Poster abstracts

Poster number 48 submitted by Blake Szkoda

Characterization of a Transcription Factor-DNA Complex Using Online Buffer Exchange Coupled to Native Mass Spectrometry

Blake E. Szkoda (The Ohio State Biochemistry Program, The Ohio State University), Angela Di Capua (Department of Chemistry & Biochemistry, The Ohio State University), Sravya Kovvali (Department of Microbiology, The Ohio State University), Joy Shaffer (Department of Chemistry and Biochemistry, The University of Texas at Dallas), Edward J. Behrman (Department of Chemistry & Biochemistry, The Ohio State University), Vicki Wysocki, Venkat Gopalan (Department of Chemistry & Biochemistry, The Ohio State University)

Abstract:
The foodborne pathogen Salmonella enterica serovar Typhimurim (Salmonella) causes approximately 94 million enteric infections and 50,000 diarrheal deaths annually worldwide.1 There are no vaccines or antibiotics that specifically combat this bacterium. During infection, Salmonella exploits fructose-asparagine (F-Asn) as a carbon and nitrogen source.2 F-Asn is a product of an Amadori rearrangement that occurs during cooking and dehydration of raw foods.3 F-Asn is metabolized by Salmonella using three enzymes and a transporter encoded by the fra operon. The roles of a periplasmic asparaginase, cytoplasmic kinase, and a cytoplasmic deglycase in F-Asn catabolism are now established.2,4 Importantly, the deglycase was identified as a promising drug target since its knock-out led to accumulation of its substrate and Salmonella self-poisoning.4 This work was undertaken to characterize gene regulation of the fra operon by FraR, the putative transcriptional factor in this locus. FraR is predicted to contain an N-terminal DNA-binding domain (DBD) and a C-terminal inducer-binding domain (IBD). We hypothesize that FraR binds to the fraB promoter in vivo and acts as a transcriptional repressor, and that binding of an inducer ligand to the FraR IBD triggers a conformational change to release the DNA from the DBD and permit transcription of the fra operon genes. This postulate was tested by first purifying recombinant FraR (post-overexpression in Escherichia coli), and then assessing FraR-DNA binding affinity (± putative inducers) with fluorescence-based gel-shift assays and native mass spectrometry (nMS). Results from our biochemical studies provide insights into FraR’s binding stoichiometry, operator sequence, and inducer identity. These findings provide a first glimpse into the regulation of Amadori metabolism in a clinically significant bacterial pathogen and uncover thematic parallels in control of gene expression during utilization of unrelated nutrients.

References:
1) Majowicz et al. (2010) Clin. Infect. Dis. 50: 882-889
2) Ali et al. (2014) PLoS Pathog. 10: e1004209
3) Wu et al. (2017) J. Food. Ag. Chem. 66: 212-217
4) Sabag-Daigle et al. (2016) Sci. Reps. 6: 1-9

Keywords: Salmonella, DNA, Native MS