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The Chlamydia muridarum plasmid revisited: new insights into growth kinetics

The Chlamydia muridarum plasmid revisited: new insights into growth kinetics
The Chlamydia muridarum plasmid revisited: new insights into growth kinetics
Background: research in chlamydial genetics is challenging because of its obligate intracellular developmental cycle. In vivo systems exist that allow studies of different aspects of basic biology of chlamydiae, the murine Chlamydia muridarum model is one of great importance and thus an essential research tool. C. muridarum carries a plasmid that has a role in virulence. Our aim was to compare and contrast the C. muridarum plasmid-free phenotype with that of a chromosomally isogenic plasmid-bearing strain, through the inclusion phase of the developmental cycle.

Methods: we measured infectivity for plasmid bearing and plasmid-cured C. muridarum by inclusion forming assays in McCoy cells and in parallel bacterial chromosome replication by quantitative PCR, throughout the developmental cycle. In addition to these studies, we have carefully monitored chlamydial inclusion formation by confocal microscopy and transmission electron microscopy. A new E.coli/chlamydial shuttle vector (pNigg::GFP) was constructed using standard cloning technology and used to transform C. muridarum for further phenotypic studies.

Results: we have advanced the definition of the chlamydial phenotype away from the simple static observation of mature inclusions and redefined the C. muridarum plasmid-based phenotype on growth profile and inclusion morphology. Our observations on the growth properties of plasmid-cured C. muridarum challenge the established interpretations, especially with regard to inclusion growth kinetics. Introduction of the shuttle plasmid pNigg::GFP into plasmid-cured C. muridarum restored the wild-type plasmid-bearing phenotype and confirmed that loss of the plasmid was the sole cause for the changes in growth and chromosomal replication.

Conclusions: accurate growth curves and sampling at multiple time points throughout the developmental cycle is necessary to define plasmid phenotypes. There are subtle but important (previously unnoticed) differences in the overall growth profile of plasmid-bearing and plasmid-free C. muridarum. We have proven that the differences described are solely due to the plasmid pNigg.
Skilton, Rachel
b02d4f32-609c-4074-b616-ec819b018dbe
Wang, Yibing
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O'neill, Colette
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Filardo, Simone
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Marsh, Peter
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Benard, Angele
313feeed-c0e7-4e8c-9e66-129d2206ce26
Thomson, Nicholas R.
5497a110-069d-4156-bccb-c77db572b1c2
Ramsey, Kyle H.
b93ffc54-69c6-4bf9-bfe5-cc078ed878fe
Clarke, Ian
ff6c9324-3547-4039-bb2c-10c0b3327a8b
Skilton, Rachel
b02d4f32-609c-4074-b616-ec819b018dbe
Wang, Yibing
f757ce83-2536-4bb2-bbaa-b5cdf64170e7
O'neill, Colette
3de0c221-6578-4a1a-96bd-2a3fba2b6193
Filardo, Simone
9215c7e9-889d-418f-b519-4d62e700d72f
Marsh, Peter
18f63c35-264b-4ec9-8505-9c57bf707ad2
Benard, Angele
313feeed-c0e7-4e8c-9e66-129d2206ce26
Thomson, Nicholas R.
5497a110-069d-4156-bccb-c77db572b1c2
Ramsey, Kyle H.
b93ffc54-69c6-4bf9-bfe5-cc078ed878fe
Clarke, Ian
ff6c9324-3547-4039-bb2c-10c0b3327a8b

Skilton, Rachel, Wang, Yibing, O'neill, Colette, Filardo, Simone, Marsh, Peter, Benard, Angele, Thomson, Nicholas R., Ramsey, Kyle H. and Clarke, Ian (2018) The Chlamydia muridarum plasmid revisited: new insights into growth kinetics. Wellcome Open Research, 3, [25]. (doi:10.12688/wellcomeopenres.13905.1).

Record type: Article

Abstract

Background: research in chlamydial genetics is challenging because of its obligate intracellular developmental cycle. In vivo systems exist that allow studies of different aspects of basic biology of chlamydiae, the murine Chlamydia muridarum model is one of great importance and thus an essential research tool. C. muridarum carries a plasmid that has a role in virulence. Our aim was to compare and contrast the C. muridarum plasmid-free phenotype with that of a chromosomally isogenic plasmid-bearing strain, through the inclusion phase of the developmental cycle.

Methods: we measured infectivity for plasmid bearing and plasmid-cured C. muridarum by inclusion forming assays in McCoy cells and in parallel bacterial chromosome replication by quantitative PCR, throughout the developmental cycle. In addition to these studies, we have carefully monitored chlamydial inclusion formation by confocal microscopy and transmission electron microscopy. A new E.coli/chlamydial shuttle vector (pNigg::GFP) was constructed using standard cloning technology and used to transform C. muridarum for further phenotypic studies.

Results: we have advanced the definition of the chlamydial phenotype away from the simple static observation of mature inclusions and redefined the C. muridarum plasmid-based phenotype on growth profile and inclusion morphology. Our observations on the growth properties of plasmid-cured C. muridarum challenge the established interpretations, especially with regard to inclusion growth kinetics. Introduction of the shuttle plasmid pNigg::GFP into plasmid-cured C. muridarum restored the wild-type plasmid-bearing phenotype and confirmed that loss of the plasmid was the sole cause for the changes in growth and chromosomal replication.

Conclusions: accurate growth curves and sampling at multiple time points throughout the developmental cycle is necessary to define plasmid phenotypes. There are subtle but important (previously unnoticed) differences in the overall growth profile of plasmid-bearing and plasmid-free C. muridarum. We have proven that the differences described are solely due to the plasmid pNigg.

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Accepted/In Press date: 8 March 2018
e-pub ahead of print date: 8 March 2018
Published date: 8 March 2018
Additional Information: This article is published in Wellcome Open Research it is a living document and may change.

Identifiers

Local EPrints ID: 419145
URI: http://eprints.soton.ac.uk/id/eprint/419145
PURE UUID: 277eabbc-7586-447b-85c0-a07c4e5cb4e8
ORCID for Ian Clarke: ORCID iD orcid.org/0000-0002-4938-1620

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Date deposited: 06 Apr 2018 16:30
Last modified: 16 Mar 2024 02:33

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Contributors

Author: Rachel Skilton
Author: Yibing Wang
Author: Colette O'neill
Author: Simone Filardo
Author: Peter Marsh
Author: Angele Benard
Author: Nicholas R. Thomson
Author: Kyle H. Ramsey
Author: Ian Clarke ORCID iD

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