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A broad-spectrum cloning vector that exists as both an integrated element and a free plasmid in Chlamydia trachomatis
Plasmid transformation of chlamydiae has created new opportunities to investigate host–microbe interactions during chlamydial infections; however, there are still limitations. Plasmid transformation requires a replicon derived from the native Chlamydia plasmid, and these transformations are species-...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Public Library of Science
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8675754/ https://www.ncbi.nlm.nih.gov/pubmed/34914750 http://dx.doi.org/10.1371/journal.pone.0261088 |
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author | Garvin, Lotisha Vande Voorde, Rebecca Dickinson, Mary Carrell, Steven Hybiske, Kevin Rockey, Daniel |
author_facet | Garvin, Lotisha Vande Voorde, Rebecca Dickinson, Mary Carrell, Steven Hybiske, Kevin Rockey, Daniel |
author_sort | Garvin, Lotisha |
collection | PubMed |
description | Plasmid transformation of chlamydiae has created new opportunities to investigate host–microbe interactions during chlamydial infections; however, there are still limitations. Plasmid transformation requires a replicon derived from the native Chlamydia plasmid, and these transformations are species-specific. We explored the utility of a broad host-range plasmid, pBBR1MCS-4, to transform chlamydiae, with a goal of simplifying the transformation process. The plasmid was modified to contain chromosomal DNA from C. trachomatis to facilitate homologous recombination. Sequences flanking incA were cloned into the pBBR1MCS-4 vector along with the GFP:CAT cassette from the pSW2-GFP chlamydial shuttle vector. The final plasmid construct, pBVR2, was successfully transformed into C. trachomatis strain L2-434. Chlamydial transformants were analyzed by immunofluorescence microscopy and positive clones were sequentially purified using limiting dilution. PCR and PacBio-based whole genome sequencing were used to determine if the plasmid was maintained within the chromosome or as an episome. PacBio sequencing of the cloned transformants revealed allelic exchange events between the chromosome and plasmid pBVR2 that replaced chromosomal incA with the plasmid GFP:CAT cassette. The data also showed evidence of full integration of the plasmid into the bacterial chromosome. While some plasmids were fully integrated, some were maintained as episomes and could be purified and retransformed into E. coli. Thus, the plasmid can be successfully transformed into chlamydia without a chlamydial origin of replication and can exist in multiple states within a transformed population. |
format | Online Article Text |
id | pubmed-8675754 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-86757542021-12-17 A broad-spectrum cloning vector that exists as both an integrated element and a free plasmid in Chlamydia trachomatis Garvin, Lotisha Vande Voorde, Rebecca Dickinson, Mary Carrell, Steven Hybiske, Kevin Rockey, Daniel PLoS One Research Article Plasmid transformation of chlamydiae has created new opportunities to investigate host–microbe interactions during chlamydial infections; however, there are still limitations. Plasmid transformation requires a replicon derived from the native Chlamydia plasmid, and these transformations are species-specific. We explored the utility of a broad host-range plasmid, pBBR1MCS-4, to transform chlamydiae, with a goal of simplifying the transformation process. The plasmid was modified to contain chromosomal DNA from C. trachomatis to facilitate homologous recombination. Sequences flanking incA were cloned into the pBBR1MCS-4 vector along with the GFP:CAT cassette from the pSW2-GFP chlamydial shuttle vector. The final plasmid construct, pBVR2, was successfully transformed into C. trachomatis strain L2-434. Chlamydial transformants were analyzed by immunofluorescence microscopy and positive clones were sequentially purified using limiting dilution. PCR and PacBio-based whole genome sequencing were used to determine if the plasmid was maintained within the chromosome or as an episome. PacBio sequencing of the cloned transformants revealed allelic exchange events between the chromosome and plasmid pBVR2 that replaced chromosomal incA with the plasmid GFP:CAT cassette. The data also showed evidence of full integration of the plasmid into the bacterial chromosome. While some plasmids were fully integrated, some were maintained as episomes and could be purified and retransformed into E. coli. Thus, the plasmid can be successfully transformed into chlamydia without a chlamydial origin of replication and can exist in multiple states within a transformed population. Public Library of Science 2021-12-16 /pmc/articles/PMC8675754/ /pubmed/34914750 http://dx.doi.org/10.1371/journal.pone.0261088 Text en https://creativecommons.org/publicdomain/zero/1.0/This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 (https://creativecommons.org/publicdomain/zero/1.0/) public domain dedication. |
spellingShingle | Research Article Garvin, Lotisha Vande Voorde, Rebecca Dickinson, Mary Carrell, Steven Hybiske, Kevin Rockey, Daniel A broad-spectrum cloning vector that exists as both an integrated element and a free plasmid in Chlamydia trachomatis |
title | A broad-spectrum cloning vector that exists as both an integrated element and a free plasmid in Chlamydia trachomatis |
title_full | A broad-spectrum cloning vector that exists as both an integrated element and a free plasmid in Chlamydia trachomatis |
title_fullStr | A broad-spectrum cloning vector that exists as both an integrated element and a free plasmid in Chlamydia trachomatis |
title_full_unstemmed | A broad-spectrum cloning vector that exists as both an integrated element and a free plasmid in Chlamydia trachomatis |
title_short | A broad-spectrum cloning vector that exists as both an integrated element and a free plasmid in Chlamydia trachomatis |
title_sort | broad-spectrum cloning vector that exists as both an integrated element and a free plasmid in chlamydia trachomatis |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8675754/ https://www.ncbi.nlm.nih.gov/pubmed/34914750 http://dx.doi.org/10.1371/journal.pone.0261088 |
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