Weaver, Sara J. and Ortega, Davi R. and Sazinsky, Matthew H. and Dalia, Triana N. and Dalia, Ankur B. and Jensen, Grant J. (2020) CryoEM structure of the type IVa pilus secretin required for natural competence in Vibrio cholerae. Nature Communications, 11 . Art. No. 5080. ISSN 2041-1723. PMCID PMC7545093; PMC7591482. doi:10.1038/s41467-020-18866-y. https://resolver.caltech.edu/CaltechAUTHORS:20201008-083809896
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Abstract
Natural transformation is the process by which bacteria take up genetic material from their environment and integrate it into their genome by homologous recombination. It represents one mode of horizontal gene transfer and contributes to the spread of traits like antibiotic resistance. In Vibrio cholerae, a type IVa pilus (T4aP) is thought to facilitate natural transformation by extending from the cell surface, binding to exogenous DNA, and retracting to thread this DNA through the outer membrane secretin, PilQ. Here, we use a functional tagged allele of VcPilQ purified from native V. cholerae cells to determine the cryoEM structure of the VcPilQ secretin in amphipol to ~2.7 Å. We use bioinformatics to examine the domain architecture and gene neighborhood of T4aP secretins in Proteobacteria in comparison with VcPilQ. This structure highlights differences in the architecture of the T4aP secretin from the type II and type III secretion system secretins. Based on our cryoEM structure, we design a series of mutants to reversibly regulate VcPilQ gate dynamics. These experiments support the idea of VcPilQ as a potential druggable target and provide insight into the channel that DNA likely traverses to promote the spread of antibiotic resistance via horizontal gene transfer by natural transformation.
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| Additional Information: | © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Received 06 March 2020. Accepted 15 September 2020. Published 08 October 2020. Cryo Electron microscopy was performed in the Beckman Institute Resource Center for Transmission Electron Microscopy at Caltech. Dr. Songye Chen and Dr. Andrey Malyutin assisted with data collection. Dr. Spiros D. Garbis, Dr. Annie Moradian, Dr. Michael Sweredoski, and Dr. Brett Lomenick at the Caltech Proteome Exploration Laboratory (PEL) performed and analyzed mass spectrometry results. Dr. Naima Sharaf, Jeffery Lai, and Prof. Doug Rees provided invaluable advice on membrane protein biochemistry and instrumentation. Jane Ding and Welison Floriano provided computational support. Dr. Debnath Ghosal, Dr. Mohammed Kaplan, Dr. Catherine Oikonomou, Dr. Lauren Ann Metskas, Dr. Christopher Barnes, Claudia Jette, and Andrew Schacht provided feedback and advice. This work was supported in part by National Institutes of Health grant AI127401 to G.J.J. and National Institutes of Health grant R35GM128674 to A.B.D. Author Contributions. S.J.W. conceptualized the project, expressed and purified the protein, prepared samples for cryoEM, collected cryoEM data, processed cryoEM data, assisted in atomic model building, interpreted results, designed figures, and wrote the paper. D.R.O. designed research, performed sequence analyses, wrote software, collected sequence data, interpreted results, designed figures, and contributed text to the paper. M.H.S. purified protein, assisted with cryoEM sample prep and data collection, built the atomic model, interpreted results, and provided feedback on the paper. T.N.D. engineered the V. cholerae constructs, performed microbial assays, and interpreted results. A.B.D. conceptualized the project, obtained funding, engineered the V. cholerae constructs, performed microbial assays, interpreted results, designed figures, and provided feedback on the paper. G.J.J. conceptualized the project, obtained funding, interpreted results, and provided feedback on the paper. Data availability. Data supporting the findings of this paper are available from the corresponding author upon reasonable request. The cryoEM reconstruction and model have been deposited in the Electron Microscopy Data Bank (https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-21559) and the Protein Data Bank (PDB 6W6M). Code availability. The scripts underlying the bioinformatics analysis are available at https://gitlab.com/jensenlab/seccomp. The alignment and the phylogenetic tree are available in Supplementary Data 2, while the thresholds used in the genehood.io analysis are in Supplementary Data 3. Other data are available from the corresponding author upon reasonable request. The authors declare no competing interests. Peer review information. Nature Communications thanks the anonymous reviewers for their contributions to the peer review of this work. Peer review reports are available. | |||||||||||||||
| Errata: | Weaver, S.J., Ortega, D.R., Sazinsky, M.H. et al. Publisher Correction: CryoEM structure of the type IVa pilus secretin required for natural competence in Vibrio cholera. Nat Commun 11, 5533 (2020). https://doi.org/10.1038/s41467-020-19389-2 | |||||||||||||||
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| PubMed Central ID: | PMC7545093; PMC7591482 | |||||||||||||||
| DOI: | 10.1038/s41467-020-18866-y | |||||||||||||||
| Record Number: | CaltechAUTHORS:20201008-083809896 | |||||||||||||||
| Persistent URL: | https://resolver.caltech.edu/CaltechAUTHORS:20201008-083809896 | |||||||||||||||
| Official Citation: | Weaver, S.J., Ortega, D.R., Sazinsky, M.H. et al. CryoEM structure of the type IVa pilus secretin required for natural competence in Vibrio cholerae. Nat Commun 11, 5080 (2020). https://doi.org/10.1038/s41467-020-18866-y | |||||||||||||||
| Usage Policy: | No commercial reproduction, distribution, display or performance rights in this work are provided. | |||||||||||||||
| ID Code: | 105918 | |||||||||||||||
| Collection: | CaltechAUTHORS | |||||||||||||||
| Deposited By: | George Porter | |||||||||||||||
| Deposited On: | 08 Oct 2020 16:39 | |||||||||||||||
| Last Modified: | 12 Feb 2022 00:45 |
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