Bacteria build elaborate nanomachines on their cell surface to interact with their environment and cause disease. Each machine must be assembled from specific parts, in the proper order, and in precise amounts. Here we propose to study the mechanism by which a structural protein of the flagellar filament, Hag, homeostatically controls its own synthesis and governs its own assembly in Bacillus subtilis. Hag is part of a three-node negative feedback loop including FliW and the highly conserved post-transcriptional regulator of virulence gene expression, CsrA. We will determine the mechanism by which FliW antagonizes CsrA and we will determine the NMR solution structures of CsrA and FliW, separately and in complex. We will study an internal feedback loop in which the flagellin transcript helps maintain CsrA-FliW stoichiometry. We will measure the cytological parameters of filament length and elongation rate, determine how feedback regulation contributes to each parameter, and explore homeostatic regulation in another structural protein, the FlgE flagellar hook subunit. Our work will establish new paradigms for the post-transcriptional regulation of nanomachine assembly and provide new insights into the origin and evolution of virulence gene regulators. We will advance B. subtilis as a model organism for flagellar research and we use B. subtilis as a genetic platform for the expression and study of heterologous regulators from distantly related pathogenic bacteria. Finally, we have isolated drugs that antagonize CsrA when added to whole cells will characterize the anti-CsrA drugs as candidate antibacterial therapeutics.
We will study how the flagellar structural subunit flagellin homeostatically restricts its own synthesis and assembly in Bacillus subtilis by focusing on each component of the three-node negative feedback loop CsrA- FliW-Hag.
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