4.3 Article

Practical observations on the use of fluorescent reporter systems in Clostridioides difficile

出版社

SPRINGER
DOI: 10.1007/s10482-021-01691-8

关键词

C; difficile; Fluorescence; Autofluorescence; HupA; Microscopy

资金

  1. Netherlands Organization for Scientific Research (NWO) [864.10.003]
  2. Leiden University Medical Center

向作者/读者索取更多资源

This study characterized commonly used fluorescent systems in Clostridioides difficile cells and compared their applicability as transcriptional or translational reporters in microscopy. The results showed that SNAP(Cd) is the most suitable candidate for live-cell imaging in C. difficile to date.
Fluorescence microscopy is a valuable tool to study a broad variety of bacterial cell components and dynamics thereof. For Clostridioides difficile, the fluorescent proteins CFPopt, mCherry(Opt) and phiLOV2.1, and the self-labelling tags SNAP(Cd) and HaloTag, hereafter collectively referred as fluorescent systems, have been described to explore different cellular pathways. In this study, we sought to characterize previously used fluorescent systems in C. difficile cells. We performed single cell analyses using fluorescence microscopy of exponentially growing C. difficile cells harbouring different fluorescent systems, either expressing these separately in the cytosol or fused to the C-terminus of HupA, under defined conditions. We show that the intrinsic fluorescence of C. difficile cells increases during growth, independent of sigB or spo0A. However, when C. difficile cells are exposed to environmental oxygen autofluorescence is enhanced. Cytosolic overexpression of the different fluorescent systems alone, using the same expression signals, showed heterogeneous expression of the fluorescent systems. High levels of mCherry(Opt) were toxic for C. difficile cells limiting the applicability of this fluorophore as a transcriptional reporter. When fused to HupA, a C. difficile histone-like protein, the fluorescent systems behaved similarly and did not affect the HupA overproduction phenotype. The present study compares several commonly used fluorescent systems for application as transcriptional or translational reporters in microscopy and summarizes the limitations and key challenges for live-cell imaging of C. difficile. Due to independence of molecular oxygen and fluorescent signal, SNAP(Cd) appears the most suitable candidate for live-cell imaging in C. difficile to date.

作者

我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。

评论

主要评分

4.3
评分不足

次要评分

新颖性
-
重要性
-
科学严谨性
-
评价这篇论文

推荐

暂无数据
暂无数据