Transposon mutagenesis used to observe factors essential to growth using 5-deoxy-D-ribose
Loading...
Date
2025-05
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
The Ohio State University
Abstract
S-adenosyl-L-methionine (SAM) is a compound used by all organisms for various cellular processes. When SAM undergoes radical SAM reactions, a deoxynucleoside known as 5’-deoxyadenosine (5dAdo) is produced. SAM may also be converted into another nucleoside known as 5’ methylthioadenosine (MTA), which is sulfur-containing. In Extraintestinal Pathogenic E. coli (ExPEC), MTA and 5dAdo are further used by the cell as a carbon source through a metabolic pathway called the dihydroxyacetone phosphate (DHAP) shunt. By studying which genes are required for the use of 5dAdo and MTA for growth by ExPEC strains we can gain a new perspective on how bacteria can grow and proliferate in a variety of different environments. Initially, it was observed that the byproducts of SAM metabolism, MTA and 5dAdo, were metabolized to the precursor metabolite DHAP by the DHAP shunt. What was unknown was if any other factors affected the cell’s ability to use the DHAP shunt for growth. Building a library of isolates using random transposon mutagenesis allowed us to scan the E. coli genome and identify these factors. The pRL27 plasmid vector containing the Tn5 transposable element made this study possible. Using certain selectivity elements of the transposon such as Kanamycin resistance and pir-dependent replication site allowed us to keep track of the Tn5 element. We were able to isolate a total of 36 isolates that showed significant differences in growth patterns on glucose versus 5-deoxyribose, a metabolite of 5dAdo metabolism in E. coli. Of these isolates, the majority of them had Tn5 insertions in genes relating to transporters and lipopolysaccharide synthesis. This transposon mutagenesis library will show us what other genes are required for use of the DHAP shunt for growth. In the future, these results may help researchers and medical professionals understand how ExPEC can utilize the DHAP shunt as a carbon salvage pathway to survive in various areas of the nutrient-scarce environments in the body.
Description
Keywords
Carbon salvaging in Extraintestinal Pathogenic Escerichia coli, Dihydroxyacetone Phosphate Shunt