The circadian rhythm of UV resistance in Synechococcus is dependent on the kai gene

Initially, the time-dependent variation in UV resistance of Synechococcus during the LD cycle was investigated. Briefly, cells were exposed to short-wavelength UV-C light (254 nm, 500 J/m², approximately 30 J/ m²·s) every 6 h during one 12-h:12-h LD cycle after synchronization of the circadian clock by two LD cycles. Then, the cells were cultured under continuous light (LL) conditions to quantify viability against UV-C at each time point (Fig 1A, for details, see Materials and Methods). As shown in Fig 1B, growth of the WT strain was severely inhibited when cells were subjected to UV-C at dusk. When UV-C irradiation was applied at subjective midnight (ZT 18: ZT = zeitgeber time, referring to time in h during the LD cycle, hour 0 refers to the light onset in LD), the growth rate partially recovered to ~40% of that without UV-C (Fig 1B and 1C). The kaiABC-deficient (ΔkaiABC) strain also showed diurnal UV-C resistance, as growth was recovered more evidently when UV-C was administrated at ZT 18.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

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.

https://doi.org/10.1371/journal.pgen.1009230.g001

Then, we examined if the UV-C resistance rhythm persists under LL condition. Cells were exposed to UV-C for the indicated times under LL (UV+L: UV with light exposure condition) after two LD cycles and then returned to LL (Fig 1D). As shown in Fig 1E, the WT strain had no circadian rhythm of UV-C resistance, as values were almost constant throughout the circadian cycle, whereas ΔkaiABC cells appeared to be less UV-C resistant than the WT cells throughout LL (Fig 1E and 1F; S1A and S1B Fig). Even though the conditions before the time of UV exposure at 0, 6 and 12 hours were the same for both LD and LL conditions, the results of the growth test were different. This is because that growth was affected by the presence or absence of the subsequent darkness. Therefore, circadian rhythms were not evident under this experimental condition. Nevertheless, there are two hints of possible involvement of circadian regulation in UV resistance: (i) the magnitude of UV-C resistance differed between the WT and ΔkaiABC strains under the LL condition, and (ii) the growth rate after UV-C irradiation at ZT 18 under LD cycles also differed between the two strains. It should be noted that UV-C irradiation did not cause a phase shift of the circadian clock under the experimental condition (S1C Fig). Compared with the results shown in Fig 1, UV-C resistance is highly affected by darkness before and/or after UV irradiation. As shown in Fig 1B, the duration of dark exposure before or after UV irradiation differed at each time point.

To avoid this difference, UV-C resistance profiles were examined in response to LL with UV pulses immediately followed by exposure to darkness for 6 h (UV+D: UV-C with dark exposure condition, Fig 2A). Surprisingly, the results shown in Fig 2B and 2C clearly indicate that growth was inhibited rhythmically in the WT cells only when UV-C irradiation was administrated at subjective dusk under the UV+D condition. This result strongly suggests that Synechococcus exhibits circadian UV-C resistance rhythm. The “UV-”control samples shown in Fig 2A–2C were cultured under LL conditions after entraining the clock to two LD cycles. We confirmed that the time-dependent variation of growth was not caused by darkness alone, as shown in S2 Fig. When the growth of cells was compared under UV+D condition or 6-h darkness only condition without UV at different time (0, 12, and 24 h in LL), dark exposure for 6 h alone did not cause time-dependent difference in growth, whereas UV+D at hour 12 in LL significantly reduced. To avoid a possibility that this rhythmicity observed in the spot assay was due to a methodological artifact, we tested several methods for measuring cell survivability or growth. Since colony-forming units after stresses are often used for measuring survivability, we validated if rhythmicity was reproduced by counting colony-forming units in several independent experiments. As shown in S3 Fig, survivability was confirmed to fluctuate in a circadian fashion after UV-C irradiation (S3A–S3C Fig). However, considering that Synechococcus is a planktonic cyanobacterium, one can still question if formation of colonies might be affected by several uncontrollable stresses and the forming units would not always correlate well with the numbers of viable cells. Therefore, some researchers prefer measuring growth in liquid media to monitor stress resistance profiles after the stress condition. Thus, we also measured growth of WT cells in liquid media after the UV+D treatment at hour 0 or 12 in the light. While cell growth was evidently confirmed after UV-C administration at hour 0, it was significantly reduced when UV was applied at hour 12, and the cell density did not recover during incubation for at least 80 h (S3D Fig). Although we tried evaluation of cell viability following UV irradiation in liquid media using vital dye, but it was difficult due to high autofluorescence of the cells. Therefore, as a second choice, we observed autofluorescence as an index of cell viability. In unicellular cyanobacteria, autofluorescence due to photosystem has been used as a convenient index of cell viability [23]. Thus, to estimate the viability of cells administrated with UV+D, we observed red autofluorescence of UV-irradiated cells under the microscope at 60 hours after the UV+D treatment. As shown in S3E Fig, despite severely limited growth after UV+D at hour L12, cells did not show defective morphology nor significant bleaching in autofluorescence, same as after UV+D at L0 (S3E Fig). Thus, UV+D at hour 12 reduced cell propagation without severe chlorophyll degradation and morphological changes. These results in liquid media are consistent with the results shown above using spotting test on solid media. Therefore, the observed rhythm in UV resistance cannot be due to methodological artifacts in measuring the resistance. Hereafter, we mainly used the spot assays owing to its easy handling for a number of mutants and time-points at once. Under UV+D condition, we also tested the response to UV-B (312 nm) irradiation, as more natural environmental condition. Since UV-B is more abundant in the natural environment, we wanted to confirm the results obtained with UV-C are related to the behavior of cyanobacteria under natural settings. As shown in S4A and S4B Fig, the WT cells showed higher resistance against UV-B irradiated at dawn (hour 0 in the light) than that at subjective dusk (hour 12 in the light). This time dependency is essentially consistent with the experiments with UV-C irradiation. Therefore, we suggest that the results obtained with UV-C irradiation were essentially the same with UV-B.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

