Background: Phytochromes are red/far-red photoreceptors using a bilin chromophore. Results: Compared with Cph1, the Cph2 bilin-binding site differs around the propionates, but utilizes an otherwise conserved tongue for sealing the chromophore from solvent. Conclusion:The tongue signals via a tryptophan switch within the tongue-GAF domain interface. Significance: The first structure of a Cph2-type phytochrome indicates a common mechanism for photoswitching in all canonical phytochromes.

SummaryCph2 from the cyanobacterium Synechocystis sp. PCC 6803 is a hybrid photoreceptor that comprises an N-terminal module for red/far-red light reception and a C-terminal module switching between a blue-and a green-receptive state. This unusual photoreceptor exerts complex, light quality-dependent control of the motility of Synechocystis sp. PCC 6803 cells by inhibiting phototaxis towards blue light. Cph2 perceives blue light by its third GAF domain that bears all characteristics of a cyanobacteriochrome (CBCR) including photoconversion between green-and blueabsorbing states as well as formation of a bilin species simultaneously tethered to two cysteines, C994 and C1022. Upon blue light illumination the CBCR domain activates the subsequent C-terminal GGDEF domain, which catalyses formation of the second messenger c-di-GMP. Accordingly, expression of the CBCR-GGDEF module in Dcph2 mutant cells restores the blue light-dependent inhibition of motility. Additional expression of the N-terminal Cph2 fragment harbouring a red/far-red interconverting phytochrome fused to a c-di-GMP degrading EAL domain restores the complex behaviour of the intact Cph2 photosensor. c-di-GMP was shown to regulate flagellar and pilibased motility in several bacteria. Here we provide the first evidence that this universal bacterial second messenger is directly involved in the light-dependent regulation of cyanobacterial phototaxis.

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Cyanobacterial phytochromes are a diverse family of light receptors controlling various biological functions including phototaxis. In addition to canonical bona fide phytochromes of the well characterized Cph1/plant-like clade, cyanobacteria also harbor phytochromes that absorb green, violet or blue light. The Synechocystis PCC 6803 Cph2 photoreceptor, a phototaxis inhibitor, is unconventional in bearing two distinct chromophore-binding GAF domains. Whereas the C-terminal GAF domain is most likely involved in blue-light perception, the first two domains correspond to a Cph1-like photosensory module lacking the PAS domain. Biochemical and spectroscopic studies show that this region switches between red (P(r) ) and far-red (P(fr) ) absorbing states. Unlike Cph1, the P(fr) state of Cph2 decays rapidly in darkness. Mutations close to the PCB chromophore further destabilize the P(fr) state without drastically affecting the spectroscopic features such as the quantum efficiency of P(r) →P(fr) conversion, fluorescence, or the Resonance-Raman signature of the chromophore. Overall, the PAS-less photosensory module of Cph2 resembles Cph1 including its mode of isomerisation, but the P(fr) state is unstable.

Phosphorylation-dependent interactions play crucial regulatory roles in all domains of life. Forkhead-associated (FHA) and von Willebrand type A (vWA) domains are involved in several phosphorylation-dependent processes of multiprotein complex assemblies. Although well-studied in eukaryotes and bacteria, the structural and functional contexts of these domains are not yet understood in Archaea. Here, we report the structural base for such an interacting pair of FHA and vWA domain-containing proteins, ArnA and ArnB, in the thermoacidophilic archaeon Sulfolobus acidocaldarius , where they act synergistically and negatively modulate motility. The structure of the FHA domain of ArnA at 1.75 Å resolution revealed that it belongs to the subclass of FHA domains, which recognizes double-pSer/pThr motifs. We also solved the 1.5 Å resolution crystal structure of the ArnB paralog vWA2, disclosing a complex topology comprising the vWA domain, a β-sandwich fold, and a C-terminal helix bundle. We further show that ArnA binds to the C terminus of ArnB, which harbors all the phosphorylation sites identified to date and is important for the function of ArnB in archaellum regulation. We also observed that expression levels of the archaellum components in response to changes in nutrient conditions are independent of changes in ArnA and ArnB levels and that a strong interaction between ArnA and ArnB observed during growth on rich medium sequentially diminishes after nutrient limitation. In summary, our findings unravel the structural features in ArnA and ArnB important for their interaction and functional archaellum expression and reveal how nutrient conditions affect this interaction.