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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-...

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Autores principales: Garvin, Lotisha, Vande Voorde, Rebecca, Dickinson, Mary, Carrell, Steven, Hybiske, Kevin, Rockey, Daniel
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2021
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.
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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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