Extending the tools of single-molecule fluorescence imaging to problems in microbiology

Single-molecule fluorescence microscopy enables non-invasive, high-sensitivity, high-resolution imaging, and this direct, quantitative method has recently been extended to understanding organization, dynamics and cooperativity of macromolecules in prokaryotes. In this issue of Molecular Microbiology, Bakshi et al. (2012) examine fluorescently labelled ribosomes and RNA polymerase (RNAP) in live Escherichia coli cells. By localizing individual molecules with 30 nm scale accuracy, they resolve the spatial distribution of RNAP (and thus of the E. coli nucleoid) and of the ribosomes, measure diffusion rates, and sensitively count protein copy numbers. This work represents an exciting achievement in terms of applying biophysical methods to live cells and quantitatively answering important questions in physiologically relevant conditions. In particular, the authors directly relate the positions, dynamics, and numbers of ribosomes and RNAP to transcription and translation in E. coli. The results indicate that, since the ribosomes and the nucleoid are well segregated, translation and transcription must be predominantly uncoupled. As well, the radial extension of ribosomes and RNAP to the cytoplasmic membrane is consistent with the hypothesis of transertion (simultaneous insertion of membrane proteins upon translation).