Molecular mechanisms underlying polarity control and regulation of type IVa pilus activity during cyanobacterial phototaxis

Cyanobacteria are photosynthetic prokaryotes that display phototaxis, the ability to move toward or away from light, thereby optimizing their position in the environment. This behavior relies on integrating multiple light signals into the motility machinery and on the coordinated action of type IVa pili (T4aP). Although cyanobacterial phototaxis has been studied for decades, the molecular mechanisms that connect sensory inputs to T4aP activity and polarity are still not fully understood. This thesis explores these mechanisms in the model cyanobacterium Synechocystis sp. PCC 6803, focusing on three key aspects of phototaxis regulation: PatA-type response regulators (RR), the Tax1 chemotaxis-like system, and the circadian clock factor CikA. In the first part, the evolutionary origin, localization, and molecular function of PatA-type RRs were examined. These RRs contain an N-terminal PATAN (N-terminus of the Anabaena protein PatA) domain and a C-terminal receiver (REC) domain. Phylogenetic analyses showed that they evolved from cyanobacterial chemosensory systems. Fluorescence microscopy and yeast two-hybrid assays revealed that PatA-type RRs localize to the cytoplasmic membrane, where they interact specifically with the pilus assembly ATPase PilB1 and with the N-terminal cytoplasmic domain of the platform protein PilC. Importantly, truncated variants containing only the PATAN domain were sufficient to reverse phototactic orientation, indicating that this domain functions as the principal output module coupling sensory pathways to pilus extension. The second part of this work examined the molecular mechanism of the Tax1 chemotaxis- like system, which is one of the cyanobacterial chemosensory systems and controls positive phototaxis. In vitro phosphotransfer assays showed that the histidine kinase PixL phosphorylates two RRs: PixG (PatA-type) and PixH (CheY-type). Analysis of the localization and function of both wild-type and phosphorylation-deficient variants of PixG and PixH revealed that phosphorylated PixG interacts with PilB1 and accumulates at the leading pole of moving cells to promote pilus assembly and forward movement. Conversely, PixH acts as a negative factor of PixG phosphorylation, thereby counteracting PixG activity and inhibiting positive phototaxis. Additionally, gene mutation analysis indicated that the C-terminal REC domain of PixL is crucial for positive phototaxis, as it influences PixL kinase activity. This division of roles between PixG and PixH demonstrates how the Tax1 system uses two RRs to coordinate polarity and movement direction. Together, these results point to similarities in how phototactic and chemotactic bacteria regulate twitching motility, while also highlighting the main output role of PatA-type RR in light-dependent motility. The final section explores the role of the quinone-binding circadian clock factor CikA. Analyses of knockout mutants lacking different combinations of photoreceptors revealed that PixJ1, UirS, and PixD are all essential for switching between positive and negative phototaxis; however, none appears to function as the primary sensor of light direction. Instead, the findings suggest additional signaling inputs that may come from the photosynthetic electron transport chain. Supporting this, the Synechocystis homolog of CikA was found to interact directly with PilB1 and PilC. These results suggest that CikA might detect plastoquinone redox signals and relay them to the motility apparatus, potentially providing a link between photosynthetic activity, circadian regulation, and phototaxis. In summary, this thesis provides new insight into how phototaxis is regulated in Synechocystis. It identifies the PATAN domain as a key output module, explains the roles of two RRs within the Tax1 system, and demonstrates that CikA connects photosynthetic signals to the T4aP activity. Since PATAN domains frequently co-occur with chemotaxis-like signaling systems, these findings not only clarify cyanobacterial phototaxis but also suggest broader principles of signal transduction and polarity regulation in prokaryotes.