Combining two optimized and affordable methods to assign chemoreceptors to a specific signal

• Published in: Analytical biochemistry Vol. 620; p. 114139
• Main Authors: Boyeldieu, Anne, Ali Chaouche, Amine, Méjean, Vincent, Jourlin-Castelli, Cécile
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.05.2021; Elsevier Masson
• Subjects: Agarose-in-plug bridge assay; Bacterial chemotaxis; Life Sciences; MBP chimeric Proteins; Methyl-accepting chemotaxis protein; Signal detection; Thermal shift assay; Bacterial chemotaxis; Agarose-in-plug bridge assay; Signal detection; Methyl-accepting chemotaxis protein; Thermal shift assay; MBP chimeric Proteins; Bacterial chemotaxis Signal detection Methyl-accepting chemotaxis protein Thermal shift assay Agarose-in-plug bridge assay MBP chimeric Proteins
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2021.114139

Chemotaxis allows bacteria to detect specific compounds and move accordingly. This pathway involves signal detection by chemoreceptors (MCPs). Attributing a chemoreceptor to a ligand is difficult because there is a lot of redundancy in the MCPs that recognize a single ligand. We propose a methodology to define which chemoreceptors bind a given ligand. First, an MCP is overproduced to increase sensitivity to the ligand(s) it recognizes, thus promoting accumulation of cells around an agarose plug containing a low attractant concentration. Second, the ligand-binding domain (LBD) of the chemoreceptor is fused to maltose-binding protein (MBP), which facilitates purification and provides a control for a thermal shift assay (TSA). An increase in the melting temperature of the LBD in the presence of the ligand indicates that the chemoreceptor directly binds it. We showed that overexpression of two Shewanella oneidensis chemoreceptors (SO_0987 and SO_1056) promoted swimming toward an agarose plug containing a low concentration of chromate. The LBD of each of the two chemoreceptors was fused to MBP. A TSA revealed that only the LBD from SO_1056 had its melting temperature increased by chromate. In conclusion, we describe an efficient approach to define chemoreceptor-ligand pairs before undertaking more-sophisticated biochemical and structural studies. [Display omitted] •Strategy for assigning chemoreceptor(s) to a ligand.•Overexpression of chemoreceptor(s) increases chemotactic sensitivity towards ligand.•Ligand-binding domain of chemoreceptor(s) fused to the maltose-binding protein.•Assessment of ligand binding to chemoreceptor(s) by thermal shift assays.