Spatio-temporal coherence of circadian clocks and temporal control of differentiation in Anabaena filaments

Abstract

Circadian clock arrays in multicellular filaments of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 display remarkable spatio-temporal coherence under nitrogen-replete conditions. To shed light on the interplay between circadian clocks and the formation of developmental patterns, we followed the expression of a clock-controlled gene under nitrogen deprivation, at the level of individual cells. Our experiments showed that differentiation into heterocysts took place preferentially within a limited interval of the circadian clock cycle, that gene expression in different vegetative intervals along a developed filament was discoordinated, and that the circadian clock was active in individual heterocysts. Furthermore, Anabaena mutants lacking the kaiABC genes encoding the circadian clock core components produced heterocysts but failed in diazotrophy. Therefore, genes related to some aspect of nitrogen fixation, rather than early or mid-heterocyst differentiation genes, are likely affected by the absence of the clock. A bioinformatics analysis supports the notion that RpaA may play a role as master regulator of clock outputs in Anabaena , the temporal control of differentiation by the circadian clock and the involvement of the clock in proper diazotrophic growth. Together, these results suggest that under nitrogen-deficient conditions, the clock coherent unit in Anabaena is reduced from a full filament under nitrogen-rich conditions to the vegetative cell interval between heterocysts. IMPORTANCE Circadian clocks, from unicellular organisms to animals, temporally align biological processes to day and night cycles. We study the dynamics of a circadian clock-controlled gene at the individual cell level in the multicellular filamentous cyanobacterium Anabaena , under nitrogen-stress conditions. Under these conditions, some cells along filaments differentiate to carry out atmospheric nitrogen fixation and lose their capability for oxygenic photosynthesis. We found that clock synchronization is limited to organismic units of contiguous photosynthetic cells, contrary to nitrogen-replete conditions in which clocks are synchronized over a whole filament. We provided evidence that the circadian clock regulates the process of differentiation, allowing it to occur preferentially within a limited time window during the circadian clock period. Lastly, we present evidence that the signal from the core clock to clock-regulated genes is conveyed in Anabaena as in unicellular cyanobacteria.