Chemoenzymatic Labeling of Proteins for Imaging in Bacterial Cells
Reliable methods to determine the subcellular localization of bacterial proteins are needed for the study of prokaryotic cell biology. We describe here a simple and general technique for imaging of bacterial proteins in situ by fluorescence microscopy. The method uses the eukaryotic enzyme N-myristoyltransferase to modify the N-terminus of the protein of interest with an azido fatty acid. Subsequent strain-promoted azide–alkyne cycloaddition allows conjugation of dyes and imaging of tagged proteins by confocal fluorescence microscopy. We demonstrate the method by labeling the chemotaxis proteins Tar and CheA and the cell division proteins FtsZ and FtsA in Escherichia coli. We observe distinct spatial patterns for each of these proteins in both fixed and live cells. The method should prove broadly useful for protein imaging in bacteria.
© 2016 American Chemical Society. Received: July 16, 2016; Published: November 10, 2016. The authors thank B. M. Babin, S. E. Stone, L. J. Dooling, K. P. Yuet, K. S. Burke, J. D. Bagert, and C. Kulkarni for insightful discussions. S.H.H. thanks D. K. Romney, S. Brinkmann-Chen, A. Collazo, S. Hess, A. Moridan, M. Shahgholi, C. Neubauer, and N. Dalleska for advice. S.H.H. was supported in part by a National Science Foundation Graduate Research Fellowship. This work was supported by the Caltech Center for Environmental Microbial Interactions, the Jacobs Institute for Molecular Engineering for Medicine, and the Institute for Collaborative Biotechnologies through Grant W911NF-09-0001 from the U.S. Army Research Office. The authors declare no competing financial interest.
Supplemental Material - ja6b07067_si_001.pdf