Analysis of stress-induced small proteins in <i>Escherichia coli</i> reveals that YoaI mediates cross-talk between distinct signaling systems

Bacterial small proteins (≤50 amino acids) are an emerging class of regulators that modulate the activity of signaling networks that enable bacterial adaptation to stress. The <i>Escherichia coli</i> genome encodes at least 150 small proteins, most of which are functionally uncharacterized. We identified and characterized 17 small proteins induced in <i>E</i>. <i>coli</i> during magnesium (Mg²⁺) starvation using ribosome profiling, RNA sequencing, and transcriptional reporter assays. Several of these were transcriptionally activated by the PhoQ-PhoP two-component signaling system, which is crucial for Mg²⁺ homeostasis. Deletion or overexpression of some of these small proteins led to growth defects and changes in cell size under low-Mg²⁺ conditions, indicating physiological roles in stress adaptation. The small transmembrane protein YoaI, which was transcriptionally induced by the phosphate-responsive PhoR-PhoB signaling pathway, increased in abundance under Mg²⁺ limitation independently of <i>yoaI</i> transcription or PhoQ-PhoP signaling. YoaI activated a third signaling system, EnvZ-OmpR, which mediates responses to osmotic stress. Overall, this study establishes an initial framework for understanding how small proteins contribute to bacterial stress adaptation by facilitating cross-talk between different signaling systems. Our results suggest that these proteins play broader roles in coordinating stress responses, reflecting the interconnected nature of cellular stress networks rather than strictly compartmentalized pathways responding to specific stressors.