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

Abstract

SummarySignaling networks in bacteria enable environmental sensing and adaptation to challenging conditions by activating specific genes that help counteract stressors. Small proteins (≤ 50 amino acids long) are a rising class of bacterial stress response regulators.Escherichia coliencodes 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 inE. coli, advancing our understanding of small protein regulators of cellular signaling.HighlightsRibo-RET identifies 17 small proteins induced under low Mg2+stress inE. coliMany of these proteins are transcriptionally activated by PhoQP signaling systemHalf of the stress-induced small proteins localize to the membraneDeletion or overexpression of specific small proteins affects growth under stressSmall protein YoaI connects PhoR-PhoB and EnvZ-OmpR signaling networksGraphical abstract