Application of the fluorescence-activating and absorption-shifting tag (FAST) for flow cytometry in methanogenic archaea.

Abstract

Methane-producing archaea play a crucial role in the global carbon cycle and are used for biotechnological fuel production. Methanogenic model organisms such as Methanococcus maripaludis and Methanosarcina acetivorans are biochemically characterized and can be genetically engineered using a variety of molecular tools. Methanogens anaerobic lifestyle and autofluorescence, however, restrict the use of common fluorescent reporter proteins (e.g., GFP and derivatives) which require oxygen for chromophore maturation. Here, we employ the tandem activation and absorption-shifting tag protein 2 (tdFAST2) which is fluorescent when the cell-permeable fluorescent ligand (fluorogen) 4-hydroxy-3,5-dimethoxybenzylidene rhodanine (HBR-3,5DOM) is present. tdFAST2 expression in M. acetivorans and M. maripaludis is not cytotoxic and tdFAST2:HBR-3,5DOM fluorescence can be clearly distinguished from the autofluorescence. In flow cytometry experiments, mixed methanogen cultures can be clearly distinguished which allows high-throughput investigations of dynamics within single and mixed cultures. ImportanceMethane-producing archaea play an essential role in the global carbon cycle and have a high potential for biotechnological applications such as biofuel production, carbon dioxide capture, and in electrochemical systems. The oxygen sensitivity and high autofluorescence hinder the use of common fluorescent proteins to study methanogens. By using the tdFAST2:HBR-3,5DOM fluorescence, which is functional also under anaerobic conditions and distinguishable from the autofluorescence, real-time reporter studies and high-throughput investigation of dynamics within (mixed) cultures via flow cytometry are possible. This will accelerate the exploitation of the methanogens biotechnological potential.