Department of Plant and Microbial Biology, University of Minnesota, Twin Cities, St. Paul, Minnesota, USA.
J Bacteriol. 2022 Jul 19;204(7):e0012022. doi: 10.1128/jb.00120-22. Epub 2022 Jun 3.
Live-cell fluorescence imaging of methanogenic archaea has been limited due to the strictly anoxic conditions required for growth and issues with autofluorescence associated with electron carriers in central metabolism. Here, we show that the fluorescence-activating and absorption-shifting tag (FAST) complexed with the fluorogenic ligand 4-hydroxy-3-methylbenzylidene-rhodanine (HMBR) overcomes these issues and displays robust fluorescence in Methanococcus maripaludis. We also describe a mechanism to visualize cells under anoxic conditions using a fluorescence microscope. Derivatives of FAST were successfully applied for protein abundance analysis, subcellular localization analysis, and determination of protein-protein interactions. FAST fusions to both formate dehydrogenase (Fdh) and F-reducing hydrogenase (Fru) displayed increased fluorescence in cells grown on formate-containing medium, consistent with previous studies suggesting the increased abundance of these proteins in the absence of H. Additionally, FAST fusions to both Fru and the ATPase associated with the archaellum (FlaI) showed a membrane localization in single cells observed using anoxic fluorescence microscopy. Finally, a split reporter translationally fused to the alpha and beta subunits of Fdh reconstituted a functionally fluorescent molecule via bimolecular fluorescence complementation. Together, these observations demonstrate the utility of FAST as a tool for studying members of the methanogenic archaea. Methanogenic archaea are important members of anaerobic microbial communities where they catalyze essential reactions in the degradation of organic matter. Developing additional tools for studying the cell biology of these organisms is essential to understanding them at a mechanistic level. Here, we show that FAST, in combination with the fluorogenic ligand HMBR, can be used to monitor protein dynamics in live cells of M. maripaludis. The application of FAST holds promise for future studies focused on the metabolism and physiology of methanogenic archaea.
由于产甲烷古菌的生长需要严格的缺氧条件，以及与中心代谢电子载体相关的自发荧光问题，因此对其进行活细胞荧光成像一直受到限制。在这里，我们展示了与荧光激活和吸收移位标签（FAST）复合的荧光发生配体 4-羟基-3-甲基苯亚甲基罗丹宁（HMBR）克服了这些问题，并在 Methanococcus maripaludis 中显示出强大的荧光。我们还描述了一种在缺氧条件下使用荧光显微镜观察细胞的机制。FAST 的衍生物成功地应用于蛋白质丰度分析、亚细胞定位分析和蛋白质-蛋白质相互作用的测定。FAST 与甲酸脱氢酶（Fdh）和 F 型还原氢化酶（Fru）的融合蛋白在含有甲酸的培养基中生长的细胞中显示出增强的荧光，这与先前的研究一致，即这些蛋白质在没有 H 的情况下丰度增加。此外，FAST 与 Fru 和菌毛相关的 ATP 酶（FlaI）的融合蛋白在使用缺氧荧光显微镜观察时，在单个细胞中显示出膜定位。最后，翻译融合到 Fdh 的α和β亚基的分裂报告子通过双分子荧光互补重新构建了功能荧光分子。总之，这些观察结果表明 FAST 是研究产甲烷古菌的有用工具。产甲烷古菌是厌氧微生物群落中的重要成员，它们在有机物质的降解中催化重要反应。开发更多用于研究这些生物细胞生物学的工具对于在机制水平上理解它们至关重要。在这里，我们展示了 FAST 与荧光发生配体 HMBR 结合，可用于监测 M. maripaludis 活细胞中的蛋白质动态。FAST 的应用有望为未来研究产甲烷古菌的代谢和生理学提供帮助。
