Fig 1.
Diurnal variation of UV-C resistance of Synechococcus.
(A) A schematic representation of the experimental schedule. Yellow and black bars indicate the light (L) and dark (D) periods, respectively. The cells were spotted onto agar plates, synchronized to two 12-h:12-h LD cycles and then irradiated with UV-C at each time point (arrowhead). (B) Growth of UV-C-irradiated cells (WT, wild-type; ΔkaiABC, kaiABC-deficient strain). Each image represents a spot assay to assess growth following UV irradiation at each time point of the 12-h LD cycle. Representative data of three independent experiments are shown. (C) Densitometric analysis of the growth test shown in Fig 1B. The timing of UV-C irradiation is shown on the horizontal axis, whereas the densitometric value of the spots as an index of cell growth and representing UV-C resistance of each strain is shown on the vertical axis. The value was normalized to that of the negative control (without UV; n = 3). Error bars represent standard deviation. The UV resistance in the WT and ΔkaiABC strains at ZT 18 significantly differ. *P < 0.01 (Student's t-test). (D) A schematic representation of the experimental schedule (UV+L condition). Each symbol is the same as in Fig 1A. After UV-C irradiation, the cells were exposed to continuous light. (E) Growth of the UV-C irradiated cells (WT, wild-type; ΔkaiABC, kaiABC-deficient strain). Each image represents a spot assay to assess growth following UV irradiation at each time point (upper label) under UV+L condition. Representative data of three independent experiments are shown. (F) Densitometric analysis of the growth test shown in Fig 1E. Each axis and normalization are the same as in Fig 1C. Detailed data used for figures on this article are provided in S1 Dataset.
Fig 2.
Synechococcus shows circadian variation of resistance to UV-C immediately followed by exposure to darkness.
(A) A schematic representation of the experimental schedule (UV+D condition). Each symbol is the same as in Fig 1A. Cells were acclimated to 6 h of darkness after UV-C irradiation. (B) Growth of UV-C-irradiated cells. Each image represents a spot assay to assess growth following UV irradiation at each time point (upper label) under the UV+D condition. Representative data of three independent experiments are shown. (C) Densitometric analysis of the growth test in Fig 2B. The timing of UV irradiation is shown on the horizontal axis, and the relative UV resistance is shown on the vertical axis, as in Fig 1C (n = 3). Error bars represent standard deviation. (D) Time profiles of UV-C resistance in periodic mutants. Each panel represents the results in control strain (Top), kaiCA87V mutant (middle), kaiCF470 mutant (bottom), respectively. Free-running period of each strain is noted in upper left of each panel. Densitometric data of UV resistance are represented with violet lines and filled circles (n = 3). The timing of UV irradiation is shown on the horizontal axis, and the relative UV resistance is shown on the vertical axis on the left. Bioluminescence rhythms of each strain are also shown with open circles. Each strain harbored the PkaiBC::luxAB reporter cassette, and the bioluminescence rhythms were measured under continuous light (LL) condition without UV. The levels of bioluminescence are shown on the vertical axis on the right.
Fig 3.
Importance of DNA photorepair activity and its circadian independency.
(A) Growth of UV-C-irradiated cells of each mutant strain under the UV+D condition. Δphr, phr-deficient strain. In the Δphr; Ptrc::phr strain, phr was expressed under the trc promoter with a phr-deficient background. In the Δphr; Ptrc::phr strain, ectopic phr expression at the leaky level without IPTG was sufficient to recover the circadian rhythm of UV-C resistance. Representative data of three independent experiments are shown. (B) Genomic DNA damage, as quantified by ELISA using a CPD-specific antibody. Genomic DNA was extracted from cells after UV irradiation at hour 0 or 12 in the light. The cells were harvested immediately after UV irradiation, (control sample: UV+) and exposed to the light for 6 h (light repairing sample: UV+6L) or the dark for 6 h (inhibiting repair sample: UV+6D). The vertical axis shows signal value obtained by CPD ELISA. In each case, the CPD level was normalized to samples without UV irradiation, which were assigned signal values of 1 (n = 2). Each plot indicates the result of two independent experiments, and bars indicate the mean values.
Fig 4.
Isolated mutants showed loss of circadian UV-C resistance.
