Endogenous clock-mediated regulation of intracellular oxygen dynamics is essential for diazotrophic growth of unicellular cyanobacteria

Abstract

The discovery of nitrogen fixation in unicellular cyanobacteria provided the first clues for the existence of a circadian clock in prokaryotes. However, recalcitrance to genetic manipulation barred their use as model systems for deciphering the clock function. Here, we explore the circadian clock in the now genetically amenable Cyanothece 51142, a unicellular, nitrogen-fixing cyanobacterium. Unlike non-diazotrophic clock models, Cyanothece 51142 exhibits conspicuous self-sustained rhythms in various discernable phenotypes, offering a platform to directly study the effects of the clock on the physiology of an organism. Deletion of kaiA, an essential clock component in the cyanobacterial system, impacted the regulation of oxygen cycling and hindered nitrogenase activity. Our findings imply a role for the KaiA component of the clock in regulating the intracellular oxygen dynamics in unicellular diazotrophic cyanobacteria and suggest that its addition to the KaiBC clock was likely an adaptive strategy that ensured optimal nitrogen fixation as microbes evolved from an anaerobic to an aerobic atmosphere under nitrogen constraints.

Fig. 1. Growth comparison of the WT and ∆kaiA strains of Cyanothece 51142 in nitrogen-deficient media.

Self-sustained endogenous rhythms of ~24 h are reflected in growth under CL(a). Rhythm imposed by 12:12 LD cycles (b). The CL curve is an average of three independent runs of the WT and 5 independent runs of the mutant. The LD curve is an average of 3 independent runs of the WT and the mutant. Black bars represent dark phase of growth.

Fig. 2. Sustained endogenous rhythm of growth in Cyanothece 51142.

Representative curves showing a comparison of the endogenous rhythms of the WT and ∆kaiA strains grown under different LD regimes – 12:12 (a), 16:8 (b), 20:4 (c), and 8:16 (d) followed by CL growth. Representative growth curves from multiple runs of the WT and mutant are presented. The black bar represents a dark phase of growth.

Fig. 3. Comparison of nitrogenase activity in the WT and ∆kaiA strains of Cyanothece 51142.

The activity of samples grown under different light regimes [12:12 (a), 16:8 (b), 20:4 (c), CL (d)] is expressed as % WT activity under 12:12 LD growth. Error bars correspond to standard deviations from at least 3 biological replicates (12:12 LD, n = 4), (16:8 LD, n = 5) and (20:4 LD and CL, n = 3) The letters a, b denote statistically different values of μ for Air and c, d indicate statistical significance for Argon (p < 0.05), while same letters a or c indicate that the difference in values of μ is insignificant (p ≥ 0.05).

Fig. 4. Cellular oxygen dynamics in the WT and ∆kaiA strains.

Rhythms in growth and DO levels coincide with external LD cycles (green arrow) in the WT (a) and ∆kaiA (b). Rhythms are sustained in the WT after cells are transitioned to CL (green arrow) but not in the mutant (orange arrow). When grown under CL the WT (c) exhibits distinct endogenous rhythms in growth which coincide with oxygen cycling (green arrow). The rhythms are lost in ∆kaiA (d) where only a few random drops in oxygen levels are seen (orange arrow). Representative data from multiple runs are presented. 3 independent datasets for growth under CL (showing variability) for the WT and mutant have been provided as raw data (Source Data).

Fig. 5. Western blot analysis of the NifD protein in WT (A, C) and ∆kaiA (B, D) cells grown under CL and incubated for nitrogen fixation assay in air (A, B) or argon (C, D).

Total cellular protein was probed with NifD antibody. Experiments were independently repeated three times with similar results. Cyanothece 51142 NifD protein was expressed in E.coli and purified protein was used as a positive control (E). Arrow showing 54 kD NifD specific band in the samples. The image is a superimposition of the 700 channel on the chemiluminescence channel recorded on a Li-Cor ImageQuant LAS-4000 imager. The Bio-Rad Precision Plus Dual Color Protein Standard was used as the molecular weight marker.

Fig. 6. Schematics showing the importance of KaiA in regulating cellular oxygen dynamics in unicellular diazotrophic cyanobacteria.

KaiA is essential for maintaining strong oscillations in oxygen levels which in turn is crucial for nitrogenase function. In the absence of KaiA the clock functions like a prokaryotic hour glass clock (kaiBC clock) and exhibits little rhythmicity.