Two- and Three-Dimensional Single-Molecule Super-Resolution Imaging in Live Bacteria Cells

Abstract

Single-molecule imaging has extended the resolution of fluorescence microscopy down to the nanometer scale. This super-resolution technique is non-invasive, tolerates simple sample preparation, and takes advantage of high-specificity labeling schemes. We will discuss recent studies focused on imaging protein structure and dynamics in live bacterial cells, with attention to the particular challenges that this system presents: the organisms are small, have short cell cycles, live in specific environments, and their organization is relatively poorly understood. We have developed methods to extend the capabilities of single-molecule fluorescence microscopy to study motion and localization in live Caulobacter crescentus, Vibrio cholerae, and Bacteroides thetaiotaomicron.FtsZ is an essential protein that polymerizes at the mid-cell, recruits the division machinery, and may generate constrictive forces necessary for cytokinesis. Based on astigmatism and on the natural dynamics of the protein, we resolve in two and three dimensions the midplane Z-ring formed by FtsZ, in C. crescentus. We further apply live-cell single-molecule imaging to two prokaryotes of biomedical interest, V. cholerae, and the gut symbiont B.thetaiotaomicron. The V. cholerae protein TcpP is a rare example of a membrane-bound transcription factor, and we have investigated the dynamics and localization of TcpP, its binding partner ToxR, and the toxT gene they regulate, in order to elucidate the mechanism of membrane-bound transcription activation. The starch utilization system (Sus) allows B. thetaiotaomicron to catabolize complex carbohydrates, and we have investigated the response of Sus proteins to stimuli with live anaerobic cell single-molecule imaging.