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), kaiC^A87V mutant (middle), kaiC^F470 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 P_kaiBC::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.

https://doi.org/10.1371/journal.pgen.1009230.g002

Importantly, in the ΔkaiABC strain, the rhythm of UV resistance was abolished with constitutively lower levels (approximately trough levels of the WT strain, Fig 2C). Thus, UV resistance of Synechococcus exhibits a Kai-based circadian rhythm when UV irradiation is immediately followed by subsequent exposure to darkness. In addition, we changed timing or duration of dark exposure after UV irradiation at hour 12 in LL to test the critical parameters in the UV+D condition to affect cell growth (S4C and S4D Fig). The results showed that cell growth was permitted by light exposure for >1 h after UV irradiation before dark exposure (S4C Fig), compared with the standard UV+D condition. Moreover, the longer the exposure to darkness immediately after UV irradiation, the more severe the effect was observed on survival after UV irradiation (S4D Fig). The result shows darkness for at least 3 h was sufficient to observe the UV resistance rhythm under our experimental conditions. The efficient length of darkness can also be influenced by UV intensity.

The period of UV resistance rhythm is consistent with the period of endogenous clock

In general, circadian rhythm refers to the phenomenon under continuous conditions. Considering UV-C irradiation and subsequent darkness as a kind of stress, the time-dependent cycle to the stress can fit the classical example of circadian rhythms. However, the above-mentioned UV-C resistance rhythm accompanies with an external dark pulse under UV+D condition. Thus, one may doubt whether this process is actually related to the circadian rhythm or is more related to difference in metabolism during light/dark cycles. In order to support that the UV-C resistance rhythm under UV+D condition is a bona fide free-running circadian rhythm derived from the endogenous clock, we performed experiments using previously reported short-period mutant strains mapped on kaiC. If the rhythm is an autonomous oscillation by the circadian clock (in other word, that is independent of the external dark pulse), the period of the UV-C resistance rhythm should fluctuate according to the lengths of endogenous (free-running) period. The kaiC^A87V and kaiC^F470Y mutations have been shown to shorten the free-running period of KaiC phosphorylation rhythm in vitro and the kaiBC promoter activity rhythm monitored with bioluminescence with a period length of 22 and 17, respectively [9,13]. Short period bioluminescence profiles of these mutant strains were confirmed as shown in Fig 2D. Importantly, each mutant also shortened the period of UV-C resistance rhythm and its length correlated well with their each free-running period even in the presence of the external darkness under the UV+D condition (Fig 2D). These results established that the observed UV-C resistance rhythm under UV+D condition is a bona fide time-dependent response derived from the endogenous clock.

The photorepair reaction is involved in UV-C resistance of Synechococcus

The role of dark exposure after UV-C irradiation in the modification of UV-C resistance was investigated. Major DNA damage caused by UV irradiation results in cyclobutane pyrimidine dimers (CPDs) formation [24,25]. CPDs are removed via multiple pathways, such as the photorepair and nucleotide excision repair (NER) processes [20,25]. In the unicellular cyanobacterium Synechocystis sp. PCC 6803, the photorepair reaction catalyzed by CPD photolyase, which is activated by visible light, plays a major role in CPD removal [26,27]. Synechococcus harbors the phr gene (Synpcc7942_0112), which encodes a homolog of CPD photolyase. It was reported that Synechococcus Phr binds to CPD-like DNA [28,29], and heterologous expression of the Synechococcus phr gene in Escherichia coli improves UV resistance [30]. Thus, the phr gene of Synechococcus is expected to encode a bona fide photolyase enzyme. We expected that dark exposure after UV-C irradiation (UV+D condition) would prevent the Phr-based photorepair reaction (S5A and S5B Fig for schematic conditions to be compared). In other words, we hypothesized that the results shown in Fig 2 would be interpreted as a clock-controlled response of cells against inhibition of the photorepair reaction.

