The Chlamydia muridarum plasmid revisited : new insights into growth kinetics

Wellcome Open Research

article

Rachel J. Skilton¹  Yibing Wang²  Colette O'Neill¹  Simone Filardo³  Peter Marsh⁴  Angèle Bénard⁵  Nicholas R. Thomson⁵  Kyle H. Ramsey⁷  Ian N. Clarke¹ 

[1] Molecular Microbiology Group, Faculty of Medicine, University of Southampton, Southampton General Hospital;Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington;Department of Public Health and Infectious Diseases, Section of Microbiology, Sapienza University;Public Health England, Public Health Laboratory Southampton, Southampton General Hospital;Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus;London School of Hygiene and Tropical Medicine;Department of Microbiology & Immunology, Chicago College of Osteopathic Medicine, Midwestern University

关键词: Chlamydia muridarum;    plasmid;    phenotype;    inclusion;    transformation;    Nigg;   

DOI  :  10.12688/wellcomeopenres.13905.1

学科分类：内科医学

来源: Wellcome

【 摘 要 】

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 murineChlamydia 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 theC. 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-curedC. 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 newE.coli/chlamydial shuttle vector (pNigg::GFP) was constructed using standard cloning technology and used to transformC. 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 theC. muridarum plasmid-based phenotype on growth profile and inclusion morphology. Our observations on the growth properties of plasmid-curedC. muridarum challenge the established interpretations, especially with regard to inclusion growth kinetics. Introduction of the shuttle plasmid pNigg::GFP into plasmid-curedC. 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-freeC. muridarum.  We have proven that the differences described are solely due to the plasmid pNigg.

【 授权许可】

CC BY