs. Y Displacement-KT2440 with 25mM Benzoate

Mid Logarithmic Phase

Late Logarithmic Phase

accumulated distance vs frame number
Accumulated Distance vs. Frame Number

Without

Chemoattractant

With 15mM Succinate

With 25mM Benzoate

slide22
Project Contribution

Previous Research

  • Collective analysis
  • Swarm Plates
  • Individual bacterium analysis
  • Video Microscopy
  • Growth Curve Parameter Comparison
conclusion
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

future work
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
acknowledgments
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
references
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.