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Real-time microscopy and physical perturbation of bacterial pili using maleimide-conjugated molecules


Bacteria use surface-exposed, proteinaceous fibers called pili for diverse behaviors, including horizontal gene transfer, surface sensing, motility, and pathogenicity. Visualization of these filamentous nanomachines and their activity in live cells has proven challenging, largely due to their small size. Here, we describe a broadly applicable method for labeling and imaging pili and other surface-exposed nanomachines in live cells. This technique uses a combination of genetics and maleimide-based click chemistry in which a cysteine substitution is made in the major pilin subunit for subsequent labeling with thiol-reactive maleimide dyes. Large maleimide-conjugated molecules can also be used to physically interfere with the dynamic activity of filamentous nanomachines. We describe parameters for selecting cysteine substitution positions, optimized labeling conditions for epifluorescence imaging of pilus fibers, and methods for impeding pilus activity. After cysteine knock-in strains have been generated, this protocol can be completed within 30 min to a few hours, depending on the species and the experiment of choice. Visualization of extracellular nanomachines such as pili using this approach can provide a more comprehensive understanding of the role played by these structures in distinct bacterial behaviors.

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Fig. 1: Pili can be visualized with maleimide-reactive fluorescent dyes.
Fig. 2: Accessible residues are unique to each pilin.
Fig. 3: Cysteine substitution and fluorescent maleimide labeling allows visualization of different classes of pili.
Fig. 4: Observation of pili is affected by multiple variables.
Fig. 5: Pilus retraction can be blocked using different maleimide conjugates.

Data availability

The data that support this study are available from the corresponding author upon reasonable request.


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We thank M. D. Koch, A. M. Randich, and M. Jacq for critical feedback on the manuscript. This work was supported by grant R35GM122556 from the National Institutes of Health and by a Canada 150 Research Chair in Bacterial Cell Biology to Y.V.B., by grants R35GM12867 and AI118863 from the National Institutes of Health to A.B.D., and by National Science Foundation Fellowship 1342962 to C.K.E.

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Authors and Affiliations



C.K.E. and Y.V.B conceived the study. C.K.E. and T.N.D. performed the experiments. C.K.E., A.B.D., and Y.V.B. analyzed the data. C.K.E. wrote the manuscript with help from A.B.D. and Y.V.B.

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Correspondence to Yves V. Brun.

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Ellison, C.K., Dalia, T.N., Dalia, A.B. et al. Real-time microscopy and physical perturbation of bacterial pili using maleimide-conjugated molecules. Nat Protoc 14, 1803–1819 (2019).

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