Determining effect of phosphorylation on global regulators in E. coli

During typical growth, a bacterial culture transitions from low cell density through a phase of rapid cell division and exponential doubling before entering stationary phase. The transition to stationary phase requires numerous physiological adaptations, including responses to reduced nutrient availability, accumulation of waste products, depletion of cofactors, and remodelling of the nucleoid. This process involves extensive changes in gene expression at the transcriptional, post-transcriptional, and post translationallevels.

Recent advances in bacterial phosphoproteomics suggest that some of these regulatory shifts may be mediated by protein phosphorylation—particularly on global transcriptional regulators and nucleoid-associated proteins. However, the exact cellular mechanisms governing these processes, as well as the functional impact of individual phosphosites, remain largely unknown.

In this project, the student will focus on two key bacterial regulators, Dps and Lrp, which play central roles during the transition into stationary phase. Both proteins contain phosphosites located in functionally important regions that influence their DNA-binding activity or degradation.

The project will involve investigating the impact of phosphorylation on their DNA-binding properties in vitro by expressing phosphorylated protein variants using genetic code expansion methods and performing DNA binding assays. The student will then learn how to generate genomic mutants in E. coli and use these strains to assess the in vivo effects of the respective phosphosites. Finally, the project will include examining the impact of phosphorylation on Dps degradation using standard protein analysis techniques, including Western blotting, while Lrp may be further investigated using more comprehensive in vitro DNA-binding assays and next-generation sequencing–based approaches.

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