Localization of Proteins Within Intact Bacterial Cells Using Fluorescent Protein Fusions

Abstract

The advent of genetically encoded fluorescent protein (FP) reporters has revolutionized the ability to track localization of specific proteins in individual cells. Being small and generally lacking structures observable by electron microscopy, bacterial cells have particularly benefited from these technologies, which have demonstrated their high degree of protein organization. To track a protein of interest, the gene encoding the protein needs to be first fused to a gene encoding an FP, with special care to preserve function of the target protein. The localization of the target fusion protein can then be observed either in fixed cells, which preserve the localization at the moment of fixation, or in live cells. Imaging of live cells allows a protein to be tracked spatially as well as temporally. Image analysis and basic quantitation of fluorescence intensities can then be done with freely available software. The locations of multiple proteins can be monitored simultaneously using multiple fluorescent protein tags and can be verified by immunofluorescence if needed. The same specimens can, if desired, be imaged at higher resolution and/or in three dimensions using deconvolution or super-resolution methods. This chapter focuses on the basic methods for localizing proteins fused to FPs in bacteria.