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Retraction of DNA-bound type IV competence pili initiates DNA uptake during natural transformation in Vibrio cholerae


Natural transformation is a broadly conserved mechanism of horizontal gene transfer in bacterial species that can shape evolution and foster the spread of antibiotic resistance determinants, promote antigenic variation and lead to the acquisition of novel virulence factors. Surface appendages called competence pili promote DNA uptake during the first step of natural transformation1; however, their mechanism of action has remained unclear owing to an absence of methods to visualize these structures in live cells. Here, using the model naturally transformable species Vibrio cholerae and a pilus-labelling method, we define the mechanism for type IV competence pilus-mediated DNA uptake during natural transformation. First, we show that type IV competence pili bind to extracellular double-stranded DNA via their tip and demonstrate that this binding is critical for DNA uptake. Next, we show that type IV competence pili are dynamic structures and that pilus retraction brings tip-bound DNA to the cell surface. Finally, we show that pilus retraction is spatiotemporally coupled to DNA internalization and that sterically obstructing pilus retraction prevents DNA uptake. Together, these results indicate that type IV competence pili directly bind to DNA via their tip and mediate DNA internalization through retraction during this conserved mechanism of horizontal gene transfer.

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Fig. 1: The tips of type IV competence pili directly bind to DNA.
Fig. 2: Type IV competence pilus dynamic activity and DNA binding are critical for DNA internalization.
Fig. 3: Pilus retraction is required for DNA uptake.
Fig. 4: Residual retraction in ΔpilT mutants allows for low rates of transformation.


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We thank A. Camilli, F. Yildiz, D. Kearns, N. Greene, C. Berne and B. LaSarre for critical comments on the manuscript. We also thank members of the Biais lab, L. Khosla, R. Rayyan and A. Ratkiewicz for assistance with micropillar assays. This work was supported by grant R35GM122556 from the National Institutes of Health to Y.V.B., by grant AI118863 from the National Institutes of Health to A.B.D., by the National Science Foundation fellowship 1342962 to C.K.E. and by grant AI116566 from the National Institutes of Health to N.B.

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Authors and Affiliations



C.K.E. and A.B.D. designed and coordinated the overall study. A.B.D., C.K.E., T.N.D., J.C.-Y.W., A.V.C. and N.B. performed the experiments. Y.V.B., A.B.D., C.K.E., T.N.D. and N.B. analysed and interpreted the data. C.K.E. and A.B.D. wrote the manuscript with help from Y.V.B.

Corresponding author

Correspondence to Ankur B. Dalia.

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The authors declare no competing interests.

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Supplementary information

Supplementary Information

Supplementary Discussion, Supplementary Figures 1–15, Supplementary Tables 1–3.

Reporting Summary

Supplementary Video 1

pilA-Cys ΔpilT cell with labelled pili bound to labelled DNA.

Supplementary Video 2

Sheared labelled pilus bound to labelled DNA.

Supplementary Video 3

Sheared labelled pilus bound to labelled DNA.

Supplementary Video 4

pilA-Cys cells extending and retracting labelled pili.

Supplementary Video 5

pilA-Cys cell with labelled pili retracting while bound to labelled DNA.

Supplementary Video 6

pilA-Cys cell with labelled pili retracting while bound to labelled DNA.

Supplementary Video 7

pilA-Cys VC0859K148Q VC0858R165Q cells extending and retracting labelled pili.

Supplementary Video 8

pilA-Cys VC0858R168Q cells extending and retracting labelled pili.

Supplementary Video 9

Formation of ComEA-mCherry focus after retraction of labelled pilus by a pilA-Cys cell.

Supplementary Video 10

pilA-Cys cells extending and retracting pili co-labelled with dye and biotin-maleimide.

Supplementary Video 11

pilA-Cys cells blocked for pilus retraction after pili colabelled with dye and biotin-maleimide were treated with neutravidin.

Supplementary Video 12

pilA-Cys ΔpilT cell with labelled pili retracting a pilus.

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Ellison, C.K., Dalia, T.N., Vidal Ceballos, A. et al. Retraction of DNA-bound type IV competence pili initiates DNA uptake during natural transformation in Vibrio cholerae. Nat Microbiol 3, 773–780 (2018).

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