The ATP-dependent cytoplasmic protease Lon has critical functions in protein quality control and cellular regulation in organisms across the three domains of life. In the opportunistic pathogen Pseudomonas aeruginosa, lon loss-of-function mutants exhibit multiple phenotypic defects in motility, virulence, antibiotic tolerance and biofilm formation. However, only a couple of native substrate proteins of Lon are described in P. aeruginosa until now and most of the phenotypes associated with Lon remain unexplained. Here, we searched for novel Lon substrates in P. aeruginosa by analyzing proteome-wide changes in protein levels and stabilities following lon overexpression. Our search yielded a large number of putative Lon substrates with diverse cellular functions, including metabolic enzymes, stress proteins and a significant fraction of motility-related proteins. In vitro degradation assays confirmed the metabolic protein SpeH, the heat shock protein IbpA as well as seven proteins involved in flagella- and type IV pilus-mediated motility as novel substrates of Lon. The new motility-associated substrates include both key regulators of motility (FliA, RpoN, AmrZ) as well as structural flagellar components (FliG, FliS and FlgE). Further, by isolating suppressor mutations bypassing the motility defect of lon- cells, we reveal that Lon-dependent degradation of the specific substrate SulA, a cell division inhibitor, is crucial for ensuring proper cell division and motility under optimal conditions. In sum, our work highlights Lon’s regulatory role in degrading functional proteins involved in critical cellular processes and contributes to a better molecular understanding of the pathways underlying Pseudomonas pathogenicity.

The Lon protease is a highly conserved protein degradation machine that contributes to protein quality control and regulatory processes in species from the three domains of life. The opportunistic pathogen Pseudomonas aeruginosa as well as a group of related bacteria possess, in addition to the canonical Lon protease, a second Lon protease named AsrA (Aminoglycoside-induced Stress Response ATP-dependent protease). While this AsrA protease was shown to be upregulated under aminoglycoside stress, it remains poorly studied regarding its biochemical activity, its specific substrates and cellular roles. Here, we show that AsrA is an active ATP-dependent protease that, despite structural and biochemical similarities to Lon, regulates its own group of substrates and possesses distinct cellular roles. Using a proteome-wide screen for substrate proteins, we identified the first specific substrates of this protease. One of these substrates is the anti-activator QslA, a critical regulator of the quorum sensing cascade, and we demonstrate that AsrA controls quorum sensing genes via this regulator. We further reveal a small protein Icp as a potential regulator of AsrA activity and we show that survival of an asrA- mutant strain is significantly impaired under heat-shock conditions. Together, our study characterizes AsrA as a novel type of Lon-like protease and reveals this protease as a critical regulator of quorum sensing and stress survival.

The Lon protease is a highly conserved protein degradation machine that has critical regulatory and protein quality control functions in cells from the three domains of life. Here, we report the discovery of a α-proteobacterial heat shock protein, LarA, that functions as a dedicated Lon regulator. We show that LarA accumulates at the onset of proteotoxic stress and allosterically activates Lon-catalysed degradation of a large group of substrates through a five amino acid sequence at its C-terminus. Further, we find that high levels of LarA cause growth inhibition in a Lon-dependent manner and that Lon-mediated degradation of LarA itself ensures low LarA levels in the absence of stress. We suggest that the temporal LarA-dependent activation of Lon helps to meet an increased proteolysis demand in response to protein unfolding stress. Our study defines a regulatory interaction of a conserved protease with a heat shock protein, serving as a paradigm of how protease activity can be tuned under changing environmental conditions.