Abstract

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.