When the phr gene was nullified, UV-C resistance of the mutant (Δphr) strain was severely lowered at all time points under UV+L conditions (S5C Fig). Thus, the growth of Synechococcus after UV-C irradiation is greatly dependent on the photorepair pathway by CPD photolyase, as with other cyanobacteria. As expected, the Δphr strain did not recover after UV-C exposure under the UV+D condition (Fig 3A).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

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; P_trc::phr strain, phr was expressed under the trc promoter with a phr-deficient background. In the Δphr; P_trc::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.

https://doi.org/10.1371/journal.pgen.1009230.g003

Next, the role of Kai-based time-dependent Phr activity on the circadian variation of UV-C resistance of Synechococcus was investigated. According to the results of our previous DNA microarray study (14), the accumulation of phr mRNA level did not show a circadian rhythm. Hence, a strain was constructed in which phr was expressed by an isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible heterologous trc promoter with a phr-deficient background (Δphr; ox-phr strain). The results showed that the circadian rhythm of UV-C resistance was rescued by continuous (leaky) phr expression even in the absence of IPTG (Fig 3A). The same result was confirmed in the presence of 100 μM IPTG (S5D and S5E Fig). These results negate the possibility that the circadian rhythm of phr expression is involved in the rhythmic control of UV resistance. However, it is possible that the UV-C resistance rhythm is generated by posttranscriptional regulation or posttranslational modification of Phr.

Thus, variations in DNA repair activity of cells exposed to UV-C at subjective dawn vs. dusk were examined. WT cells were exposed to UV at hour 0 or 12 in LL and then divided into three groups. The first group was immediately harvested as a UV+ control sample, whereas the second group was cultured in the light for 6 h and then harvested (light repairing sample, shown as UV+6L in Fig 3B), and the third group was harvested after acclimation to darkness for 6 h (inhibition of DNA repair sample, shown as UV+6D in Fig 3B). Genomic DNA was extracted from each group, and the CPD content was quantified using an ELISA with a specific antibody against CPD. The results confirmed that UV-C irradiation increased CPD levels by four- to fivefold, as compared to the non-UV control, regardless of the timing of UV-C administration (0 or 12 h of light) (Fig 3B). When cells were maintained in the light after UV-C exposure, the CPD level decreased to the control level, while dark exposure failed to reduce it, regardless of the timing of UV-C irradiation (Fig 3B). These observations are consistent with the results of the present study that UV resistance of Synechococcus is highly dependent on the Phr-mediated CPD photorepair pathway. Nevertheless, time-independent photorepair activity, as shown in Fig 3B, cannot explain the time-dependent resistance against UV (Fig 2). Thus, CPD photolyase activity following dark acclimation is not likely a major target of circadian control that gives rise to the rhythm of UV-C resistance. Instead, rhythmic control of UV-C resistance is overt when CPD photorepair is partially inhibited by the dark but is masked when CPD photorepair activity is intact without darkness.

Then, we expected that the circadian clock would control (i) UV-induced DNA damage, with the exception of the CPD and CPD photolyase-independent DNA repair processes, and/or (ii) UV-induced damage other than DNA damage and the related repair mechanism. In the case of (i), (6–4) photolyase is not likely involved, since in the Synechococcus genome, only the phr gene harbors a photolyase domain. Generally, photolyases have high substrate specificity and bind to either CPD or the (6–4) photoproduct in a structure-specific manner [31]. The crystal structure of the Synechococcus Phr protein has been shown to bind a CPD analog [28]. Also, the histidine residue, which is essential for the catalytic activity of (6–4) photolyase [32], is not conserved in the Synechococcus Phr protein.

Nucleotide excision repair (NER) is a representative photorepair-independent DNA repair mechanism. Inactivation of the uvrA gene, which participates in the NER process, moderately reduced UV-C resistance to ~50% of that in the wild type strain under the UV+L condition (S5F and S5H Fig). More significantly, UV-C resistance was dramatically lowered in the uvrA strain under the UV+D condition (S5G and S5I Fig). Nevertheless, an attenuated time-dependent variation in UV-C resistance still remained (S5I Fig, see the magnified line), suggesting that uvrA is important for the magnitude of UV resistance but not essential to drive the rhythmicity. Our previous DNA microarray analysis indicated that the uvrA mRNA expressed in a kai-dependent circadian fashion as many other genes (S5J Fig, [14]). However, its amplitude is low with peak-to-trough ratio of ~1.4, which would be insufficient to drive a high amplitude protein abundance rhythm. Although we cannot rule out the possibility that clock-controlled NER somewhat supports and amplify UV resistance rhythms when CPD photolyase activity is partially inhibited by dark exposure, further studies are needed to validate this possibility.

