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

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 genetic engineering and fluorescent labeling of 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.

Structure-Function Analysis of the TssL Cytoplasmic Domain Reveals a New Interaction between the Type VI Secretion Baseplate and Membrane Complexes.

The type VI secretion system (T6SS) is a multiprotein complex that delivers toxin effectors in both prokaryotic and eukaryotic cells. It is constituted of a long cytoplasmic structure-the tail-made of stacked Hcp hexamers and wrapped by a contractile sheath. Contraction of the sheath propels the inner tube capped by the VgrG spike protein toward the target cell. This tubular structure is built onto an assembly platform-the baseplate-that is composed of the TssEFGK-VgrG subunits. During the assembly process, the baseplate is recruited to a trans-envelope complex comprising the TssJ outer membrane lipoprotein and the TssL and TssM inner membrane proteins. This membrane complex serves as a docking station for the baseplate/tail and as a channel for the passage of the inner tube during sheath contraction. The baseplate is recruited to the membrane complex through multiple contacts including interactions of TssG and TssK with the cytoplasmic loop of TssM and of TssK with the cytoplasmic domain of TssL, TssLCyto. Here, we show that TssLCyto interacts also with the TssE baseplate subunit. Based on the available TssLCyto structures, we targeted conserved regions and specific features of TssLCyto in enteroaggregative Escherichia coli. By using bacterial two-hybrid analysis and co-immunoprecipitation, we further show that the disordered L3-L4 loop is necessary to interact with TssK and that the L6-L7 loop mediates the interaction with TssE, whereas the TssM cytoplasmic loop binds the conserved groove of TssLCyto. Finally, competition assays demonstrated that these interactions are physiologically important for T6SS function.