POSTERS - BLAST X - University of Utah

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