As an alternative possibility, with the exception of the DNA repair process (case ii), the circadian clock might control the resistance mechanism against reactive oxygen species induced by UV light. We examined if sensitivity to H₂O₂ varied at hour 0 and 12 in the light. Cell suspensions were treated with H₂O₂ at each time point, incubated for 6 h either in the light or dark, and then inoculated onto solid media to quantify the growth rate. As shown in S6 Fig, the growth profiles of the cells did not significantly vary, regardless of the timing of H₂O₂ administration or subsequent light/dark exposure. Although it remains a room to test more reactive ROS such as superoxide or singlet oxygen, it was difficult to directly identify factors that control the circadian rhythm of UV resistance. Therefore, genetic screening was performed to identify such factors.

Tn-5-based screening identified genes involved in UV-C resistance control

To elucidate the mechanisms underlying circadian rhythms in UV-C resistance, mutants were isolated from the randomly transposon (Tn5)-inserted mutant library, which abolished the low UV-C resistance profile, when UV was irradiated at hour 12 in the light under the UV+D condition (Fig 2A, see Materials and Methods, for experimental schedule). Of 5,000 clones that were screened, four mutant strains were isolated with greater UV resistance at hour 12 than the WT strain, thereby showing attenuated circadian variation (Fig 4A). Two mutations (mutants 2 and 3) were mapped to the coding region of sasA and a region upstream of rpaA (presumably the rpaA promoter region), respectively (Fig 4B). More detailed information of Tn-5 insertion sites was shown in S7A Fig. The KaiC-binding histidine kinase SasA and its cognate response regulator RpaA constitute a major circadian output mediator that drives genome-wide circadian transcriptional orchestration [15–17]. Both mutants 2 and 3 abolished the bioluminescence rhythm to monitor kaiBC promoter activity under continuous light conditions (S7B and S7C Fig) at constitutively low levels, as reported previously for knockout strains which deleted each gene [15,16]. These results further support that the circadian rhythm of UV-C resistance is dependent on the transcriptional output of the clock via the SasA-RpaA system. Disruption of sasA and rpaA has been known to arrest the transcriptional rhythm at subjective dawn-like state under standard continuous light conditions [15,16,33]. By contrast, disruption of another clock-related histidine kinase gene, cikA, shifts the transcriptional profile to the subjective dusk-like state, while low amplitude rhythm remains [33,34]. Thus, we prepared two strains either deficient of sasA or cikA for UV-C resistance assay under the UV+D condition. As shown in Fig 4C and 4D, UV-C resistance rhythm was abolished in both sasA and cikA strains with opposite profiles. The sasA mutant maintained constitutively higher UV-C resistance level (comparable to the peak level of the rhythm in the wild type strain. On the contrary, the cikA strain showed constitutively lowered UV-C resistance throughout the circadian cycle (comparable to the trough level of the wild type rhythm. This opposite effect is well correlated with the opposite functions of SasA and CikA: SasA activates RpaA phosphorylation to enhance transcription of genes peaking at subjective dusk, while CikA dephosphorylates RpaA [34]. Roughly speaking, above-mentioned results suggest that UV-C resistance is lowered when expression of subjective dusk genes is relatively enhanced, such as any circadian time in the cikA and kaiABC-null mutant strains, and subjective dusk in the wild type strain. In both sasA and rpaA strains, expression of subjective dusk genes are constitutively suppressed and the magnitude of UV-C resistance is maintained at high levels.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

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.

https://doi.org/10.1371/journal.pgen.1009230.g004

The other two of four isolated mutations (mutants 1 and 4, shown in Fig 4A) which did not show attenuated UV-C resistance at hour 12 in the light were mapped to different positions of the same gene, glgP, which encodes glycogen phosphorylase (Fig 4B). Glycogen phosphorylase is an enzyme that degrades glycogen and produces glucose-1-phosphate [35]. To confirm the results observed with the Tn-5-insertion mutants, strain without any glgP coding sequence was newly constructed. This strain showed constitutively higher UV-C resistance under the UV+D conditions throughout the circadian cycle, phenocopying the sasA strain (Fig 4C and 4D). Interestingly, according to our previous microarray analysis [14], glgP is a typical high-amplitude clock-controlled gene which peaks at subjective dusk in LL, while it is constitutively maintained at moderate levels in the kaiABC-null mutant (Fig 4E). Accordingly, ChIP-seq analysis by Markson et al. [17] has identified this gene as a RpaA-binding target, which is directly activated by RpaA. Thus, glgP expression is strongly downregulated in either rpaA or sasA strain, and upregulated constitutively in cikA strain (Fig 4E). Thus, all these results do not contradict with the hypothesis that UV-C resistance is lowered when glgP expression is relatively enhanced. In the wild type strain, glycogen accumulation fluctuates in a circadian fashion, with a maximum at dusk and a minimum at dawn in LL [36]. This is most likely due to anti-phasic expression and/or enzymatic activities of enzymes for glycogen degradation and synthesis. To be more precise, the expression of glycogen degradation enzyme genes such as glgP peaks at dusk via the SasA-RpaA-mediated pathway, while the expression of glycogen synthesis genes such as glgC peaks at dawn. In other words, glycogen degradation activity peaks at the time of the maximum glycogen content, while the glycogen synthetic activity is higher at the time of the minimum glycogen level. Thus, the activity of degradation and synthesis of glycogen do not coincide with the accumulation of glycogen at that time, and the fluctuation of glycogen accumulation delays by ~12 hours relative to that of the enzymatic activity. Therefore, even though glycogen content peaks at dusk, it decreases over the following 12 hours due to increased degradation activity and decreased synthetic activity.

