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A genetic system for targeted mutations to disrupt and restore genes in the obligate bacterium, Ehrlichia chaffeensis
Obligate intracellular bacteria (obligates) belonging to Rickettsiales and Chlamydiales cause diseases in hundreds of millions of people worldwide and in many animal species. Lack of an efficient system for targeted mutagenesis in obligates remains a major impediment in understanding microbial patho...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5693922/ https://www.ncbi.nlm.nih.gov/pubmed/29150636 http://dx.doi.org/10.1038/s41598-017-16023-y |
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author | Wang, Ying Wei, Lanjing Liu, Huitao Cheng, Chuanmin Ganta, Roman R. |
author_facet | Wang, Ying Wei, Lanjing Liu, Huitao Cheng, Chuanmin Ganta, Roman R. |
author_sort | Wang, Ying |
collection | PubMed |
description | Obligate intracellular bacteria (obligates) belonging to Rickettsiales and Chlamydiales cause diseases in hundreds of millions of people worldwide and in many animal species. Lack of an efficient system for targeted mutagenesis in obligates remains a major impediment in understanding microbial pathogenesis. Challenges in creating targeted mutations may be attributed to essential nature of majority of the genes and intracellular replication dependence. Despite success in generating random mutations, a method that works well in creating mutations in specific genes of interest followed by complementation remains problematic for obligates and is a highly sought-after goal. We describe protocols to generate stable targeted mutations by allelic exchange in Ehrlichia chaffeensis, an obligate intracellular tick-borne bacterium responsible for human monocytic ehrlichiosis. Targeted mutations in E. chaffeensis were created to disrupt two genes, and also to restore one gene by another allelic exchange mutation leading to the restoration of transcription and protein expression from the inactivated gene and the recovered organisms also express mCherry, which distinguishes from the wild type. We expect that the methods developed are broadly applicable to other obligates, particularly to rickettsial pathogens, to routinely perform targeted mutations to enable studies focused on protein structure-function analyses, host-pathogen interactions and in developing vaccines. |
format | Online Article Text |
id | pubmed-5693922 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-56939222017-11-27 A genetic system for targeted mutations to disrupt and restore genes in the obligate bacterium, Ehrlichia chaffeensis Wang, Ying Wei, Lanjing Liu, Huitao Cheng, Chuanmin Ganta, Roman R. Sci Rep Article Obligate intracellular bacteria (obligates) belonging to Rickettsiales and Chlamydiales cause diseases in hundreds of millions of people worldwide and in many animal species. Lack of an efficient system for targeted mutagenesis in obligates remains a major impediment in understanding microbial pathogenesis. Challenges in creating targeted mutations may be attributed to essential nature of majority of the genes and intracellular replication dependence. Despite success in generating random mutations, a method that works well in creating mutations in specific genes of interest followed by complementation remains problematic for obligates and is a highly sought-after goal. We describe protocols to generate stable targeted mutations by allelic exchange in Ehrlichia chaffeensis, an obligate intracellular tick-borne bacterium responsible for human monocytic ehrlichiosis. Targeted mutations in E. chaffeensis were created to disrupt two genes, and also to restore one gene by another allelic exchange mutation leading to the restoration of transcription and protein expression from the inactivated gene and the recovered organisms also express mCherry, which distinguishes from the wild type. We expect that the methods developed are broadly applicable to other obligates, particularly to rickettsial pathogens, to routinely perform targeted mutations to enable studies focused on protein structure-function analyses, host-pathogen interactions and in developing vaccines. Nature Publishing Group UK 2017-11-17 /pmc/articles/PMC5693922/ /pubmed/29150636 http://dx.doi.org/10.1038/s41598-017-16023-y Text en © The Author(s) 2017 Open Access 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/. |
spellingShingle | Article Wang, Ying Wei, Lanjing Liu, Huitao Cheng, Chuanmin Ganta, Roman R. A genetic system for targeted mutations to disrupt and restore genes in the obligate bacterium, Ehrlichia chaffeensis |
title | A genetic system for targeted mutations to disrupt and restore genes in the obligate bacterium, Ehrlichia chaffeensis |
title_full | A genetic system for targeted mutations to disrupt and restore genes in the obligate bacterium, Ehrlichia chaffeensis |
title_fullStr | A genetic system for targeted mutations to disrupt and restore genes in the obligate bacterium, Ehrlichia chaffeensis |
title_full_unstemmed | A genetic system for targeted mutations to disrupt and restore genes in the obligate bacterium, Ehrlichia chaffeensis |
title_short | A genetic system for targeted mutations to disrupt and restore genes in the obligate bacterium, Ehrlichia chaffeensis |
title_sort | genetic system for targeted mutations to disrupt and restore genes in the obligate bacterium, ehrlichia chaffeensis |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5693922/ https://www.ncbi.nlm.nih.gov/pubmed/29150636 http://dx.doi.org/10.1038/s41598-017-16023-y |
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