Translation profiling of stress-induced small proteins reveals a novel link among signaling systems

Signaling networks allow adaptation to stressful environments by activating genes that counteract stressors. Small proteins (≤ 50 amino acids long) are a rising class of stress response regulators. Escherichia coli encodes over 150 small proteins, most of which lack phenotypes and their biological roles remain elusive. Using magnesium limitation as a stressor, we identify stress-induced small proteins using ribosome profiling, RNA sequencing, and transcriptional reporter assays. We uncover 17 small proteins with increased translation initiation, several of them transcriptionally upregulated by the PhoQ-PhoP two-component signaling system, crucial for magnesium homeostasis. Next, we describe small protein-specific deletion and overexpression phenotypes, underscoring their physiological significance in low magnesium stress. Most remarkably, we elucidate an unusual connection via a small membrane protein YoaI, between major signaling networks - PhoR-PhoB and EnvZ-OmpR in E. coli, advancing our understanding of small protein regulators in cellular signaling.