Knowledge Bank

The dihydroxyacetone phosphate (DHAP) shunt is a carbon and/or sulfur metabolism pathway found in bacteria that can metabolize the nucleosides 5’-methylthioadenosine and 5’-deoxyadenosine. Both compounds lead to DHAP which can enter central carbon metabolism. 5’-methylthioadenosine also leads to (2-methylthio)acetaldehyde for sulfur metabolism and 5’-deoxyadenosine also leads to acetaldehyde for carbon metabolism as well. The DHAP shunt pathway genes in Escherichia coli include a kinase, isomerase, and aldolase, all of which are expressed together as a single polycistronic message. In E. coli, the DHAP shunt is associated with Extraintestinal Pathogenic E. coli (ExPEC) strains, not intestinal pathogenic or commensal strains of E. coli. ExPEC strains are responsible for various extraintestinal illnesses, including urinary tract infections, sepsis, and meningitis. After further investigation, the DHAP shunt was identified through sequence homology in several additional pathogenic species. However, the physiological role of this pathway is unknown in many bacteria. To fill this knowledge gap, this project aims to investigate the transcriptional regulation of the DHAP shunt operon in E. coli and the function of DHAP shunt homologs in other identified pathogenic species. The first project investigates the promoter region proposed to be responsible for transcribing the DHAP shunt at different levels when the cell experiences changes in carbon availability. We observe two promoters for the DHAP shunt genes based on preliminary data. Future work is required to identify the regulation of transcription from each of these two promoters. Identifying regulatory elements of the DHAP shunt promoter in ExPEC strains could advance studies on how this new region influences gene expression, shedding light on the regulatory and adaptive mechanisms of carbon metabolism. The second project endeavors to provide initial characterization of the DHAP shunt enzyme homologs from additional pathogenic species. E. coli Rosetta cells transformed with pET28 carrying a DHAP shunt gene homolog was used to recombinantly synthesize His-tagged proteins that were purified by nickel affinity chromatography. The purified proteins were analyzed for thermal stability via Differential Scanning Fluorometry (DSF) to characterize the homogeneity of the protein population. Future studies will characterize the catalytic properties of the DHAP enzyme homologs from pathogenic species in the metabolism of 5’-methylthioadenosine and 5’-deoxyadenosine. Ultimately, these two projects investigating the regulation and characterization of DHAP shunt pathways will enhance our understanding of the physiological role of the DHAP shunt pathway in diverse bacteria species.