Biosensors-Based In Vivo Quantification of 2-Oxoglutarate in Cyanobacteria and Proteobacteria

Abstract

2-oxoglutarate (α-ketoglutarate; 2-OG) is an intermediate of the Krebs cycle, and constitutes the carbon skeleton for nitrogen assimilation and the synthesis of a variety of compounds. In addition to being an important metabolite, 2-OG is a signaling molecule with a broad regulatory repertoire in a variety of organisms, including plants, animals, and bacteria. Although challenging, measuring the levels and variations of metabolic signals in vivo is critical to better understand how cells control specific processes. To measure cellular 2-OG concentrations and dynamics, we designed a set of biosensors based on the fluorescence resonance energy transfer (FRET) technology that can be used in vivo in different organisms. For this purpose, we took advantage of the conformational changes of two cyanobacterial proteins induced by 2-OG binding. We show that these biosensors responded immediately and specifically to different 2-OG levels, and hence allowed to measure 2-OG variations in function of environmental modifications in the proteobacterium Escherichia coli and in the cyanobacterium Anabaena sp. PCC 7120. Our results pave the way to study 2-OG dynamics at the cellular level in uni- and multi-cellular organisms.

Keywords

• biosensor
• cyanobacteria
• <i>Escherichia coli</i>
• FRET
• 2-oxoglutarate