Scaffold-Scaffold Interaction Facilitates Cell Polarity Development in Caulobacter crescentus.

Cell polarity development is the prerequisite for cell differentiation and generating biodiversity. In the model bacterium Caulobacter crescentus, the polarization of the scaffold protein PopZ during the predivisional cell stage plays a central role in asymmetric cell division. However, our understanding of the spatiotemporal regulation of PopZ localization remains incomplete. In the current study, a direct interaction between PopZ and the new pole scaffold PodJ is revealed, which plays a primary role in triggering the new pole accumulation of PopZ. The coiled-coil 4-6 domain in PodJ is responsible for interacting with PopZ in vitro and promoting PopZ transition from monopolar to bipolar in vivo. Elimination of the PodJ-PopZ interaction impairs the PopZ-mediated chromosome segregation by affecting both the positioning and partitioning of the ParB-parS centromere. Further analyses of PodJ and PopZ from other bacterial species indicate this scaffold-scaffold interaction may represent a widespread strategy for spatiotemporal regulation of cell polarity in bacteria. IMPORTANCE Caulobacter crescentus is a well-established bacterial model to study asymmetric cell division for decades. During cell development, the polarization of scaffold protein PopZ from monopolar to bipolar plays a central role in C. crescentus asymmetric cell division. Nevertheless, the spatiotemporal regulation of PopZ has remained unclear. Here, we demonstrate that the new pole scaffold PodJ functions as a regulator in triggering PopZ bipolarization. The primary regulatory role of PodJ was demonstrated in parallel by comparing it with other known PopZ regulators, such as ZitP and TipN. Physical interaction between PopZ and PodJ ensures the timely accumulation of PopZ at the new cell pole and the inheritance of the polarity axis. Disruption of the PodJ-PopZ interaction impaired PopZ-mediated chromosome segregation and may lead to a decoupling of DNA replication from cell division during the cell cycle. Together, the scaffold-scaffold interaction may provide an underlying infrastructure for cell polarity development and asymmetric cell division.

Regulation of the activity of the bacterial histidine kinase PleC by the scaffolding protein PodJ.

Scaffolding proteins can customize the response of signaling networks to support cell development and behaviors. PleC is a bifunctional histidine kinase whose signaling activity coordinates asymmetric cell division to yield a motile swarmer cell and a stalked cell in the gram-negative bacterium Caulobacter crescentus. Past studies have shown that PleC's switch in activity from kinase to phosphatase correlates with a change in its subcellular localization pattern from diffuse to localized at the new cell pole. Here we investigated how the bacterial scaffolding protein PodJ regulates the subcellular positioning and activity of PleC. We reconstituted the PleC-PodJ signaling complex through both heterologous expressions in Escherichia coli and in vitro studies. In vitro, PodJ phase separates as a biomolecular condensate that recruits PleC and inhibits its kinase activity. We also constructed an in vivo PleC-CcaS chimeric histidine kinase reporter assay and demonstrated using this method that PodJ leverages its intrinsically disordered region to bind to PleC's PAS sensory domain and regulate PleC-CcaS signaling. Regulation of the PleC-CcaS was most robust when PodJ was concentrated at the cell poles and was dependent on the allosteric coupling between PleC-CcaS's PAS sensory domain and its downstream histidine kinase domain. In conclusion, our in vitro biochemical studies suggest that PodJ phase separation may be coupled to changes in PleC enzymatic function. We propose that this coupling of phase separation and allosteric regulation may be a generalizable phenomenon among enzymes associated with biomolecular condensates.

Design of a Histidine Kinase FRET Sensor to Detect Complex Signal Integration within Living Bacteria.

Histidine kinases (HK) switch between conformational states that promote kinase and phosphatase activities to regulate diverse cellular processes. Past studies have shown that these functional states can display heterogeneity between cells in microbial communities and can vary at the subcellular level. Methods to track and correlate the kinase conformational state with the phenotypic response of living bacteria cells will offer new opportunities to interrogate bacterial signaling mechanisms. As a proof of principle, we incorporated both mClover3 (donor) and mRuby3 (acceptor) fluorescent proteins into the Caulobacter crescentus cell-cycle HK CckA as an in vivo fluorescence resonance energy transfer (FRET) sensor to detect these structural changes. Our engineered FRET sensor was responsive to CckA-specific input signals and detected subcellular changes in CckA signal integration that occurs as cells develop. We demonstrated the potential of using the CckA FRET sensor as an in vivo screening tool for HK inhibitors. In summary, we have developed a new HK FRET sensor design strategy that can be adopted to monitor in vivo changes for interrogation of a broad range of signaling mechanisms in living bacteria.

Manipulation of Bacterial Signaling Using Engineered Histidine Kinases.

Two-component systems allow bacteria to respond to changes in environmental or cytosolic conditions through autophosphorylation of a histidine kinase (HK) and subsequent transfer of the phosphate group to its downstream cognate response regulator (RR). The RR then elicits a cellular response, commonly through regulation of transcription. Engineering two-component system signaling networks provides a strategy to study bacterial signaling mechanisms related to bacterial cell survival, symbiosis, and virulence, and to develop sensory devices in synthetic biology. Here we focus on the principles for engineering the HK to identify unknown signal inputs, test signal transmission mechanisms, design small molecule sensors, and rewire two-component signaling networks.