The Regulation, Characterization and Supporting Factors of the Dihydroxyacetone Phosphate Shunt in Extraintestinal Pathogenic E. coli
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Date
2024-12
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The Ohio State University
Abstract
Extraintestinal pathogenic E. coli (ExPEC) are Gram-negative bacteria that possess the ability to metabolize carbon sources other than glucose. Approximately 42% of sequenced ExPEC isolates have a metabolic pathway called the dihydroxyacetone phosphate (DHAP) shunt which allows them to utilize 5’-deoxynucleosides and 5-deoxypentoses as growth substrates. These include 5’-deoxyadenosine (5’dAdo), 5-deoxy-D-ribose (5dR), 5’-methylthioadenosine (MTA), and 5-methylthio-D-ribose (MTR), which can serve as sole carbon sources for growth.11 In commensal E. coli and ExPEC strains that do not possess the DHAP shunt, 5’dAdo and MTA are initially cleaved by Pfs, a conserved MTA/5’dAdo nucleosidase to salvage the adenine and form 5dR and MTR, respectively, which are excreted from the cell. However, in ExPEC strains that possess the DHAP shunt, 5dR and MTR are further metabolized to DHAP, allowing ExPEC to use these sugars in central carbon metabolism as growth substrates. To identify conditions under which the DHAP Shunt genes are expressed for use of 5dR as a growth substrate, and to identify the promoter region for the DHAP shunt gene cluster and conditions for which the DHAP shunt genes are expressed, ExPEC strain ATCC 25922 was transformed with reporter plasmids containing different DNA fragment lengths of the upstream region from the DHAP shunt gene cluster fused to the lacZ gene. Subsequent LacZ activity assays showed that expression of the DHAP shunt is repressed by glucose and activated by the presence of 5dR, and that the promoter region for the DHAP shunt genes appear to be located within ~125 nucleotides immediately upstream of the first DHAP shunt gene. Additionally, a random Tn5 transposon mutagenesis library was conducted using the ExPEC strain ATCC 25922 in order to further study the DHAP shunt and identify any other genes that are required for E. coli to grow using 5dR as the sole carbon source. Results showed that mutations in genes related to the lipopolysaccharide layer, the DHAP shunt, and several other genes annotated as transporters and transcriptional regulators reduce or prevent growth on 5dR. Future studies will seek to confirm the location of promoter regions and other regulatory factors related to the DHAP shunt gene cluster as well as examine the ability of ExPEC strains to grow with other non-glucose carbon sources.
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Keywords
Microbiology, Dihydroxyacetone Phosphate Shunt, Escherichia Coli, Bacteria, Biology