It should be noted that nullification of either sasA or rpaA is known to reduce glycogen content, while that of cikA over-accumulate it via genome-wide transcriptional control [33,36,37]. Therefore, any of the mutations identified here would give rise to abnormalities in temporal dynamics of glycogen metabolism. Interestingly, glycogen content was lower in the rpaA strain, despite the fact that the maximum catalytic activity of enzymes involved in glycogen biosynthesis are not reduced the rpaA strain [37]. We suggest that the intracellular state of the rpaA strain is arrested at the dawn phase state of the wild strain, where glycogen synthetic enzyme may not catalyze its reactions since some steps for glycogen synthesis such as gluconeogenesis pathway are depleted. In the rpaA strain, energy charge (relative levels of ATP, ADP and AMP in the intracellular adenine nucleotide pool) strikingly drops when cells are exposed to the dark, compared with the wild type strain [37]. While restoration of energy charge by forced glucose uptake alone seems not sufficient to recover higher glycogen content [37], ATP synthesis may also be a rate-limiting factor for glycogen synthesis in the rpaA strain. In any cases, this background indicates that it is not easy to estimate the intracellular physiological state, including the activity of glycogen degradation and synthesis, based solely on the amount of glycogen content at each time. The link between UV resistance and glycogen metabolism suggested by the screening would be either the result of the glycogen content itself, the effect of the activity of glycogen degradation by glgP, or the effect of the downstream pentose phosphate circuit and the TCA cycle.

Involvement of the glycogen metabolic gene in UV-C resistance control

We aimed to clarify the relationship between UV resistance and glycogen metabolism. A simplified diagram of the glycogen-related metabolic pathways is shown in Fig 5A. GlgP plays an important role in glycogen degradation and is involved in major metabolic pathways, such as the glycolysis and oxidative pentose phosphate pathways, which are affected by glycogen catabolism [35,38]. Thus, we evaluated whether the glgP strain actually lowered glycogen degradation activity under our experimental conditions. Initially, we monitored glycogen accumulation levels in the wild type strain in LL, which showed an overt circadian rhythm peaking at subjective dusk (S8A Fig) [33]. Glycogen is believed to be important as a carbohydrate reservoir, mainly for survival under ATP-limited dark condition in the absence of photosynthesis activity [36]. Therefore, we tested glycogen consumption profile during 6-h of darkness applied at subjective dawn and dusk. Dark exposure starting at hour 0 maintained glycogen content at constitutively low level (Fig 5B), comparable to the trough level of the circadian glycogen rhythm in LL (S8A Fig). On the other hand, dark exposure at hour 12 triggered more rapid glycogen degradation leading to ~45% within 6 h (Fig 5B). More importantly, ΔglgP strain did not show significant glycogen degradation during 6-h dark exposure, regardless of the time when the cells were exposed to darkness (Fig 5B). These results suggest that the higher UV-C resistance is accompanied by lower glycogen degradation activity, such as in the wild type strain at subjective dawn and in the glgP strain throughout the circadian cycle.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

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 P_trc::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.

