Circadian clock proteins KaiB and Rbp2 of Synechococcus elongatus display oscillations in their subcellular localization patterns.

Circadian rhythms generated by molecular clocks time a diverse array of physiological cycles in many organisms. Cyanobacteria are currently the only prokaryotic system with a robust and rigorously tested circadian clock, which is carried out by KaiA, KaiB, and KaiC core oscillator proteins. This KaiABC oscillator drives global rhythms in gene expression, compaction of the chromosome, natural transformation, and timing of cell division in the model organism Synechococcus elongatus PCC 7942. The oscillator proteins have been previously shown to undergo changes in their subcellular localization patterns, where KaiA and KaiC are diffuse throughout the cell during the day and localize to or near one pole of the cell at night. However, functional fluorescent fusions of KaiB to track subcellular localization could not be obtained. More recently, we have described the identification of Rbp2 as a member of the extended clock network that associates with KaiC in a localized state. However, the subcellular localization patterns of KaiB and Rbp2 remain uncharacterized, and the mechanism driving the dynamic localization of circadian proteins remains undescribed. Here, we describe the patterns of KaiB and Rbp2 subcellular localization over the course of the day. Specifically, we demonstrate that KaiB and Rbp2 form polar foci and co-localize with KaiC at night using both traditional fusions to fluorescent proteins and endogenously expressed antibodies. We show that the RNA-binding activity of Rbp2 is necessary for the robust formation of Rbp2 foci. We propose a model by which Rbp2 drives circadian protein localization via its RNA binding activity.IMPORTANCECircadian rhythms driven by a circadian clock are required to time a wide range of physiological and metabolic processes. In cyanobacteria, the circadian clock is required for optimal fitness in environmental cycles. Where the clock proteins are located within the cell and how this helps them to synchronize with the environment and time rhythmic behaviors is incompletely understood. Here, we demonstrate that recently identified extended clock protein Rbp2 and core oscillator protein KaiB localize to the cell poles at night and co-localize with other members of the clock complex. We also demonstrate that the RNA binding activity of Rbp2 is necessary for the polar localization of Rbp2. Moreover, the use of endogenously expressed antibodies introduces a new and robust method for imaging challenging proteins in bacteria that is applicable across various fields.