Secreted retropepsin-like enzymes are essential for stress tolerance and biofilm formation in .

Proteases regulate important biological functions. Here, we present the structural and functional characterization of three previously uncharacterized aspartic proteases in . We show that these proteases have structural hallmarks of retropepsin peptidases and play redundant roles for cell survival under hypoosmotic stress conditions. Consequently, we named them retropepsin-like osmotic stress tolerance peptidases (Rlo). Our research shows that while Rlo proteases are homologous to RimB, an aspartic peptidase involved in rhizosphere colonization and plant infection, they contain N-terminal signal peptides and perform distinct biological functions. Mutants lacking all three secreted Rlo peptidases show defects in antibiotic resistance, biofilm formation, and cell morphology. These defects are rescued by mutations in the inactive transglutaminase transmembrane protein RloB and the cytoplasmic ATP-grasp protein RloC, two previously uncharacterized genes in the same operon as one of the Rlo proteases. These studies identify Rlo proteases and operon products as critical factors in clinically relevant processes, making them appealing targets for therapeutic strategies against infections.IMPORTANCEBacterial infections have become harder to treat due to the ability of pathogens to adapt to different environments and the rise of antimicrobial resistance. This has led to longer illnesses, increased medical costs, and higher mortality rates. The opportunistic pathogen is particularly problematic because of its inherent resistance to many antibiotics and its capacity to form biofilms, structures that allow bacteria to withstand hostile conditions. Our study uncovers a new class of retropepsin-like proteases in that are required for biofilm formation and bacterial survival under stress conditions, including antibiotic exposure. By identifying critical factors that determine bacterial fitness and adaptability, our research lays the foundation for developing new therapeutic strategies against bacterial infections.