https://doi.org/10.1371/journal.pgen.1009230.g005

To further analyze a link between UV resistance and glycogen metabolism, additional mutant strains lacking either the glgC or glgX gene were also generated. The glgC and glgX genes encode a glycogen synthesis enzyme (glucose-1-phosphate adenylyl transferase) and degradation enzyme (isoamylase), respectively (Fig 5A) [35]. Disruption of the glgX gene is expected to reduce glycogen consumption, similar to the phenotype of the glgP strain with lower UV-C resistance. However, UV-C resistance of the glgX strain did not significantly change under the UV+D condition (S8B Fig), suggesting a possibility that GlgP constitutes a major glycogen degradation pathway. This possibility is consistent with our glycogen degradation assay in ΔglgP strain (Fig 5B). Therefore, disruption of glgX alone may be insufficient to reduce glycogen consumption. A strain with doubly disruption of both glgP and glgX genes was generated, which had essentially the same UV-C resistance property as the glgP strain (S8B Fig), which further supports the primary function of GlgP in glycogen degradation. Nullification of glgC is known to inactivate glycogen synthesis and leads to the constitutive loss of glycogen accumulation throughout the day [33]. Nullification of glgC is reported to severely restrict growth under 12-h:12-h LD cycles even without UV irradiation [39]. Assuming that circadian alternation in glycogen degradation is important for rhythmic UV-C resistance, the loss of glycogen synthesis by disruption of the glgC gene would abolish the circadian control of UV-C resistance. Under the UV+D condition, UV-C resistance was largely reduced in the glgC strain (S8B and S8C Fig). While the UV-resistance rhythm was not completely abolished, the amplitude was much lower than the wild type strain (S8C and S8D Fig). It should be noted that glgC disruption is known to enhance the magnitude of phase shifts against dark pulses due to an altered metabolic state, especially in the dark, whereas the amplitude of circadian transcriptional cycle in LL is less affected [33]. These observations further support a link between glycogen metabolism and UV resistance under the UV+D condition.

Inhibition of ATP synthesis in darkness recovered the attenuated UV-C resistance at subjective dusk

When UV resistance is reduced in the wild type strain (subjective dusk), glycogen degradation activity is elevated (Fig 5B and [36]). However, as discussed earlier, glycogen accumulation itself also fluctuates in a time-dependent manner, and it remains unclear whether the accumulation itself or the degradation activity affects UV resistance. Thus, we hypothesized that increased de novo energy production due to glycogen degradation via enhancing catabolic pathways would reduce UV-C resistance. We evaluated the effect of de novo energy production via glycogen degradation in the dark, especially ATP synthesis, on UV-C resistance under UV+D condition. As shown in Fig 5C, following UV irradiation at hour 12 in LL, we treated WT cells with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) as an uncoupler to inhibit ATP synthesis in darkness (for details, see Materials and Methods). Cells without CCCP suppressed cell growth after UV+D treatment (Fig 5D), same as shown in S2D Fig. On the other hand, CCCP addition did not restrict growth regardless of the presence or absence of UV-C irradiation (Fig 5D). It should be noted in the absence of UV, transient CCCP administration under our experimental condition did not affect growth profile much, comparable to control cells without CCCP. In other word, growth of the UV irradiated cells was rescued by CCCP treatment (Fig 5D). These results are consistent with the hypothesis that de novo ATP synthesis in the dark after UV-C irradiation severely limited growth, and the circadian fluctuation in ATP availability via glycogen degradation would drive the rhythm in UV resistance. This is also consistent with the reduced energy charge in the rpaA strain when the cells are exposed to the dark [37], remaining higher UV resistance as discussed above. Although ATP production is highly active in the light, UV resistance does not appear to be low, as shown in the WT strain under UV+L conditions. We believe that the higher UV resistance in the light is due to the higher DNA damage repair activity of photolyase, which masks the effects of UV irradiation. The very high UV sensitivity in the photolyase deficient strain under UV+L condition (S5C Fig) can be interpreted that this strain is highly sensitive to UV in the light when ATP production is highly active without being masked by the photolyase activity.

Forced incorporation of glucose constitutively lowered UV-C resistance

In order to validate whether changes in further downstream catabolic pathways affect UV-C resistance, we used a strain that was genetically modified to uptake exogenous glucose directly into the cells, as reported previously [40]. It should be noted that we also considered another possibility to use strains overexpressing the glgP and glgC genes to activate glycogen catabolism. However, overexpression strains may cause unexpected side effects. More importantly, it is obscure whether overexpression of metabolic enzymes really activates the magnitude of targeted metabolic pathways, considering the amount of the carbon source itself may not change. On the other hand, it has been shown in the forced glucose uptake strain that glucose is incorporated into OPPP and the Emden-Meyerhof pathway (EMP) to be used as bona fide carbon source (Fig 5E, [41]). In this strain, the glcP gene, which encodes a glucose transporter derived from another cyanobacterium, was expressed by the trc promoter in the presence of IPTG. In the dark, the growth of Synechococcus cells is suppressed. By contrast, forced glucose uptake enables the growth of glcP-expressing cells even in the dark [40]. The effect of forced activation of the glucose catabolism pathway on the circadian rhythm of UV-C resistance rhythm with dark exposure was investigated using the mutant and WT strains. As shown in Fig 5E, the addition of glucose and IPTG constitutively lowered UV-C resistance of the glucose uptake strain, whereas there was no change in the absence of glucose. UV-C resistance of the WT strain did not change regardless of the presence or absence of glucose (Fig 5E).

