Redox crisis underlies conditional light–dark lethality in cyanobacterial mutants that lack the circadian regulator, RpaA

Significance

The evolution of photosynthetic cyanobacteria under 24-h cycles of light and darkness selected for a robust circadian clock. Understanding how cyanobacteria integrate circadian clock signals with natural light–dark cycles to control metabolism is critical, because these organisms are central to global carbon cycling and hold promise for development of renewable energy. Here we assess how the circadian transcription factor regulator of phycobilisome association A (RpaA) influences metabolism as a cyanobacterium goes through a light-to-dark transition. The data show that RpaA plays a key role in maintaining metabolic stability during the night period. Additionally, RpaA is important in controlling redox balance, which in turn is very important for regulating metabolism at night.

Abstract

Cyanobacteria evolved a robust circadian clock, which has a profound influence on fitness and metabolism under daily light–dark (LD) cycles. In the model cyanobacterium Synechococcus elongatus PCC 7942, a functional clock is not required for diurnal growth, but mutants defective for the response regulator that mediates transcriptional rhythms in the wild-type, regulator of phycobilisome association A (RpaA), cannot be cultured under LD conditions. We found that rpaA-null mutants are inviable after several hours in the dark and compared the metabolomes of wild-type and rpaA-null strains to identify the source of lethality. Here, we show that the wild-type metabolome is very stable throughout the night, and this stability is lost in the absence of RpaA. Additionally, an rpaA mutant accumulates excessive reactive oxygen species (ROS) during the day and is unable to clear it during the night. The rpaA-null metabolome indicates that these cells are reductant-starved in the dark, likely because enzymes of the primary nighttime NADPH-producing pathway are direct targets of RpaA. Because NADPH is required for processes that detoxify ROS, conditional LD lethality likely results from inability of the mutant to activate reductant-requiring pathways that detoxify ROS when photosynthesis is not active. We identified second-site mutations and growth conditions that suppress LD lethality in the mutant background that support these conclusions. These results provide a mechanistic explanation as to why rpaA-null mutants die in the dark, further connect the clock to metabolism under diurnal growth, and indicate that RpaA likely has important unidentified functions during the day.