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 Tue. Morning Session<br />
THE ROLE OF POSITIVE FEEDBACK IN CONTROLLING FLAGELLA ASSEMBLY<br />
DYNAMICS<br />
Supreet Saini 1 , Christy Aldridge 2 , Jonathan Brown 2 , Philip Aldridge 2 , Christopher Rao 1<br />
1<br />
Department <strong>of</strong> Chemical and Biomolecular Engineering, <strong>University</strong> <strong>of</strong> Illinois, Urbana, IL 61801,<br />
United States<br />
2<br />
Institute for Cell and Molecular Biosciences, Newcastle <strong>University</strong>, Framlington Place,<br />
Newcastle upon Tyne NE2 4HH, United Kingdom<br />
Flagellar assembly in Salmonella enterica serovar Typhimurium (S. typhimurium)<br />
proceeds in a sequential manner, starting from the base <strong>of</strong> the flagella and concluding at the<br />
filament tip. A key regulatory step in the assembly process is the σ 28 -FlgM checkpoint, which<br />
prevents the activation <strong>of</strong> σ 28 -dependent Pclass3 promoters prior to completion <strong>of</strong> the hook basal<br />
body. This regulatory checkpoint is typically assumed to involve a binary decision process:<br />
either proceed with Pclass3 gene expression or not, depending on the state <strong>of</strong> assembly.<br />
However, mathematical modeling suggests that this binary checkpoint may in fact result in more<br />
subtle, rheostat-like control. σ 28 is involved in a positive feedback loop, as ιτ positively regulates<br />
its own expression along with the expression <strong>of</strong> FliZ, an activator <strong>of</strong> Pclass2 gene expression. In<br />
addition, the ability <strong>of</strong> σ 28 to regulate gene expression depends on the concentration <strong>of</strong> FlgM in<br />
the cell. This suggests that the response <strong>of</strong> the σ 28 positive feedback loop is tuned by late<br />
protein secretion. In fact, our modeling and experimental results suggests that σ 28 and FlgM are<br />
not only involved in establishing the binary checkpoint between Pclass2 and Pclass3 gene<br />
expression but are also involved in fine tuning the relative timing <strong>of</strong> expression. In particular, the<br />
positive feedback loop involving σ 28 and FliZ establishes the delay between Pclass2 and Pclass3<br />
gene expression.<br />
In this talk, we will discuss our recent work investigating the positive feedback loops<br />
involving σ 28 and FliZ. We have recently shown that FliZ is an FlhD4C2-dependent activator <strong>of</strong><br />
Pclass2 gene expression. In addition, our results indicate the FliZ speeds up the induction <strong>of</strong> Pclass2<br />
genes in a secretion-dependent manner. With regards to σ 28 , we have found that autoregulation<br />
controls the relative timing <strong>of</strong> gene expression. In cells lacking FlgM, both Pclass2 and Pclass3<br />
genes are induced at the same times. Conversely, when the rate <strong>of</strong> FlgM secretion is reduced,<br />
the delay between Pclass2 and Pclass3 gene expression is exaggerated. These results suggest that<br />
timing is responsive to the rate <strong>of</strong> late protein secretion. Moreover, mathematical modeling<br />
predicts that this control is due to autoregulation by σ 28 . To test this model, we have rewired the<br />
flagellar gene circuit by replacing the native PfliA promoter with Pclass1/Pclass2/Pclass3 promoters.<br />
Consistent with the model predictions, these promoter replacement experiments show that<br />
autoregulation plays a key role in enforcing the timing <strong>of</strong> flagellar gene expression. Last, we also<br />
investigated gene expression dynamics at single-cell resolution, and our preliminary results<br />
suggest that the dynamics may exhibit bistability, consistent with control involving feedback.<br />
Collectively, our results suggest that the regulation <strong>of</strong> flagellar gene expression is complex and<br />
involves multiple layers <strong>of</strong> control.<br />
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