Super-Resolution Microscopy and Tracking of DNA-Binding Proteins in Bacterial Cells

Abstract

The ability to detect individual fluorescent molecules inside living cells has enabled a range of powerful microscopy techniques that resolve biological processes on the molecular scale. These methods have also transformed the study of bacterial cell biology, which was previously obstructed by the limited spatial resolution of conventional microscopy. In the case of DNA-binding proteins, super-resolution microscopy can visualize the detailed spatial organization of DNA replication, transcription, and repair processes by reconstructing a map of single-molecule localizations. Furthermore, DNA-binding activities can be observed directly by tracking protein movement in real time. This allows identifying subpopulations of DNA-bound and diffusing proteins, and can be used to measure DNA-binding times in vivo. This chapter provides a detailed protocol for super-resolution microscopy and tracking of DNA-binding proteins in Escherichia coli cells. The protocol covers the construction of cell strains and describes data acquisition and analysis procedures, such as super-resolution image reconstruction, mapping single-molecule tracks, computing diffusion coefficients to identify molecular subpopulations with different mobility, and analysis of DNA-binding kinetics. While the focus is on the study of bacterial chromosome biology, these approaches are generally applicable to other molecular processes and cell types.

Keywords: DNA repair; DNA-binding proteins; Escherichia coli; Lambda red recombination; Single-molecule imaging; Single-particle tracking; Super-resolution fluorescence microscopy.

Fig. 1. Example images of a PALM recording.

The transmitted light image shows live E. coli cells immobilized on an agarose pad. Cells are expressing a fusion of DNA polymerase 1 (Pol1) with PAmCherry. Example frames of a PALM movie show isolated fluorescent spots of single Pol1-PAmCherry molecules. Different molecules become photoactivated in different frames such that their precise positions can be recorded over time. Scale bars: 1 μm.

Fig. 2. Super-resolution image reconstruction.

(a) Localizations of Pol1-PAmCherry from a PALM recording of 7.500 frames (example frames in Fig. 1) are displayed as a scatter plot. (b) Localizations mapped onto the transmitted light image of cells. (c) Histogram visualization: Localizations were binned into a two-dimensional grid of subpixels (38 nm × 38 nm, i.e. 4 × 4 subpixels per original pixel). The number of localizations per bin is represented according to the colours in the scale bar. (d) Gaussian kernel visualization: The image is reconstructed by summing normalized Gaussian kernels with 40 nm standard deviation (equivalent to the localization precision) centred on the localizations. The boxed regions are shown magnified. Scale bars: 1 μm.

Fig. 3. Photoactivated single-molecule tracking analysis.

Tracks of Pol1-PAmCherry molecules were generated from the localization data in Fig. 2 and shown on a transmitted light image of cells. The histograms show the distribution of apparent diffusion coefficients. Live cells were treated with the DNA-damaging agent methyl methanesulfonate (100 mM MMS) for 20 min before imaging. Base-excision repair of MMS damage creates gapped DNA substrates to which Pol1 binds for DNA repair synthesis. Single-molecule tracking of Pol1 allows identifying such events by the low apparent diffusion coefficient. (a) Data for all tracks with at least 5 localizations. (b) Detecting individual bound molecules by plotting only tracks with an apparent diffusion coefficient below 0.15 μm²/s. (c) All observed tracks, colour coded by their classification as bound or mobile, in red and blue, respectively. Scale bars: 1 μm.