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2002
LSU-HHMI Summer Undergraduate Research Program |
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 Elizabeth
Baldwin (Alan J. Biel, LSU Dept. of Biological Sciences)
Effects of Overexpression of Aminolevulinate Synthase on
Tetrapyrrole Biosynthesis in Rhodobacter capsulatus
Rhodobacter capsulatus, purple non-sulfur photosynthetic bacterium,
uses the common tetrapyrrole pathway to synthesize four different
tetrapyrroles: bacteriochlorophyll, heme, vitamin B12 and siroheme.
When growing photosynthetically, bacteriochlorophyll accounts
for more than 98% of all the tetrapyrroles synthesized. Its
synthesis is regulated by oxygen, so that when R. capsulatus
is growing aerobically, bacteriochlorophyll is not synthesized.
It was previously demonstrated that oxygen regulates the intracellular
levels of porphobilinogen, the first intermediate committed
to tetrapyrrole biosynthesis. It was suggested that regulation
of porphobilinogen levels was accomplished by a combination
of feedback inhibition of aminolevulinate synthase and diversion
of aminolevulinate from the common tetrapyrrole pathway by aminolevulinate
dehydrogenase.
One way to test this suggestion would be to increase the intracellular
levels of aminolevulinate and measure carbon-flow over the common
tetrapyrrole pathway under different oxygen tensions. The plasmid
pCAP40, which carries the R. capsulatus hemA gene, was introduced
into strain AJB530. This strain has a block in cytochrome c
biogenesis, and as a result excretes coproporphyrin and protoporphyrin.
As expected, the level of aminolevulinate synthase, the enzyme
coded for by the hemA gene, was two-fold higher in the strain
carrying pCAP40.
Carbon-flow over the common tetrapyrrole pathway was measured
by growing strain pCAP40/AJB530 for 2 generations under either
3% or 23% oxygen. Total porphyrin production was measured and
compared to AJB530 carrying the vector pRK404. Initial results
indicate that the increased aminolevulinate synthase levels
resulted in slightly elevated porphyrin accumulation under high
oxygen, and had no effect on porphyrin accumulation under low
oxygen tensions. This suggests that oxygen-mediated regulation
of the common tetrapyrrole pathway can be altered by increasing
the intracellular level of aminolevulinate, and indicates that
this is the major step at which carbon-flow over this pathway
is regulated.
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