Bacterial strains and culture conditions

All strains were modified from the pseudo-wild-type (WT) S. elongatus PCC 7942, designated as strain NUC42 [9], which contained the P_kaiBC::luxAB bioluminescence reporter segment at a targeting site NS I. The derivatives used in this study are listed in S1 Table. To construct plasmids to disrupt the Synechococcus genes phr, glgP, glgC, glgX, and uvrA, the 500-bp upstream region of each target gene, an antibiotic resistance gene, and the 500-bp downstream region of each target gene were amplified and assembled by polymerase chain reaction (PCR), then cloned into the pGEM-T easy vector (Promega Corporation, Madison, WI, USA). The previously constructed plasmids pDkaiABC, pAM2176, and pAM2152 were used to disrupt the clock-related genes kaiABC, sasA, and cikA, respectively [9,52]. To yield short-period mutant strains and its negative control (rescued kaiC^WT), we re-introduced kaiABC and mutated kaiABC genes into kaiABC-null background by previously constructed plasmids and its derivative (pCkaiABC [9], pIL788 (equivalent to pCkaiABC^A87V) and pIL782 (equivalent to pCkaiABC^F470Y)). pIL788 and pIL782 were constructed by introducing mutated kaiC DNA fragment into EcoRV-BspEI sites of pCkaiABC. To yield strains expressing phr from the trc promoter at a targeting site NS II, a DNA fragment containing the phr open reading frame was amplified by PCR using WT Synechococcus genomic DNA as a template and then cloned into pNS2KmTΔHincII-Ptrc [53]. pAL46, a plasmid for constructing the glucose uptake strain, was provided by Prof. Shota Atsumi of the University of California, Davis (Davis, CA, USA). The glcP gene expressed by the trc promoter was introduced into the NS I site using the plasmid pAL46. Detailed information of pAL46 was described previously [40]. All strains were generated by natural transformation with plasmid DNA [54]. Cells were grown at 30°C with illumination of ~40 μmol/m²·s on either BG-11 liquid or solid (containing 1.5% agar) media supplemented with appropriate antibiotics.

Bioluminescence assay

The P_kaiBC::luxAB bioluminescence profile was monitored on solid media with photomultiplier tubes under continuous light conditions after synchronization of the cells to a 12-h:12-h LD) cycle, as described previously [55].

Evaluation of UV resistance by spot assays to assess growth

In order to evaluate UV resistance, each Synechococcus strain was cultured in BG-11 liquid media, diluted to an optical density at 730 nm (OD₇₃₀) of 0.2. The aliquots were further diluted with BG-11 media to 1:100, and 2 μL of each dilution was spotted onto solid BG-11 media without antibiotics. Cells were synchronized to twice 12-h:12-h light:dark (LD) cycles, and then released into each experimental condition as described in the text. UV exposure was initiated just before the dark/light transitions under diurnal condition (ZT = 0, 12, 24) and UV+D condition. UV-C irradiation was performed using a UV Crosslinker (model, CL-1000; Analytic Jena AG, Jena, Germany) equipped with a discharge tube with a main wavelength of 254 nm. The irradiation intensity was set at 500 J/m². UV-B irradiation was performed using UV transilluminator (PI-20.AB; BIO CRAFT, Tokyo, Japan) equipped with a discharge tube with a main wavelength of 312 nm. UV-B intensity was measured by a digital UV radiometer (Radiometer Sensor, UVX-31; Analytic Jena AG, Jena, Germany). The UV-irradiated cells on agar plates were exposed to darkness and then to light for the “UV+D” condition experiments or cultured without dark exposure in the light for the “UV+L” experiments. Cellular growth was evaluated by densitometric analysis of each spot captured using a single-lens reflex camera (Pentax model K20D; Ricoh Company, Ltd., Tokyo, Japan). Quantification of UV resistance (growth index) was performed using ImageJ (https://imagej.nih.gov/ij/) as described in [56]. UV resistance of each strain was normalized to the densitometric value of a corresponding negative control strain without UV exposure. The quantified densitometric value (n = 3) were obtained from experiments performed at the same time using three independently pre-cultured cells. We have confirmed that at least two or more completely independent experiments (independently precultured cells to be analyzed on different plates at different time) obtained essentially similar results.

Evaluation of UV resistance and effect of ATP synthesis inhibitor by monitoring growth in liquid cultures

In order to evaluate UV resistance in liquid culture, each Synechococcus strain was cultured in BG-11 liquid media, diluted to an optical density at 730 nm (OD₇₃₀) of 0.075. Cells in liquid media were entrained to two 12-h:12-h light:dark (LD) cycles in 6-well or 24-well plastic plates with shaking, and then released into each experimental condition as described in the text. UV-C irradiation was performed using a UV Crosslinker as described above in spot assays and the irradiation intensity was set at 500 J/m². For evaluating the growth of cells, the absorbance at 730 nm was measured by microplate reader (Powerscan HT; DS Pharma Biomedical, Ltd., Osaka, Japan). In order to evaluate the effects of blocking ATP synthesis, CCCP (Carbonyl cyanide 3-chlorophenylhydrazone, C2759, Sigma-Aldrich) was added to the cultures at a final concentration of 10 μM following UV irradiation at hour 0 or 12 in LL conditions. For negative control, the same volume of ethanol instead of CCCP was added. Cultures were exposed to darkness for 6 h and CCCP treatment was terminated by twice washing of cells with fresh BG-11 media.

