In-depth characterization of the metabolic effect of the bacterial alamorne ppGpp

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Project Summary Bacterial cells are extremely efficient in adapting to environmental stresses. As a prime example, they synthesize a second messenger called ppGpp in response to starvation. Accumulation of ppGpp in bacteria arrests growth and reprograms cellular physiology to promote survival. These effects of ppGpp is required for antibiotic persistence that allows bacteria to survive the treatment of antibiotics to which they do not encode genetic resistance. At molecular levels, ppGpp reprograms gene expression, and targets many other proteins involved in translation and small-molecule metabolism. As a preliminary study, I synthesized a photo-crosslinking probe of ppGpp and used this probe to capture and identify about 30 new ppGpp-binding proteins in E. coli. Recently, a ppGpp riboswitch has been discovered in Gram-positive organisms. Intriguingly, E. coli transcriptome has a rich secondary-structure landscape, but the capability of these secondary structures in binding to small-molecule metabolite has not been explored. Therefore, in Aim 1, I will use a photo-crosslinking approach to capture binding partners of ppGpp from E. coli transcriptome, and identify these transcripts using RNA-seq. Hit interactions will be validated biophysically, and their putative effects on translation efficiency or the transcript stability will be examined in vivo. Additionally, in my preliminary study, I also compared metabolite profiles in E. coli before and shortly after ppGpp induction, and found strong perturbation of dozens of essential metabolites. This perturbation of cellular metabolism goes much beyond known direct effects of ppGpp. It is unclear how ppGpp induction drives the inhibition of various metabolic pathways to effectively arrest bacterial growth and promote persistence. In Aim 2, I hypothesize that “ppGpp-sensitive” metabolites, i. e., those whose levels perturbed by ppGpp induction, serve as a proxy of ppGpp to regulate cellular metabolism. I will seek discovering protein-metabolite interactions responsible for this indirect effect of ppGpp. To this end, I will use time-resolved metabolomics to identify candidate metabolites and enzymes whose levels/activities perturbed contemporaneously with ppGpp accumulation. I will then screen for protein-metabolite interactions among these candidates using an ultrafiltration-based assay. Briefly, I will purify each protein of interest (POI) to high homogeneity. Then, I will subject a mixture containing a library of all candidate metabolites and a single POI to ultrafiltration. Analyzing the filtrate using MS should reveal a decrease of the POI’s cognate ligands. I will validate any novel interactions identified using biochemical, structural, and genetic approaches. Together, the proposed research will explore two new aspects of ppGpp signaling, namely RNA targeting and indirect effects on cellular metabolism via ppGpp-sensitive metabolites. This study may lead to the discovery of key regulatory interactions required for bacterial persistence, and these interactions may serve as targets for anti-persistence drug design.