The signal processing capabilities of bacterial signaling networks offer immense potential for advanced phospho-signaling systems for synthetic biology. Emerging models suggest that complex development may require interconnections between what were once thought to be isolated signaling arrays. For example, Caulobacter crescentus achieves the feat of asymmetric division by utilizing a novel pseudokinase DivL, which senses the output of one signaling pathway to modulate a second pathway. It has been proposed that DivL reverses signal flow by exploiting conserved kinase conformational changes and protein−protein interactions. We engineered a series of DivL-based modulators to synthetically stimulate reverse signaling of the network in vivo. Stimulation of conformational changes through the DivL signal transmission helix resulted in changes to hallmark features of the network: C. crescentus motility and DivL accumulation at the cell poles. Additionally, mutations to a conserved PAS sensor transmission motif disrupted reverse signaling flow in vivo. We propose that synthetic stimulation and sensor disruption provide strategies to define signaling circuit organization principles for the rational design and validation of synthetic pathways.

Bacteria respond to environmental and cellular cues both through isolated signaling events between one sensor histidine kinase and its response regulator, and through more interconnected arrays. Caulobacter crescentus achieves asymmetric division through a network of histidine kinases, and here we interrogate a novel DivL pseudokinase reverse signaling mechanism that enables productive cross-talk across the network. A leucine zipper fusion method was used to synthetically stimulate reverse signaling between the sensor and kinase domains and directly test if reverse signaling could modulate the signaling network in vivo. Stimulation of sensor-kinase helix conformational changes resulted in changes in C. crescentus motility and DivL accumulation at the cell poles. The repurposed roles of the sensor domain in these processes were evaluated. We demonstrate that a domain of unknown function that binds to two scaffolding proteins, and two conserved signaling domains are employed as modulators of an active kinase. We propose that reversed signaling may be widely used across signaling enzymes.