Microscopy and image acquisition

UV-irradiated cells in liquid cultures under UV+D condition were prepared as described above. UV-irradiated cells were mounted between cover glass and imaged with a confocal laser scanning microscope (FV1000; Olympus, Japan) equipped with 40x objective lens. Image acquisition were done on FV10-ASW software applied to this system. Autofluorescent images of Synechococcus cells were collected after 559 nm excitation using a 550–600 nm emission window. Differential interference contrast images were obtain using TD1 channel in FV10-ASW.

DNA repair activity detection with a cyclobutane pyrimidine dimer (CPD)-specific enzyme-linked immunosorbent assay (ELISA)

Briefly, Synechococcus cells at an OD₇₃₀ of ~0.3 were grown in BG-11 liquid media in a continuous culture system in a 1.5-L flat-bottom glass bottle at 30°C with irradiation of 40 μmol/m²·s generated by white fluorescent lamps. Cells were sampled at hour 0 or 12 in LL, irradiated with UV-C, as described above, and then harvested and stored at –80°C until assayed. Genomic DNA was extracted from the harvested cells using the phenol–chloroform extraction method and Cica Geneus DNA Prep Kit (for plants; Kanto Kagaku, Tokyo, Japan). CPD contained in the genomic DNA was detected and quantified using the High-Sensitivity CPD ELISA Kit (Cosmo Bio Co., Ltd., Tokyo, Japan) in accordance with the manufacturer’s protocol.

Screening and identification of UV-resistant mutants

WT Synechococcus was transformed with a genomic DNA library fused to a Tn5-derived transposon with the kanamycin resistance gene [57] in order to introduce random mutations. This genomic DNA library was provided by Dr. Mitsunori Katayama of Nihon University (Tokyo, Japan). Transformants were streaked onto solid BG-11 plates containing kanamycin. After synchronizing the cells to two 12-h:12-h LD cycles, transformant cells were exposed to the light for 12 h, irradiated with UV-C as mentioned above, exposed to the dark for 6 h, and then returned to the light. Several days after UV irradiation, among 5,000 transformant clones screened, 18 colonies formed on the plates were initially picked up as possible mutants with greater UV resistance. Four out of the 18 clones were confirmed to show elevated survival rates after the UV+D treatment at ZT 12 by spot tests. All the four clones are reported in the present paper. Genomic DNA was extracted from each of the four mutant strains, and the transposon insertion sites were identified by inverse PCR and sequence analysis.

Evaluation of H₂O₂ resistance

In order to evaluate H₂O₂ resistance, WT Synechococcus cells were cultured in liquid BG-11 media and diluted to an OD₇₃₀ of 0.2. Cells in liquid media were entrained to two 12-h:12-h light:dark (LD) cycles in 24-well plastic plates with shaking, and H₂O₂ was added to the cultures at a final concentration of 0.01–10 mM at hour 0 or 12 in LL conditions. Following the addition of H₂O₂, each cell suspension was exposed to light or dark for 6 h to imitate the UV+L or UV+D condition. Then, 2 μL aliquot was spotted onto solid media and cultured under LL conditions. The experimental schedule is shown in S6A Fig. Cellular growth was evaluated by densitometric analysis of each spot.

Quantification of glycogen content

Synechococcus cells at an OD₇₃₀ of ~0.3 were grown in BG-11 liquid media at 30°C with air bubbling and irradiation with 40 μmol/m²·s of white fluorescent lamps, and were entrained to two 12-h:12-h light:dark (LD) cycles. At hour 0 or 12 in LL, cells were exposed to darkness and harvested at hour 0, 1, 3 and 6 in darkness for extracting and quantifying the glycogen in the cells. Harvested cells were resuspended with sterilized water and disrupted with 0.1 mm glass beads using a Multi-beads Shocker (Yasui Kikai, Osaka, Japan). The homogenates were boiled for 10 min to inactive the endogenous enzymes, and then insoluble fraction was removed by centrifugation. Glycogen content of supernatant was measured using the Glycogen Colorimetric/Fluorometric Assay Kit (BioVision, Inc., US) in accordance with the manufacturer’s protocol. For colorimetric assay, the absorbance at 570 nm was measured with a microplate reader (Powerscan HT; DS Pharma Biomedical, Ltd., Osaka, Japan). Glycogen contents were determined using a standard curve and normalized by OD₇₃₀ units.