Omega stabilizes RNA polymerase condensates and contributes to cellular fitness during acid stress.

In eukaryotes, organization of the nucleolus is tightly linked to ribosomal RNA (rRNA) synthesis. External stresses, such as heat shock and acidosis, induce a transition of the nucleolus from a liquid- to solid-like state. Bacterial RNA polymerase (RNAP) condensates share similarities with the nucleolus as they colocalize with pre-rRNA synthesis and their assembly correlates with high rRNA synthesis. In addition, their organization is growth dependent, highlighted by dissolution upon nutritional stress. However, their behavior and biophysical properties during other stresses remain unknown. Here, we find that RNAP condensates persist during acid stress despite an arrest in cell growth. In contrast to fast-growth RNAP condensates, acid-stabilized RNAP condensates become insensitive to drug treatments, suggesting a change in their dynamics. We identify both passive and active mechanisms that contribute to the maintenance of RNAP condensates during acid stress: a drop in intracellular pH and the stringent response. Specifically, the omega subunit of RNAP, which contributes to (p)ppGpp binding site 1, is critical for condensate maintenance during acid stress. In contrast, we show that DksA, a major stress regulator that binds to RNAP, does not contribute to RNAP condensate stabilization. Interestingly, we find that maintenance of RNAP condensates correlates with survival during recovery from acid stress. Our work sheds light on a new aspect of bacterial stress tolerance through regulation of RNAP codensates and a new role for the omega subunit. Our data bring RNAP condensates conceptually one step closer to nucleoli.