Functional diversification of two Lon homologs enhances stress adaptation in Pseudomonas aeruginosa
TL;DR
It is shown that AsrA has evolved distinct substrate preferences and stress-responsive functions while retaining substantial functional overlap with Lon, demonstrating how duplication of a conserved protease can generate specialized regulatory functions through differential expression and substrate preference, expanding the proteolytic network that enables bacterial adaptation to stress.
Abstract
The Lon protease is a highly conserved ATP-dependent protease that contributes to protein quality control and regulatory processes across all domains of life. The opportunistic pathogen Pseudomonas aeruginosa, along with other members of the Pseudomonadales, encodes AsrA (aminoglycoside-induced stress response ATP-dependent protease), a second LonA-type protease in addition to canonical Lon. Although AsrA is upregulated by aminoglycoside stress, its biochemical activity, substrate spectrum, and cellular functions have remained elusive. Here, we demonstrate that AsrA is a temperature- and antimicrobial stress-induced protease with specialized functions and partial redundancy with Lon. Quantitative proteomics revealed a distinct AsrA substrate profile, while comparative biochemical analyses showed that AsrA and Lon share a substantial number of substrates in vitro but differ in their degradation kinetics, indicating divergent substrate preferences. Among the proteins preferentially degraded by AsrA is the quorum-sensing anti-activator QslA, and we show that AsrA-dependent QslA degradation under tobramycin stress induces quorum-sensing gene expression. Together, our findings demonstrate how duplication of a conserved protease can generate specialized regulatory functions through differential expression and substrate preference, expanding the proteolytic network that enables bacterial adaptation to stress. Significance Proteolysis is central to bacterial stress adaptation, but how expansion of protease families generates new regulatory functions remains poorly understood. The opportunistic pathogen Pseudomonas aeruginosa encodes two homologous Lon proteases, Lon and AsrA. We show that AsrA has evolved distinct substrate preferences and stress-responsive functions while retaining substantial functional overlap with Lon. Notably, AsrA directly degrades the quorum-sensing anti-activator QslA during aminoglycoside stress, revealing a previously unrecognized connection between stress-induced proteolysis and quorum-sensing regulation. Our findings demonstrate how duplication and functional diversification of homologous proteases can expand bacterial regulatory capacity and promote adaptation to environmental stress. This work provides a framework for understanding how proteolytic network diversification contributes to bacterial adaptation.