Single-molecule fluorescence analysis of transcription factors in living bacteria

Super-resolution microscopy has become an important tool to image cells at ever increasing resolution. Specifically, localization microscopy, in which single emitters are localized with high accuracy, has allowed us to follow and track single-molecules in live cells. The technique has revealed aspects in the proteins’ real-time function that were impossible to study previously. Despite their extensive use in recent years, there are still possibilities to improve and validate the technique and the resulting data. This thesis presents the first tracking photo-activation localization microscopy (tracking PALM) on three transcription factors (TFs): the lac repressor (LacI), araC protein and the cAMP receptor protein (CRP). With this work I expand the range of applicability of localization methods and increase in detail an important part of cellular function. TFs control the expression of genes so that cells can adapt to external conditions. In Escherichia coli (E. coli), previous studies have shown that more than 60% of TFs have less than 100 monomers per genome copy. For example LacI, involved in the lactose utilization control, exists in E. coli in around 40 monomers in contrast to other proteins that exist in the tens of thousands. To study such low-copy numbers we found it necessary increase the throughput of tracking PALM and this thesis summarises our efforts to increase throughput. After constructing a setup capable of tracking PALM with a 3X increased throughput, I studied the diffusion and localization of LacI and LacI relative to its operators. By studying LacI, I found that there were a number of challenges related to low copy numbers, specifically background noise that leads to localisations that can affect the results. I characterized the background tracks and tested the ability of clustering to find a number of tandem operators. This knowledge allowed us to study other TFs: araC, involved in the control of arabinose utilization which also exists in low-copy numbers but functions in multiple genes; and the CRP, which both regulates multiple sites and exists in large copy numbers. In addition I worked towards the understanding of TF binding by simulating the combination of non-specific binding and free diffusion that emulates the search process of TFs. I obtained insight into the analysis of the data generated from tracking PALM. Finally I focused on creating a strategy to use our existing data to extract blinking rates from the fluorescent protein. Our insights into TFs diffusion and binding have shown that tracking PALM can yield important insights at this regime of protein concentrations. Our work can be applied to other TFs and proteins that function in low copy numbers.