Translational profiling of stress-induced small proteins uncovers an unexpected connection among distinct signaling systems

Summary Signaling networks in bacteria enable sensing and adaptation to challenging environments by activating specific genes that help counteract stressors. Small proteins (≤ 50 amino acids long) are a rising class of bacterial stress response regulators. Escherichia coli encodes over 150 small proteins, most of which lack known phenotypes and their biological roles remain elusive. Using magnesium limitation as a stressor, we investigate small proteins induced in response to stress using ribosome profiling, RNA sequencing, and transcriptional reporter assays. We uncover 17 small proteins with increased translation initiation, a majority of which are transcriptionally upregulated by the PhoQ-PhoP two-component signaling system, crucial for magnesium homeostasis. Next, we describe small protein-specific deletion and overexpression phenotypes, which underscore the physiological significance of their expression in low magnesium stress. Most remarkably, our study reveals that a small membrane protein YoaI is an unusual connector of the major signaling networks – PhoR-PhoB and EnvZ-OmpR in E. coli , advancing our understanding of small protein regulators of cellular signaling. Highlights Ribo-RET identifies 17 small proteins induced under low Mg 2+ stress in E. coli Many of these proteins are transcriptionally activated by PhoQP signaling system Half of the stress-induced small proteins localize to the membrane Deletion or overexpression of specific small proteins affects growth under stress Small protein YoaI connects PhoR-PhoB and EnvZ-OmpR signaling networks Graphical abstract