To adapt to changing environments, bacteria have evolved numerous pathways that activate
stress response genes. In Gram-positive bacteria, the stressosome, a cytoplasmic complex,
relays external cues and activates the sigma B regulon. The stressosome is structurally
well-characterized in Bacillus, but how it senses stress remains elusive. Here, we
report a genome-wide N-terminomic approach in Listeria that strikingly led to the
discovery of 19 internal translation initiation sites and 6 miniproteins, among which
one, Prli42, is conserved in Firmicutes. Prli42 is membrane-anchored and interacts
with orthologues of Bacillus stressosome components. We reconstituted the Listeria
stressosome in vitro and visualized its supramolecular structure by electron microscopy.
Analysis of a series of Prli42 mutants demonstrated that Prli42 is important for sigma
B activation, bacterial growth following oxidative stress and for survival in macrophages.
Taken together, our N-terminonic approach unveiled Prli42 as a long-sought link between
stress and the stressosome.