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Genetic Transformation of a Clinical (Genital Tract), Plasmid-Free Isolate of Chlamydia trachomatis: Engineering the Plasmid as a Cloning Vector

Genetic Transformation of a Clinical (Genital Tract), Plasmid-Free Isolate of Chlamydia trachomatis: Engineering the Plasmid as a Cloning Vector
Genetic Transformation of a Clinical (Genital Tract), Plasmid-Free Isolate of Chlamydia trachomatis: Engineering the Plasmid as a Cloning Vector
Our study had three objectives: to extend the plasmid-based transformation protocol to a clinical isolate of C. trachomatis belonging to the trachoma biovar, to provide "proof of principle" that it is possible to "knock out" selected plasmid genes (retaining a replication competent plasmid) and to investigate the plasticity of the plasmid. A recently developed, plasmid-based transformation protocol for LGV isolates of C. trachomatis was modified and a plasmid-free, genital tract C. trachomatis isolate from Sweden (SWFP-) was genetically transformed. Transformation of this non-LGV C. trachomatis host required a centrifugation step, but the absence of the natural plasmid removed the need for plaque purification of transformants. Transformants expressed GFP, were penicillin resistant and iodine stain positive for accumulated glycogen. The transforming plasmid did not recombine with the host chromosome. A derivative of pGFP::SW2 carrying a deletion of the plasmid CDS5 gene was engineered. CDS5 encodes pgp3, a protein secreted from the inclusion into the cell cytoplasm. This plasmid (pCDS5KO) was used to transform C. trachomatis SWFP-, and established that pgp3 is dispensable for plasmid function. The work shows it is possible to selectively delete segments of the chlamydial plasmid, and this is the first step towards a detailed molecular dissection of the role of the plasmid. The 3.6 kb ?-galactosidase cassette was inserted into the deletion site of CDS5 to produce plasmid placZ-CDS5KO. Transformants were penicillin resistant, expressed GFP and stained for glycogen. In addition, they expressed ?-galactosidase showing that the lacZ cassette was functional in C. trachomatis. An assay was developed that allowed the visualisation of individual inclusions by X-gal staining. The ability to express active ?-galactosidase within chlamydial inclusions is an important advance as it allows simple, rapid assays to measure directly chlamydial infectivity without the need for plaquing, fluorescence or antibody staining.
1932-6203
e59195
Wang, Yibing
a4dc0303-7006-411e-9063-c26297fac847
Kahane, Simona
d74091ff-89e9-4c75-8a01-1da5315fb5e9
Cutcliffe, Lesley T.
88f242f4-b4f7-46d4-a1dd-c187dd60a773
Skilton, Rachel J.
b02d4f32-609c-4074-b616-ec819b018dbe
Lambden, Paul R.
4fcd536e-2d9a-4366-97c6-386e6b005698
Persson, Kenneth
9ecaea9f-c093-4d57-8bf3-99373ab580c7
Bjartling, Carina
b8c0bfea-b7dc-4605-a865-0f1f4d40685b
Clarke, Ian N.
ff6c9324-3547-4039-bb2c-10c0b3327a8b
Wang, Yibing
a4dc0303-7006-411e-9063-c26297fac847
Kahane, Simona
d74091ff-89e9-4c75-8a01-1da5315fb5e9
Cutcliffe, Lesley T.
88f242f4-b4f7-46d4-a1dd-c187dd60a773
Skilton, Rachel J.
b02d4f32-609c-4074-b616-ec819b018dbe
Lambden, Paul R.
4fcd536e-2d9a-4366-97c6-386e6b005698
Persson, Kenneth
9ecaea9f-c093-4d57-8bf3-99373ab580c7
Bjartling, Carina
b8c0bfea-b7dc-4605-a865-0f1f4d40685b
Clarke, Ian N.
ff6c9324-3547-4039-bb2c-10c0b3327a8b

Wang, Yibing, Kahane, Simona, Cutcliffe, Lesley T., Skilton, Rachel J., Lambden, Paul R., Persson, Kenneth, Bjartling, Carina and Clarke, Ian N. (2013) Genetic Transformation of a Clinical (Genital Tract), Plasmid-Free Isolate of Chlamydia trachomatis: Engineering the Plasmid as a Cloning Vector. PLoS ONE, 8 (3), e59195. (doi:10.1371/journal.pone.0059195). (PMID:23527131)

Record type: Article

Abstract

Our study had three objectives: to extend the plasmid-based transformation protocol to a clinical isolate of C. trachomatis belonging to the trachoma biovar, to provide "proof of principle" that it is possible to "knock out" selected plasmid genes (retaining a replication competent plasmid) and to investigate the plasticity of the plasmid. A recently developed, plasmid-based transformation protocol for LGV isolates of C. trachomatis was modified and a plasmid-free, genital tract C. trachomatis isolate from Sweden (SWFP-) was genetically transformed. Transformation of this non-LGV C. trachomatis host required a centrifugation step, but the absence of the natural plasmid removed the need for plaque purification of transformants. Transformants expressed GFP, were penicillin resistant and iodine stain positive for accumulated glycogen. The transforming plasmid did not recombine with the host chromosome. A derivative of pGFP::SW2 carrying a deletion of the plasmid CDS5 gene was engineered. CDS5 encodes pgp3, a protein secreted from the inclusion into the cell cytoplasm. This plasmid (pCDS5KO) was used to transform C. trachomatis SWFP-, and established that pgp3 is dispensable for plasmid function. The work shows it is possible to selectively delete segments of the chlamydial plasmid, and this is the first step towards a detailed molecular dissection of the role of the plasmid. The 3.6 kb ?-galactosidase cassette was inserted into the deletion site of CDS5 to produce plasmid placZ-CDS5KO. Transformants were penicillin resistant, expressed GFP and stained for glycogen. In addition, they expressed ?-galactosidase showing that the lacZ cassette was functional in C. trachomatis. An assay was developed that allowed the visualisation of individual inclusions by X-gal staining. The ability to express active ?-galactosidase within chlamydial inclusions is an important advance as it allows simple, rapid assays to measure directly chlamydial infectivity without the need for plaquing, fluorescence or antibody staining.

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Published date: 2013
Organisations: Clinical & Experimental Sciences

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Local EPrints ID: 350914
URI: http://eprints.soton.ac.uk/id/eprint/350914
ISSN: 1932-6203
PURE UUID: 8abdcded-d146-41ed-b89a-c2cdbb45a15b
ORCID for Ian N. Clarke: ORCID iD orcid.org/0000-0002-4938-1620

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Date deposited: 10 Apr 2013 15:20
Last modified: 15 Mar 2024 02:33

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Contributors

Author: Yibing Wang
Author: Simona Kahane
Author: Lesley T. Cutcliffe
Author: Rachel J. Skilton
Author: Paul R. Lambden
Author: Kenneth Persson
Author: Carina Bjartling
Author: Ian N. Clarke ORCID iD

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