ATP-responsive biomolecular condensates tune bacterial kinase signaling

Abstract

Maintenance of robust biochemical signaling is essential for oligotrophic bacteria that survive under fluctuating environmental challenges. We report that a polar microdomain in Caulobacter crescentus tunes a critical signaling pathway to nutrient availability using the hydrotrope effect of ATP on an intracellular biomolecular condensate. Sequestration of this signaling pathway within a specialized polar microdomain formed by two interacting membraneless organelles, stimulates the activity of a localized kinase, which is dependent on the phase transition parameters of a disordered protein component of the microdomain. Under low ATP concentrations, phase separation in the microdomain is reinforced, enhancing kinase activity, and thereby conferring control of a critical cell cycle signaling pathway to the physical properties of a unique system of membraneless organelles.

The emergence of phase separation as an organizing principle in bacteria

“…At the stalked pole, an integral membrane protein, SpmX, interacts with PopZ and localizes the kinase DivJ, thus generating another asymmetry in the cell(43,44). A recent study by Saurabh and colleagues used in vitro reconstitution in solution and on supported lipid bilayers to show that both PopZ and SpmX form condensates via LLPS(45). Furthermore, after heterologous expression of PopZ and SpmX in E. coli, it was found that diffusion of these proteins within the condensates in living cells could be modulated by changing any parameter that affects protein self-interaction (pH, osmolarity, crowding, etc.).…”

Regulation of a bacterial histidine kinase by a phase separating scaffolding protein

“…To determine the role of allostery, we found that a functional PAS sensor transmission motif was required for stimulation by PodJ (Fig. 3 Collectively these three studies suggest strategies that concentrate histidine kinases, such as phase separation, may have a generalizable effect of robustly stimulating low copy histidine kinases 9,10 .…”

“…More broadly, phase separation provides an accessible compartmentalization strategy for bacterial kingdom 41 . The three scaffolding proteins, PopZ 8 , SpmX 9 , and PodJ (Fig. 4-5), phase separate as two distinct membraneless organelles at opposite ends of the cell.…”

“…S9A). Moreover, adenosine triphosphate (ATP) has been shown to act as a biological hydrotrope that dissolves liquidliquid phase separation (LLPS) assemblies 35,9 . Similarly, we observed that the addition of 10-20 mM ATP or ADP led to a reduction in droplet size, partitioning ratio and PleC-mCherry recruitment (Fig.…”

“…Three scaffolds play roles in regulating the subcellular position of histidine kinases involved in the asymmetric cell division of the gram-negative bacteria Caulobacter crescentus: PopZ-CckA 4 , SpmX-DivJ 5 , and PodJ-PleC 6,7 . PopZ 8 and SpmX 9 phase separate as a biomolecular condensate that recruits distinct clients, including the histidine kinase DivJ and CckA, and the pseudokinase DivL at the old cell pole 4 . At the opposite new cell pole, PodJ serves as a scaffold that sequesters four distinct clients directly or indirectly: PleC, PopA, DivL and CpaE 7 .…”