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

Schnorr, W. E.; Sasazawa, M.; Dzurik, K. G.; Cho, R.; Millheim, S. L.; Millstone, J. E.; Saurabh, S.; Childers, W. S.

doi:10.1101/2025.06.27.661991

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bioRxiv · 10.1101/2025.06.27.661991

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

Schnorr, W. E.·Sasazawa, M.·Dzurik, K. G.·Cho, R.·Millheim, S. L.·Millstone, J. E.·Saurabh, S.·Childers, W. S.

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Abstract

Phase separated biomolecular condensates create subcellular niches, yet their role in client regulation remains unclear. Here, we demonstrate that Bacterial Ribonucleoprotein bodies (BR-bodies) are acidic. Using ratiometric fluorescent probes in vivo, we find BR-bodies exhibit a dense-phase pH of [~]5.1, significantly lower than the near-neutral cytoplasm. Single-molecule localization microscopy and fluorescence lifetime imaging reveals that Caulobacter crescentus BR-bodies have spatially variable and acidic nanoscale RNase E clusters. These results question the notion of homogeneous condensates, suggesting that BR-bodies exhibit structural and biochemical diversity, which may facilitate RNA processing under stress. In vitro, pH gradients observed with C-SNARF-4F and RNase E CTD-pHluorin2 deteriorate with increasing buffer concentrations. Notably, the acidic microenvironment within BR-bodies enhances PNPase activity, highlighting the significance of condensate pH regulation. These findings suggest that pH modulation is intrinsic to condensates, directly influencing biochemical reactions and offering a new strategy for designing pH-sensitive drugs to target enzymes within condensates.

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Schnorr, W. E., Sasazawa, M., Dzurik, K. G., Cho, R., Millheim, S. L., Millstone, J. E., Saurabh, S., Childers, W. S.. 2025-06-29. Bacterial Ribonucleoprotein bodies maintain an acidic pH environment as a mechanism of enzyme regulation. https://doi.org/10.1101/2025.06.27.661991

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