Single-molecule FRET studies in live bacteria

Single-molecule fluorescence and single-molecule Förster resonance energy transfer (FRET) have proven enormously successful in understanding molecular and cellular processes over the last two decades. However, in vivo single-molecule FRET studies are still very difficult, mainly because they require site-specifically labelled biomolecules with photostable organic dyes. In this work, I established single-molecule FRET studies in live bacteria and applied the developed method to study mechanisms of gene expression and gene regulation. To begin with, I optimised a recently developed internalisation method based on electroporation for the efficient loading of bacterial cells with organic dye-labelled nucleic acids and proteins while maintaining cell viability. Following these studies, I internalised labelled tRNA molecules, measured their diffusion coefficient, and showed that most tRNA molecules diffuse freely in live bacteria. I also showed that bound tRNA molecules are predominantly at the cell periphery and compete with the endogenous tRNA pool during translation using antibiotic controls and simulations. Finally, I studied transcription initiation in vivo by internalising promoter DNAs with different FRET labelling schemes reporting on individual steps in transcription initiation. Thus, I observed single-molecule FRET signatures attributed to open complex formation, DNA scrunching during initial transcription, and promoter escape, which were not present in null-promoter DNA and antibiotic controls. By carefully designing single-molecule FRET assays, I imagine single-molecule FRET studies to become a major tool in understanding protein dynamics, and elucidating mechanistic details of gene regulation processes in living cells.</p

Single-molecule fluorescence of internalised biomolecules in live bacteria

In the last two decades emerging single-molecule fluorescence tools have been developed and adapted to study individual molecules in vitro under various conditions and to reveal biochemical and cellular processes in live cells with high precision. Single-molecule investigations in vivo rely mainly on fluorescent protein (FP) fusions with low quantum yield and photostability. Methods to introduce organic-labelled fluorescent molecules into cells have been achieved for mammalian cells via microinjection (not possible with bacteria) and are limited in throughput. We have developed a physical transfection method for delivering organic-fluorophore labelled biomolecules into live bacteria for single-molecule studies. We internalised labelled dsDNA and proteins (up to 100kDa) with high efficiency whilst maintain- ing cell viability. I was able to count the number of internalised molecules through photobleaching analysis. From this analysis, I was able to tune the concentration of internalised material from high concentrations compatible with localisation-based super-resolution imaging to lower concentrations compatible with single-molecule observation. Using localisation and tracking algorithms I followed the diffusion of individual molecules for up to 10 s and monitored the tracking paths within the cellular cytoplasm. This method allows us to observe molecules for up to 10 minutes (FPs ~ 5 s) under continuous illumination opening a new time regime to study biological processes. Our approaches are general and widely applicable to different microorganisms using biomolecules labelled with organic fluorophores already available for in vitro experiments.</p

Single-molecule fluorescence of internalised biomolecules in live bacteria

In the last two decades emerging single-molecule fluorescence tools have been developed and adapted to study individual molecules in vitro under various conditions and to reveal biochemical and cellular processes in live cells with high precision. Single-molecule investigations in vivo rely mainly on fluorescent protein (FP) fusions with low quantum yield and photostability. Methods to introduce organic-labelled fluorescent molecules into cells have been achieved for mammalian cells via microinjection (not possible with bacteria) and are limited in throughput. We have developed a physical transfection method for delivering organic-fluorophore labelled biomolecules into live bacteria for single-molecule studies. We internalised labelled dsDNA and proteins (up to 100kDa) with high efficiency whilst maintain- ing cell viability. I was able to count the number of internalised molecules through photobleaching analysis. From this analysis, I was able to tune the concentration of internalised material from high concentrations compatible with localisation-based super-resolution imaging to lower concentrations compatible with single-molecule observation. Using localisation and tracking algorithms I followed the diffusion of individual molecules for up to 10 s and monitored the tracking paths within the cellular cytoplasm. This method allows us to observe molecules for up to 10 minutes (FPs ~ 5 s) under continuous illumination opening a new time regime to study biological processes. Our approaches are general and widely applicable to different microorganisms using biomolecules labelled with organic fluorophores already available for in vitro experiments.This thesis is not currently available in ORA