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

Bacteria utilize sophisticated mechanisms to detect and adapt to challenging environmental conditions by activating specific genes that help counteract stressors. Over 150 small proteins (≤ 50 amino acids long) are documented in Escherichia coli, but only a fraction of them are well-studied. Many of these proteins do not have an associated phenotype and their biological roles remain elusive. Here, we investigate small proteins induced in response to magnesium limitation 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, central for magnesium homeostasis. Next, we describe small protein-specific deletion and overexpression phenotypes, which underscore the significance of their expression to cellular physiology in low magnesium stress. Most remarkably, our study reveals the small protein YoaI as a connector of the major signaling networks – PhoR-PhoB and EnvZ-OmpR in E. coli. To investigate low magnesium stress induced small proteins in E. coli K-12 MG1655 strain, we utilized translation initiation profiling (Ribo-RET) and RNA-Seq under conditions of magnesium starvation and no stress. Code for processing the data can be found at https://github.com/yadavalli-lab/Small-proteins-induced-under-low-magnesium-stress