| Physical
and Chemical Properties of an Extracellular Factor Isolated from Zebra
Mussel Bacteria
Alissa
R. DeHaan
Gustavus
Adolphus College
St.
Peter, Minnesota
Mentor:
Dr. James Courtright
Mentor:
Dr. James Maki
Bacteria
collected from the surface of zebra mussel shells have been shown to display
inhibitory activity towards other bacterial species by the production of
an extracellular factor. Jerry Kavouras, a graduate student, collected
many bacterial species from zebra mussel shells and identified JK9 and
JK10, which exhibit the most inhibitory activity of those he isolated.
The goal of this summer research was to determine properties of the extracellular
factor and the zebra mussel bacteria.
To
determine the best method for optimal production of and response to the
extracellular factor, liquid freeze extracts were spotted on minimal medium,
R2A medium and LB medium streaked with E.coli B, E.coli K12, and B.megaterium.
Liquid freeze extracts taken from JK9 and JK10 grown on minimal medium
produced the largest, clearest zones of inhibition when spotted on B.megaterium
spread on minimal medium, so this method was used throughout all of the
experiments.
A test
for inhibition by JK9 and JK10 showed inhibition of several different bacterial
species. Testing for whether the extracellular factor has a static or cidal
effect on other bacteria revealed that at least 10 _L of extracellular
factor added to 200 _L of liquid minimal medium containing a clear suspension
of B.megaterium cells produces a static effect with some killing. B.megaterium
cells grow at a very reduced rate and number at least 24 hours after exposure
to the extracellular factor.
Numerous
chemical and physical properties of the extracellular factor were also
tested. The heat resistance test showed inhibition of B.megaterium by the
extracellular factor at temperatures up to 90°C, which indicates that
the factor is not denatured at high temperatures. Gram staining and phase
contrast microscopy showed that JK9 and JK10 are both gram positive and
are small rods. Refractible, brighter areas in the bacteria suggest that
JK9 and JK10 may be spore formers. Centrifuging the extracellular factor
of JK10 through Microsep“ tubes with different molecular weight cutoffs
gave a molecular weight estimate of 30K to 100K. Centrifuging the extracellular
factor through cationic and anionic exchange resins suggested that the
factor binds to cationic resins, and therefore has a negative charge.
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