Bacterial ribonucleoprotein bodies maintain an acidic pH environment as a mechanism of enzyme regulation.

Biomolecular condensates create distinct subcellular niches, but their chemical environments and impacts on clients remain poorly understood. Using ratiometric fluorescent probes in vivo, we find that bacterial ribonucleoprotein bodies (BR-bodies) exhibit an acidic dense phase (pH ~5.1) due to the scaffold's low pI and negative charge. Single-molecule localization microscopy and fluorescence lifetime imaging reveal spatially variable, acidic nanoscale RNase E clusters in Caulobacter crescentus BR-bodies. Reducing RNase E's negative charge yields more neutral BR-bodies, causing morphological defects and viability loss upon outgrowth from stationary phase, likely reflecting altered pH and reduced condensation. In vitro assays with C-SNARF-4F and RNase E CTD-pHluorin2 recapitulate this acidic gradient and show that the acidic microenvironment enhances PNPase activity (optimal pH 5.3-6.3). Moreover, stationary-phase BR-bodies average just below this pH at 4.8 ± 0.2, a feature consistent with arrested RNA decay observed in vitro and stationary phase RNA storage in past work.