A fluorescent reporter system for anaerobic thermophiles

In this study, we show that pFAST displays a high melting temperature (Tm) and can serve as a fluorescent marker in the thermophilic anaerobe T. kivui. We engineered a modular cloning system based on Golden Gate assembly for this relatively uncommon microbe, which allowed easy construction and introduction of pFAST-bearing shuttle plasmids. pFAST-mediated fluorescence was subsequently established at 55°C and 66°C, which was exploited to characterize promoter parts and to engineer novel Gram-positive origins of replication.
In this work, we pioneered the use of the oxygen-independent system FAST (Fluorescence Activating and absorption Shifting tag) in the thermophilic anaerobe Thermoanaerobacter kivui. We developed a modular cloning system that was used to easily clone a library of FAST expression cassettes in an E. coli—Thermoanaerobacter shuttle plasmid. FAST-mediated fluorescence was then assessed in vivo in T. kivui, and we observed bright green and red fluorescence for cells grown at 55°C. Next, we took advantage of this functional reporter system to characterize a set of homologous and heterologous promoters by quantifying gene expression, expanding the T. kivui genetic toolbox.

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