POSTERS - BLAST X - University of Utah
POSTERS - BLAST X - University of Utah
POSTERS - BLAST X - University of Utah
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<strong>BLAST</strong> X ______ Poster #1<br />
THE CHARACTERISATION OF THE DYNAMICS OF THE FliT:FliD:FlhD4C2 INTERACTION<br />
AND ITS ROLE IN REGULATING FLAGELLAR ASSEMBLY<br />
C. Aldridge 1,2 , K. Poonchareon 1,2 , S. Saini 3 , A. Soloyva 2 , M. Banfield 4 , T. Minamino 5,6 , C. V.<br />
Rao 3 , and P. D. Aldridge 1,2<br />
1: Centre for Bacterial Cell Biology, Newcastle <strong>University</strong>, Framlington Place, Newcastle upon<br />
Tyne, United Kingdom, NE2 4HH. 2: Institute for Cell and Molecular Biosciences, Newcastle<br />
<strong>University</strong>, Framlington Place, Newcastle upon Tyne, United Kingdom, NE2 4HH. 3: Department<br />
<strong>of</strong> Chemical and Biomolecular Engineering, <strong>University</strong> <strong>of</strong> Illinois at Urbana-Champaign, Urbana,<br />
Illinois, United States, 61801. 4: Dept. <strong>of</strong> Biological Chemistry, John Innes Centre, Norwich,<br />
NR4 7UH UK. 5: Graduate School <strong>of</strong> Frontier Biosciences, Osaka <strong>University</strong>, 1-3 Yamadaoka,<br />
Suita, Osaka 565-0871, Japan. 6: Dynamic NanoMachine Project, ICORP, JST, 1-3<br />
Yamadaoka, Suita, Osaka 565-0871, Japan.<br />
Each bacterial cell <strong>of</strong> Salmonella enterica serovar Typhimurium produces a discrete<br />
number <strong>of</strong> complete flagella. Flagellar gene expression in Salmonella is negatively regulated by<br />
the Type 3 secretion chaperone FliT. FliT is known to interact with the flagellar structural subunit<br />
FliD and the master transcriptional regulator FlhD4C2. In this regulatory circuit FliD is proposed<br />
to act as an anti-regulator - a regulatory role similar to that observed for FlgM inhibition <strong>of</strong> s 28<br />
activity. We were interested in determining the kinetics <strong>of</strong> the FliT:FliD:FlhD4C2 regulatory circuit<br />
and how they influence flagellar assembly. We have shown that the FliT:FliD interaction is a 1:1<br />
ratio while in solution FliT is a dimer. Surface plasma resonance (SPR) and analytical<br />
ultracentrifugation (AUC) analysis <strong>of</strong> the dynamics <strong>of</strong> the FliT:FlhD4C2 interaction showed that it<br />
was very different from FliT:FliD. A FliT:FlhD4C2 complex is only observed when excess FliT is<br />
added to FlhD4C2. AUC analysis identified that a stable intermediate <strong>of</strong> a combination <strong>of</strong> FlhD,<br />
FlhC and/or FliT exists. Our data suggests that FliT acts to dissociate the FlhD4C2 complex and<br />
that this is a transient interaction allowing for the FlhD4C2 complex to reform. This suggests that<br />
a limiting factor in the regulation <strong>of</strong> FlhD4C2 activity is the concentration <strong>of</strong> free FliT. To test this<br />
model, we have investigated the affect <strong>of</strong> overexpressing FliT on flagellar gene expression and<br />
basal body assembly.<br />
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