Genome-wide fitness assessment during diurnal growth reveals an expanded role of the cyanobacterial circadian clock protein KaiA

Abstract

The recurrent pattern of light and darkness generated by Earth's axial rotation has profoundly influenced the evolution of organisms, selecting for both biological mechanisms that respond acutely to environmental changes and circadian clocks that program physiology in anticipation of daily variations. The necessity to integrate environmental responsiveness and circadian programming is exemplified in photosynthetic organisms such as cyanobacteria, which depend on light-driven photochemical processes. The cyanobacterium PCC 7942 is an excellent model system for dissecting these entwined mechanisms. Its core circadian oscillator, consisting of three proteins, KaiA, KaiB, and KaiC, transmits time-of-day signals to clock-output proteins, which reciprocally regulate global transcription. Research performed under constant light facilitates analysis of intrinsic cycles separately from direct environmental responses but does not provide insight into how these regulatory systems are integrated during light-dark cycles. Thus, we sought to identify genes that are specifically necessary in a day-night environment. We screened a dense bar-coded transposon library in both continuous light and daily cycling conditions and compared the fitness consequences of loss of each nonessential gene in the genome. Although the clock itself is not essential for viability in light-dark cycles, the most detrimental mutations revealed by the screen were those that disrupt KaiA. The screen broadened our understanding of light-dark survival in photosynthetic organisms, identified unforeseen clock-protein interaction dynamics, and reinforced the role of the clock as a negative regulator of a nighttime metabolic program that is essential for to survive in the dark.

The circadian clock and darkness control natural competence in cyanobacteria

“…Sequencing reads of the barcodes were curated to keep only barcodes located within the middle 80% of each coding sequence; genes not represented by at least three bar codes in different positions or without at least 15 T 0 reads across replicates were removed from the analysis 26,53 . This curation resulted in 82,495 barcodes (also named strains) for the experimental conditions, 90,997 barcodes for the controls, and 99,872 barcodes for the T 0 samples distributed across 1,885 genes.…”

“…This curation resulted in 82,495 barcodes (also named strains) for the experimental conditions, 90,997 barcodes for the controls, and 99,872 barcodes for the T 0 samples distributed across 1,885 genes. For each gene, a fitness value that describes how the loss of function affects natural competence and a corresponding statistic were estimated in R as described previously 26,53 .…”

Pooled CRISPRi screening of the cyanobacteriumSynechocystissp. PCC 6803 for enhanced growth, tolerance, and chemical production

“…3 and 4). This is far fewer than in Synechococcus PCC 7942 where more than 100 genes were found with a transposon library to be important for LD growth 15 . The discrepancy could be attributed to the CRISPRi library comprising partial repressions and not gene knockouts, and that many of the genes with a fitness effect in LD are involved in alleviating light stresses experienced at dawn, making them also important for the L300 condition.…”

“…Inducible CRISPRi has been developed for several cyanobacteria strains 12,13 , but pooled sgRNA libraries have not been exploited. A transposon library in the cyanobacterium Synechococcus elongatus PCC 7942 (hereafter Synechococcus PCC 7942) was used to map essential genes in both photoautotrophic and diurnal growth 14,15 .…”

Glycogen metabolism jump-starts photosynthesis through the oxidative pentose phosphate pathway (OPPP) in cyanobacteria

“…On the other hand, the OPP pathway seems to become indispensable only after long period of dark (over 24 hours), while glycolysis might have been able to compensate for the need of reducing equivalent during short incubation in the dark(Scanlan et al, 1995). However, it is also important to know that OPP pathway is responsible for generating NADPH to serve as cofactors for certain ROS-detoxifying enzymes important for cyanobacterial survival (Welkie et al, 2018). In addition, cellular respiration is closely associated with photosynthesis.…”