Skilton Rachel J, Wang Yibing, O'Neill Colette, Filardo Simone, Marsh Peter, Bénard Angèle, Thomson Nicholas R, Ramsey Kyle H, Clarke Ian N
Molecular Microbiology Group, Faculty of Medicine, University of Southampton, Southampton General Hospital, Southampton, UK.
Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington, Seattle, WA, USA.
Wellcome Open Res. 2018 Mar 8;3:25. doi: 10.12688/wellcomeopenres.13905.1. eCollection 2018.
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.
由于衣原体具有专性胞内发育周期，衣原体遗传学研究颇具挑战性。现有多种系统可用于研究衣原体基础生物学的不同方面，其中小鼠模型尤为重要，是一种必不可少的研究工具。[该模型的衣原体]携带一种与毒力有关的质粒。我们的目的是在发育周期的包涵体阶段，比较和对比无质粒表型与染色体同基因携带质粒菌株的表型。在整个发育周期中，我们通过在 McCoy 细胞中进行包涵体形成试验来测定携带质粒和去除质粒的[衣原体]的感染性，并通过定量 PCR 并行检测细菌染色体复制情况。除了这些研究，我们还通过共聚焦显微镜和透射电子显微镜仔细监测了衣原体包涵体的形成。利用标准克隆技术构建了一种新的衣原体穿梭载体（pNigg::GFP），并用于转化[衣原体]以进行进一步的表型研究。我们对衣原体表型的定义已从简单地静态观察成熟包涵体向前推进，并且根据生长曲线和包涵体形态重新定义了基于质粒的表型。我们对去除质粒的[衣原体]生长特性的观察对既定的解释提出了挑战，特别是在包涵体生长动力学方面。将穿梭质粒 pNigg::GFP 导入去除质粒的[衣原体]中恢复了野生型携带质粒的表型，并证实质粒的丢失是生长和染色体复制变化的唯一原因。在整个发育周期的多个时间点绘制准确的生长曲线并进行采样，对于定义质粒表型是必要的。携带质粒和无质粒的[衣原体]在总体生长曲线上存在细微但重要（以前未被注意到）的差异。我们已经证明所描述的差异完全是由于质粒 pNigg 所致。
