The plasmid revisited : new insights into growth kinetics.

Research in chlamydial genetics is challenging because of its obligate intracellular developmental cycle. systems exist that allow studies of different aspects of basic biology of chlamydiae, the murine model is one of great importance and thus an essential research tool. carries a plasmid that has a role in virulence.  Our aim was to compare and contrast the plasmid-free phenotype with that of a chromosomally isogenic plasmid-bearing strain, through the inclusion phase of the developmental cycle. We measured infectivity for plasmid bearing and plasmid-cured 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 /chlamydial shuttle vector (pNigg::GFP) was constructed using standard cloning technology and used to transform for further phenotypic studies. We have advanced the definition of the chlamydial phenotype away from the simple static observation of mature inclusions and redefined the plasmid-based phenotype on growth profile and inclusion morphology. Our observations on the growth properties of plasmid-cured challenge the established interpretations, especially with regard to inclusion growth kinetics. Introduction of the shuttle plasmid pNigg::GFP into plasmid-cured 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. 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 .  We have proven that the differences described are solely due to the plasmid pNigg.