Single‐molecule tracking in live Vibrio cholerae reveals that ToxR recruits the membrane‐bound virulence regulator TcpP to the toxT promoter

Abstract

Summary Vibrio cholerae causes the human disease cholera by producing a potent toxin. The V. cholerae virulence pathway involves an unusual transcription step: the bitopic inner-membrane proteins TcpP and ToxR activate toxT transcription. As ToxT is the primary direct transcription activator in V. cholerae pathogenicity, its regulation by membrane-localized activators is key in the disease process. However, the molecular mechanisms by which membrane-localized activators engage the transcription process have yet to be uncovered in live cells. Here we report the use of super-resolution microscopy, single-molecule tracking, and gene knockouts to examine the dynamics of individual TcpP proteins in live V. cholerae cells with <40-nm spatial resolution on a 50-ms timescale. Single-molecule trajectory analysis reveals that TcpP diffusion is heterogeneous and can be described by three populations of TcpP motion: one fast, one slow, and one immobile. By comparing TcpP diffusion in wild type V. cholerae to that in mutant strains lacking either toxR or the toxT promoter, we determine that TcpP mobility is greater in the presence of its interaction partners than in their absence. Our findings support a mechanism in which ToxR recruits TcpP to the toxT promoter for transcription activation.

Calcium Enhances Bile Salt-Dependent Virulence Activation in Vibrio cholerae

“…These researchers also noted that a portion of TcpP proteins were immobile and suggested that this halting was due to TcpP binding at the toxT promoter. A mutant strain lacking the toxT promoter region had fewer immobile TcpP events (46). In our experiments, TcpP mobility was shifted under conditions that are both strongly virulence inducing and physiologically relevant (9,24,47).…”

Unveiling the Inner Workings of Live Bacteria Using Super-Resolution Microscopy

“…Still, FPs remain the most common label for singlemolecule imaging in live bacteria cells and have been used for applications as diverse as assaying the control of transcription, 65,122−124 visualizing cytoskeletal protein organization, 36,58,61,62,82,86,125−128 and examining protein dynamics in virulence pathways. 63 In contrast to FPs, small molecule dyes are far brighter, have a longer lifetime, and are significantly smaller. However, for subcellular imaging, a dye must be introduced into the cell, creating the opportunity for nonspecific binding and inhomogeneous labeling since most of these organic dyes cannot be genetically encoded (with the exception of fluorescent unnatural amino acids, discussed in the Outlook and Conclusions section).…”

“…Single-molecule trajectories and CPD analysis ( Figure 4) support a mechanistic model in which ToxR recruits TcpP to the toxT promoter for transcription activation. 63 In contrast with the pathogenic V. cholerae, bacteria in the human microbiome play important beneficial roles in health. 248 In particular, gut microbes can impact human health and nutrition by metabolizing host-indigestible carbohydrates.…”

“…180 Since the model function can contain several terms, it can account for multiple modes of motion and for immobile populations. 179,181 CPD analysis has been used to quantify protein diffusion in the E. coli membrane, 182 to reveal heterogeneous motion of the membrane-bound transcription factor TcpP in virulent V. cholerae cells, 63 and to measure the 64 Although the CPD is a highly accurate metric, determining which model function to use is perhaps the most error-prone and potentially subjective step in this data analysis process. While adding more terms will invariably produce a better fit, it is not always clear whether these additional terms have any physical meaning.…”

Optical and force nanoscopy in microbiology

“…Cell envelopes play essential roles, such as defining cell shape and division, resisting turgor pressure, providing receptor sites for viruses and antibiotics, and controlling cell adhesion and biofilm formation. At this level, optical nanoscopy continues to enable the mapping of unique membrane distribution patterns and dynamics of a variety of membrane proteins, including chemotaxis receptors 111 , sporulation complexes 112 , toxins 113 , respiratory systems 114 and surface sugar metabolizing complexes 115 , and helped elucidate possible mechanisms of how a membrane protein’s localization and dynamics are coupled to its biological function. Yet here, force nanoscopy offers unique advantages over optical nanoscopy in probing structural and biophysical properties of cell surfaces with unmatched spatiotemporal resolution.…”