Independent Promoter Recognition by TcpP Precedes Cooperative Promoter Activation by TcpP and ToxR

Cholera is a diarrhea! disease caused by the Gram-negative bacterium Vibrio cholerae. To reach the surface of intestinal epithelial cells, proliferate, and cause disease, V. cholerae tightly regulates the production of virulence factors such as cholera toxin (ctxAB) and the toxin-coregulated pilus (tcpA-F). ToxT is directly responsible for regulating these major virulence factors while TcpP and ToxR indirectly regulate virulence factor production by stimulating toxT expression. TcpP and ToxR are membranelocalized transcription activators (MLTAs) required to activate toxT expression. To gain a deeper understanding of how MLTAs identify promoter DNA while in the membrane, we tracked the dynamics of single TcpP-PAmCherry molecules in live cells using photoactivated localization microscopy and identified heterogeneous diffusion patterns. Our results provide evidence that (i) TcpP exists in three biophysical states (fast diffusion, intermediate diffusion, and slow diffusion), (ii) TcpP transitions between these different diffusion states, (iii) TcpP molecules in the slow diffusion state are interacting with the toxT promoter, and (iv) ToxR is not essential for TcpP to localize the toxT promoter. These data refine the current model of cooperativity between TcpP and ToxR in stimulating toxT expression and demonstrate that TcpP locates the toxT promoter independently of ToxR. IMPORTANCE Vibrio cholerae continues to be a public health threat throughout much of the world. Its ability to cause disease is governed by an unusual complex of regulatory proteins in the membrane of the cell, including ToxR and TcpP. These proteins collaborate to activate expression of the toxT gene, whose product activates genes for cholera toxin and other virulence factors. To study these membrane regulators, ToxR and TcpP, we applied superresolution imaging, which enables us to look at individual proteins in living cells. With this approach, we have uncovered dynamic intermolecular relationships between ToxR, TcpP, and toxT promoter DNA that dictate how toxT expression occurs. Because membrane regulators like ToxR and TcpP are broadly distributed in nature but poorly understood, this work describes mechanisms and approaches that will be of significant interest to a wide range of microbial scientists.