Validation of Missing Gene Functions in the Rhamnose

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1 Οκτ 2013 (πριν από 3 χρόνια και 6 μήνες)

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Krista Stanton


Mentor
: Dr. Aaron

A.

Best

Department of
Biology


Validation of Missing Gene Functions in the Rhamnose Metabolic Pathway of
Bacillus,
Streptomyces,
and
Salmonella


Genome sequencing projects yield vast amounts of information about microbes, yet each project
reveals a large fraction of genes with no known function.


This pool of genes represents an
opportunity to enrich our understanding of metabolic diversity in micr
obes.
Salmonella enterica
serovar Typhimurium
,
Streptomyces coelicolor

and
Bacillus subtilis

are known to utilize
rhamnose, though not all of the genes involved have been identified.


Advanced bioinformatics
genome context techniques were used to identify
candidate genes for these missing functions.


Rhamnulose
-
1
-
phosphate aldolase (
rhaD, rhaE
) and lactaldehyde dehydrogenase (
rhaZ, rhaW
)
perform the final two steps in the catabolism of rhamnose.


In
S. enterica, rhaD

is of known
function based on high seque
nce similarity with
Escherichia coli

rhaD
, but the function of
rhaZ

requires verification.


In contrast,
B. subtilis
and
S. coelicolor

are predicted to have a fused gene
product responsible for both functions, RhaE/W.


The candidate genes were cloned, sequ
enced,
and overexpressed in
E. coli
.


The recombinant proteins were purified using affinity
chromatography and used in biochemical assays to test their predicted functions.


The assays
required the reconstruction of the first two steps in the rhamnose cata
bolic pathway, the activity
of which was confirmed in a coupled assay with glycerol kinase.


Assays using the experimental
proteins RhaD/RhaZ or RhaE/W yielded inconclusive results; buffer conditions are being altered
to address potential co
-
factor require
ments.


The substrates for both the aldolase and the
dehydrogenase reactions are being synthesized to allow for direct validation of enzyme functions
and for modification of substrate concentrations for determination of kinetic properties of the
enzymes.




American Society for Microbiology Undergraduate Research Fellowship