Isolation and Characterization of Siderophores from Marine Bacteria

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Isolation and Characterization of Siderophores
from Marine Bacteria

• Makeda Tekle-Smith
• Mentor Rose Theisen
• Advisor Alison Butler
• Department Chemistry and Biochemistry

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• Siderophores are low molecular weight iron(III)
  chelators, that are produced under low iron
  conditions.

FeIII
http//oceancolor.gsfc.nasa.gov/
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Marine Siderophores

• Marine bacteria collected off of the western
  coast of Africa
• We will focus on the isolation and
  characterization of aerobactin produced by marine
  bacteria, Vibrio sp. DS40M5

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Grow the Bacteria

• Make the artificial seawater media
• ASG-Fe and ASW
• Inoculate
• Shake the culture
• Monitor the growth
• Chrome Azurol Sulfonate(CAS) test
• Optical Density Measurements

Fe(III)-CAS Fe(III)-Siderophore CAS
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Harvest the Bacteria

• Centrifuge
• Solid phase extraction
• Rotary vacuum evaporator

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Isolate and Characterize

• Reverse-Phase High
• Pressure Liquid
• Chromatography (RP-HPLC)
• Mass spectrometry
• Determines molecular weight of compounds

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HPLC Trace Monitored at 215nm

• Solvents used Nanopure water (0.05TFA) and
  methanol (0.05TFA)
• Column used C4 prep column

Peak 4 42min
Absorbance at 215nm
Peak 2 24min
Peak 3 39min
Peak 1 22min
Elution Time (min)
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Mass Spectrometry
565 MH
564 amu
Mass/Charge (m/z)
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Tandem Mass Spectrometry
205
315
565
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Photochemistry
Photochemical Reaction of Fe(III)- Aerobactin
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Conclusion

• Vibrio sp. DS40M5 grew in artificial sea water
  and produced aerobactin
• We characterized aerobactin using HPLC, mass
  spectrometry, and UV/Vis spectroscopy
• Iron(III)-aerobactin when reacted with sunlight
  forms the iron(III)-aerobactin photoproduct
• The future plans for this research are to use the
  isolated Aerobactin for further aerobic and
  anaerobic photochemistry experiments to learn
  more about siderophore-mediated iron acquisition
  by marine bacteria

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Reflections

• This experience has taught me that learning is an
  ongoing process
• Being a part of this program has shown me that
  scientific research isnt just about proving your
  hypothesis correct, it is about making
  observations and learning from the unexpected

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Acknowledgments

• Professor Alison Butler
• Department of Chemistry and Biochemistry
• The National Institutes of Health (NIH)
• The Apprentice Researchers
• California NanoSystems Institute
• Dr. Rose Theisen

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Isolation and Characterization of Siderophores
from Marine Bacteria

• Makeda Tekle-Smith
• Mentor Rose Theisen
• Advisor Alison Butler
• Department Chemistry and Biochemistry

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Why Iron?

• Respiration
• Photosynthesis
• Nitrogen fixation

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Growth Curve- Measure Optical Density at 600nm

• ASW Media

• ASG-Fe Media

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Siderophore

• Greek for iron carrier
• Low molecular weight compound
• High affinity for iron
• Produced by soil and marine bacteria in a low
  iron environment
• The siderophores bind iron and then they bring
  iron back for the bacteria to grow

Chrome Azurol Sulfonate(CAS)
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Project Goals

• The project focus is to learn more about
  siderophore-mediated iron acquisition by marine
  bacteria
• In part because there is very little iron in the
  ocean and the majority of microorganisms require
  iron for growth
• phytoplankton which are responsible for half the
  worlds fixation of carbon dioxide
• The structures of marine siderophores are very
  unusual