Detection and characterization of cyclic di-GMP-binding proteins

Nucleotide signaling plays an important role in the adjustment of microbial physiology and metabolism. These signaling systems are involved in the regulation of all physiological processes including biofilm formation, virulence, environmental survival, and phage defense. One of the most prominent nucleotide-based second messenger molecules is the ubiquitous second messenger cyclic di-GMP. Cyclic di‐GMP synthesizing and hydrolyzing proteins, GGDEF and EAL domain proteins, respectively, are widely present across bacterial phyla, numerous within genomes, and highly conserved throughout the phylogenetic tree. However, there is still limited understanding of c-di-GMP binding receptors, since they are diverse, not necessarily characterized by common signature binding motifs, and still require experimentation to be identified.Having established a novel screen for cyclic di-GMP binding proteins based on alterations in thermal stability (PISA), this project aims to identify and characterize novel c-di-GMP binding receptors of the gastrointestinal pathogen Salmonella typhimurium as a model organism. Candidate c-di-GMP binding proteins will be verified by alternative approaches. Verified receptors will be assessed using microbiology, biochemistry, molecular biology, bioinformatics, and genetic engineering approaches. While new cyclic di-GMP binding proteins will be predominantly characterized in Salmonella typhimurium, novel and improved screens for cyclic di-GMP binding proteins based on mass-spectrometry-based DRaCALA and isolation of cellular nanomachines like ribosomes will be developed and partially investigated as research resources. Successful conduction of this post-doctoral project will expand the number of c-di-GMP receptors, discover novel underlying mechanisms of regulation by c-di-GMP, and thus significantly improve our understanding of the multilayer regulation of microbial pathways that contribute to the exceptional adaptation and evolution of bacteria.