(A) Effects of UV-C irradiation on isolated mutants under the UV+D condition. Each image represents a spot assay to assess growth as shown in Fig 2B. (B) Mapping of Tn-5 insertions. The Tn-5-insertion sites of each UV resistance mutant are indicated by arrowheads. The mutations were mapped onto the open reading frames (ORFs) of the glgP and sasA genes, and the upstream region of rpaA. The function of each gene is indicated under each gene symbol. The numbers under the arrowheads indicate the positions of insertion sites when the start of the ORF of each mutated gene is assigned a positional value of 1. (C) Growth of the UV-C-irradiated cells of each re-constructed mutant strain (ΔglgP, glgP-null strain; ΔsasA, sasA-null strain; cikA-, circadian input kinase cikA-deficient strain) under the UV+D condition. Each image represents the result of a spot assay to assess the growth of each mutant following UV irradiation at each time point. Representative data of three independent experiments are shown. (D) Densitometric analysis of the growth test in Fig 4C. The timing of UV irradiation is shown on the horizontal axis, and relative UV resistance is shown on the vertical axis, as in Fig 1C (n = 3). Error bars represent standard deviation. (E) glgP mRNA accumulation profile in each mutant strain. The glgP expression level from microarray data reported in the previous studies is shown. The microarray data were extracted for ΔkaiABC and the corresponding control wild type (shown here as WT1) strains from Ito et al. [14], for ΔsasA and its control (WT2) strains from data available at (NCBI, Gene Expression Omnibus databank, #GSE28430), and ΔcikA and its control (WT3) strains from Pattanayak et al. [33]. The expression data of glgP in each WT strain were normalized to the average of 1.
Fig 5.
Glycogen and related metabolism affect circadian control of UV-C resistance.
(A) A simplified diagram of the glycogen-related metabolic pathways (G1P, glucose-1-phosphate; G6P, glucose-6-phosphate; ADP-Glc, adenosine diphosphate glucose). GlgP is the core enzyme involved in glycogen degradation. SasA and CikA activate the catabolic and anabolic metabolic pathways, respectively, through mediating the timing information from the core oscillator to transcriptional machinery to control gene expression. (B) Quantification of glycogen content in WT and ΔglgP (glgP-null) strains. Cells were exposed to darkness and then harvested at each timepoint. Left and right panels show the dynamics of glycogen content when the cells were transferred into darkness at hour 0 and 12 in LL, respectively. Hours in the dark is shown on the horizontal axis, and glycogen content is shown on the vertical axis. The glycogen contents are normalized by OD730 unit of harvested cells. (C) A schematic representation of the experiment. UV-C irradiation and dark exposure was performed at hour 12 in LL. During the darkness, CCCP was added to the culture. At the end of darkness, CCCP treatment was terminated by washing cells with fresh BG-11 media. (D) Growth curves of UV-C irradiated cells. Hours after medium exchange are shown on the horizontal axis, absorbance at 730 nm of cell culture is shown on the vertical axis as an index of cell growth (n = 3). Error bars represent standard deviation. Left panel shows the results in negative control without CCCP treatment. Right panel shows the results in the presence of 10 μM CCCP. UV- and UV+ represent with and without UV exposure, respectively. (E) Effects of UV-C irradiation on the strain genetically modified to uptake glucose. A schematic representation of modified strain and its function are illustrated (upper). In the Ptrc::glcP strain, the glucose transporter gene was expressed with a WT background. Each image represents the growth following the UV irradiation under the UV+D condition. The symbols “–” and “+” indicate the absence and presence of D-glucose, respectively. Both with and without D-glucose, experiments were performed in the presence of 1 mM IPTG. Representative data of three independent experiments are shown.
Fig 6.
Schematic diagram of a possible trade-off between UV resistance and energy metabolism in Synechococcus.
The results of the present and previous studies show an inverse correlation between UV resistance and glycogen degradation. High UV resistance at any time of the day would require continuous suppression of glycogen degradation, which must be disadvantageous to energy utilization in order to survive in the dark. Such a trade-off would give rise to the necessary use of different physiological functions in a time-dependent manner. Priority would be given to UV resistance in the daytime when UV irradiation is present, whereas energy production via glycogen degradation in the night occurs when photosynthesis is interrupted. Such a time-dependent trade-off is plausibly controlled by the output pathways mediated by the SasA and CikA from the Kai-based central oscillator.
Table 1.
Summary of UV resistance properties and growth of each strain under the LD condition.