Single-molecule super-resolution methods are ideally suited to the small size of bacterial cells, and hold particular promise for understanding processes in microbiology that have important impact on human health and disease. In bacteria, two-component signaling systems are responsible for much of the interaction between the bacterium and the outside world. These systems can sense a wide variety of stimuli and respond, often by altering gene expression. In the pathogenic Vibrio cholerae, agent of the human cholera disease, a two-component signaling system regulates the toxin synthesis pathway at the ToxR regulon. Interestingly, the integral inner-membrane protein TcpP remains membrane-bound while binding DNA to upregulate toxin expression. The mechanism of such membrane-bound transcription has yet to be elucidated in live cells. By measuring TcpP mobility from the trajectories of single molecules of this DNA transcription regulator, and by using genetic knockouts to examine the effects on TcpP motion of the TcpP interaction partner ToxR and of the toxT DNA promoter binding site, our live-cell super-resolution imaging has provided mechanistic insight into the V. cholerae virulence pathway (Haas et al., Molecular Microbiology 2014). Based on single-molecule trajectory analyses, including cumulative probability distributions, our observations support a mechanistic model in which ToxR recruits TcpP to the toxT promoter for transcription activation. This work, which informs on the mechanism by which V. cholerae regulates cholera toxin production, is timely because cholera remains a threat to health in the developing world.

Super-resolving the dynamics of a membrane-bound virulence regulator in Vibrio cholerae

249th National Meeting (2015)

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