algae/lake_proposal_outline.htm
June 12, 2001
Siderophores, such as DFB, help cells take up iron during iron limitation but they are also strong inhibitors of the photo-Fenton reaction. The photo-Fenton reaction will generate <dot>OH- in the presence of H2O2 and Fe^2+. Are there situations where cyanobacteria produce siderophores mainly to block <dot>OH- production, rather than for Fe nutritional needs? If so, this would provide an interesting insight into the molecular ecology of cyanobacteria in relationship to aquatic chemistry of natural waters.
Test Wiliamson and K Lake water for Fenton reaction potential.
add H2O2
add Fe
light absorption of the humics with and without Fe
compare response of filtered lake water samples to 20 uM FeEDTA solutions at the same pH, temperature and light
action spectra of TA production (using filters and halogen light sources)
block with DFB
Test H2O2 production in natural samples of algae.
offset method
scopoletin method
scavenging rate (destruction of added H2O2) using the O2 electrode of scopoletin
Test for siderophore production.
use CAS/HDTMA binding assay
look for water chemistry that decreases photo-Fenton reaction when added to 20 uM FeEDTA solution.
look for increased production when cells are artifically oxidatively stressed
high light - no bubbling
addition of H2O2
photo-Fenton generation of <dot>OH- using 20uM FeEDTA solution
Develop/adapt an intracellular assay for <dot>OH-
overlap this study with photoinhibition study (i.e. that Ecrit is caused by <dot>OH- damage
alternatively, could use a cell lysis prep and add back H2O2
Compare responses and sensitivities of Anabaena, Microcystis, and AFA.
light, photoinhibition, H2O2 production, H2O2 scavenging, <dot>OH-, siderophore production,
is oxidative stress a precursor to toxin production?
Biosis search on fur AND Fe limitation AND oxidative stress pulled up multiple examples in the heterotrophic eubacteria.
In some cases Fur is also regulated by two oxidative stress response regulators (Hantke 2001) and Azotobacter vinelandii continues catecholate sideerophore synthesis upto 10 uM iron to help with oxidative stress.
Multiple studies have demonstrated this linkage in the heterotrophic eubacteria. This project would look for an extremely efficient method to verify that this mechanism was working in cyanobacteria.
Possible approaches:
culture studies with added Fe and oxidative stress to demontrate the interaction
look for RNA transcripts (hybridizing to oligomer probes) under Fe and H2O2 stressors
target molecules could be Mn-SOD - sodA (Tardat and Touati 1991) or catalase katA (Horsburgh et al 2001)
best would be something like the katA-lacZ fusion described by Horsburgh et al 2001)
katG::lacZ fusion described in Gonzalez-Flecha & Demple
Revisit the Mn toxicity studies.
Mn blocks fur regulation by binding to the fur protein and hitting the iron box
see Bsat et al (abstract) for an example of how they found perR mutations by their ability to derepress mrgA in the presence of Mn ion
These genetic approaches would allow us to generalize across taxonomic groups probably much faster than doing the culture studies.
Gonzalez-Flecha & Demple (????) Genetic responses to free radicals: Homeostasis and gene control -- in v899 of Annals New York Academy of Sciences
Bsat et al http://www.bio.cornell.edu/microbio/helmann/Helmann48.html