Updated publication reference for PubMed record(s): 33320415. Using Mycoplasma pneumoniae as a model organism, we conditionally depleted the two essential ATP-dependent proteases (Lon and FtsH) of this bacterium, by engineering three strains carrying a Lon and/or FtsH inducible expression locus. An integrative comparative study combining label-free shotgun proteomics and RNA-seq allowed us to decipher the global cellular response to Lon and FtsH depletion and to define protease substrates in this genome-reduced organism.