委内瑞拉链霉菌中fbiC的缺失使青色荧光蛋白激发-发射范围内的自身荧光消失。

IF 2.6 4区 生物学 Q3 MICROBIOLOGY
Parminder Singh Mavi, Klas Flärdh
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引用次数: 0

摘要

自体荧光是生物样品荧光显微镜研究的一个障碍。在革兰氏阳性,链霉菌属的土壤细菌中,自身荧光的来源尚未被系统地研究过。在这里,我们展示了该属的模式生物,委内瑞拉链霉菌,在两种常用的荧光通道中显示自身荧光,用于观察青色和绿色/黄色荧光蛋白。我们确定了青色荧光通道中自身荧光的来源是氧化还原辅助因子420 (F420),并针对其合成来去除它。通过删除F420产生的关键生物合成基因vnz15170 (fbiC)基因,我们能够在荧光激发-发射的青色范围内建立一个无自身荧光的菌株。我们通过在fbiC缺失背景下成像mturquoise标记的极性生长相关蛋白DivIVA和细胞分裂相关蛋白FtsZ来证明该菌株的有效性。利用活细胞成像来跟踪DivIVA和FtsZ的动态,我们证明了突变菌株的信噪比有所改善。结果表明,该菌株可作为链霉菌蛋白定位可视化的合适工具,并可促进多色成像和荧光共振能量转移成像的利用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Deletion of fbiC in Streptomyces venezuelae removes autofluorescence in the excitation-emission range of cyan fluorescent protein.

Autofluorescence poses an impediment to fluorescence microscopy of biological samples. In the Gram-positive, soil-dwelling bacteria of the genus Streptomyces, sources of autofluorescence have not been examined systematically to date. Here, we show that the model organism for the genus, Streptomyces venezuelae, shows autofluorescence in two of the commonly used fluorescence channels for visualizing cyan and green/yellow fluorescent proteins. We identify the source of autofluorescence in the cyan fluorescence channel as redox cofactor factor 420 (F420) and target its synthesis to remove it. By deleting the vnz15170 (fbiC) gene, which is a key biosynthetic gene for the production of F420, we were able to create an autofluorescence-free strain in the cyan range of fluorescence excitation-emission. We demonstrate the usefulness of this strain by imaging the mTurquoise-tagged polar growth-related protein DivIVA and the cell division-related protein FtsZ in the fbiC deletion background. Using live-cell imaging to follow the dynamics of DivIVA and FtsZ, we demonstrate an improved signal-to-noise ratio in the mutant strain. We show that this strain can be a suitable tool for visualizing the localization of proteins in Streptomyces spp. and can facilitate the utilization of multi-colour imaging and fluorescence resonance energy transfer-based imaging.

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来源期刊
Microbiology-Sgm
Microbiology-Sgm 生物-微生物学
CiteScore
4.60
自引率
7.10%
发文量
132
审稿时长
3.0 months
期刊介绍: We publish high-quality original research on bacteria, fungi, protists, archaea, algae, parasites and other microscopic life forms. Topics include but are not limited to: Antimicrobials and antimicrobial resistance Bacteriology and parasitology Biochemistry and biophysics Biofilms and biological systems Biotechnology and bioremediation Cell biology and signalling Chemical biology Cross-disciplinary work Ecology and environmental microbiology Food microbiology Genetics Host–microbe interactions Microbial methods and techniques Microscopy and imaging Omics, including genomics, proteomics and metabolomics Physiology and metabolism Systems biology and synthetic biology The microbiome.
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