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-...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2021-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0261088 |
| _version_ | 1811215880739094528 |
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| author | Lotisha Garvin Rebecca Vande Voorde Mary Dickinson Steven Carrell Kevin Hybiske Daniel Rockey |
| author_facet | Lotisha Garvin Rebecca Vande Voorde Mary Dickinson Steven Carrell Kevin Hybiske Daniel Rockey |
| author_sort | Lotisha Garvin |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-04-12T06:31:03Z |
| format | Article |
| id | doaj.art-128d678cfd314e1eb5b675b9a2b1ba59 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-12T06:31:03Z |
| publishDate | 2021-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-128d678cfd314e1eb5b675b9a2b1ba592022-12-22T03:44:01ZengPublic Library of Science (PLoS)PLoS ONE1932-62032021-01-011612e026108810.1371/journal.pone.0261088A broad-spectrum cloning vector that exists as both an integrated element and a free plasmid in Chlamydia trachomatis.Lotisha GarvinRebecca Vande VoordeMary DickinsonSteven CarrellKevin HybiskeDaniel RockeyPlasmid 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.https://doi.org/10.1371/journal.pone.0261088 |
| spellingShingle | Lotisha Garvin Rebecca Vande Voorde Mary Dickinson Steven Carrell Kevin Hybiske Daniel Rockey A broad-spectrum cloning vector that exists as both an integrated element and a free plasmid in Chlamydia trachomatis. PLoS ONE |
| 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 |
| url | https://doi.org/10.1371/journal.pone.0261088 |
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