FraR, a transcriptional repressor, was postulated to regulate the metabolism of the Amadori compound fructose-asparagine (F-Asn) in the foodborne pathogen Salmonella enterica. Here, the DNA- and inducer-binding affinities and stoichiometries of FraR were determined and cross-validated by electrophoretic mobility-shift assays (EMSAs) and online buffer exchange coupled to native mass spectrometry (OBE-nMS). We demonstrate the utility of OBE-nMS to characterize protein and protein-DNA complexes that are not amenable to offline exchange into volatile buffers. OBE-nMS complemented EMSAs by revealing that FraR binds to the operator DNA as a dimer and by establishing 6-phosphofructose-aspartate as the inducer that weakens DNA binding by FraR. These results provide insights into how FraR regulates the expression of F-Asn-catabolizing enzymes and add to our understanding of the intricate bacterial circuitry that dictates utilization of diverse nutrients.

Characterization of a Salmonella Transcription Factor-DNA Complex and Identification of the Inducer by Native Mass Spectrometry

Di Capua A.;
2022

Abstract

FraR, a transcriptional repressor, was postulated to regulate the metabolism of the Amadori compound fructose-asparagine (F-Asn) in the foodborne pathogen Salmonella enterica. Here, the DNA- and inducer-binding affinities and stoichiometries of FraR were determined and cross-validated by electrophoretic mobility-shift assays (EMSAs) and online buffer exchange coupled to native mass spectrometry (OBE-nMS). We demonstrate the utility of OBE-nMS to characterize protein and protein-DNA complexes that are not amenable to offline exchange into volatile buffers. OBE-nMS complemented EMSAs by revealing that FraR binds to the operator DNA as a dimer and by establishing 6-phosphofructose-aspartate as the inducer that weakens DNA binding by FraR. These results provide insights into how FraR regulates the expression of F-Asn-catabolizing enzymes and add to our understanding of the intricate bacterial circuitry that dictates utilization of diverse nutrients.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11563/160590
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