[11]. The members of this genus are able to synthesize microcystins (MCs), a group of monocyclic heptapeptides. MCs can cause hepatotoxic diseases and liver cancer by inhibiting the activity of protein phosphatases 1 and 2A, which are key proteins in the regulation of many eukaryotic cell cycles [12,13]. Many physiological activities of cyanobacteria are controlled by its biological clock, which allows these activities to adapt to daily fluctuations in the environment [14]. Agrawal et al. [15] demonstrated that photosynthetic gene transcripts are regulated by a biological clock to achieve efficiency in M. aeruginosa. There are three clock genes, kaiA, kaiB and kaiC, in cyanobacteria, and kaiB and kaiC operate as a unit with a single promoter (independent from the kaiA transcript) [16]. Transcription from either the kaiA or kaiBC operon is under circadian feedback regulation, and circadian rhythms of expression are necessary to maintain normal circadian sustainability [16]. In addition, SasA, a histidine kinase that interacts with the KaiC protein, was found to be required for sustaining robust circadian rhythms [17]. Cell division, amino acid uptake, nitrogen fixation, respiration, and carbohydrate synthesis are also under circadian control in cyanobacteria [18–20]. More recently, Straub et al. [21] demonstrated that many metabolites are controlled by a circadian clock in M. aeruginosa. However, little is known regarding whether xenobiotics affect cell growth, photosynthesis or other metabolic pathways by changing the circadian rhythms of the clock genes or circadian genes. In this study, we comprehensively analyzed the relationship between exogenous H2O2 treatment and the circadian rhythms of the clock genes and circadian genes in M. aeruginosa to determine the effect of H2O2 on its growth and the transport of these stress signals to a related metabolite. Results The effect of H2O2 on the expression patterns of the clock genes in LD-grown cultures The abundances of kaiA, kaiB and kaiC exhibit daily rhythmic patterns in the LD cycle, with peaks after 4 h of light (L4) and troughs after 4–8 h of darkness (Figure 1). A similar oscillation pattern was also observed in the daily expression of sasA mRNA, which reached its peak or trough at the similar time as the kai genes. The diurnal rhythm amplitudes (the ratios of the peak to the trough) of kaiA, kaiB, kaiC and sasA were 3.1, 4.3, 3.5 and 3.5, respectively. To determine whether H2O2 affects the circadian system, we examined the transcripts of the clock genes (kaiA, kaiB, kaiC and sasA) after H2O2 exposure at L0 and D0. In the L0 treatment, H2O2 altered the daily transcript patterns of these clock genes, and the mRNA expression of kaiA displayed a 16-h phase- forward and a peak time shift from L4 to D8; however, the peak mRNA transcript levels of kaiA did not change significantly. The daily rhythms of kaiB, kaiC and sasA disappeared when subjected to cosine wave analysis, as described by Wu et al. [22], although there was a significant daily variation in the mRNA transcript levels of kaiC and sasA (one-way ANOVA, p,0.05). Furthermore, the peak mRNA transcript levels of kaiB, kaiC and sasA decreased to 48.8%, 57.1% and 71.4% of the control (p,0.05), respectively. However, the amplitudes of these three genes were only 2.1, 1.5 and 1.9, respectively, which are less than those of the control. In the D0 treatment, H2O2 altered the daily expression patterns of these clock genes by a 4-h phase delay and a peak time shift from L4 to L8. Interestingly, the abundance and amplitude of these genes also decreased, but their daily rhythms were maintained in an apparently circadian pattern in this treatment, as determined by cosine wave analysis and one-way ANOVA. The Effects of H2O2 on M. aeruginosa Circadian Rhythms peak transcript levels of kaiB, kaiC and sasA decreased significantly (p,0.05), resulting in levels that were 46.5%, 54.3% and 65.7% of the control, respectively. The daily amplitudes of these genes were 2.5, 2.7 and 2.3, respectively. The effect of H2O2 on the expression of photosynthesis- related genes in LD-grown cultures In cyanobacteria, oxygenic photosynthesis is a central metabolic process. To determine whether H2O2 affects the circadian system in the photosynthetic pathway, we investigated the expression of photosynthesis-related genes (psaB, psbD1 and rbcL) after H2O2 treatment. The transcript levels of the photosynthesis-related genes were light-dependent and produced circadian expression patterns (Figure 2). The peak levels of psaB, psbD1 and rbcL mRNA occurred at D0 (the end of light exposure), L4 and L8, respectively. The peak-to-trough ratios for psaB, psbD1 and rbcL were 3.7, 4.0 and 4.0, respectively. H2O2 treatment at L0 significantly decreased the mRNA abundances of three photosynthesis-related genes (p,0.05) and changed their circadian expression patterns. H2O2 treatment reduced the peak abundances of psaB, psbD1 and rbcL to 29.7%, 57.5 and 48.1% of the control, respectively. Their amplitudes were reduced to 33.0%, 57.5 and 47.5% of the control, respectively. The cosine wave analysis showed that H2O2 treatment caused the daily rhythms of psaB and rbcL to disappear, but H2O2 treatment did not alter psbD1 rhythm. H2O2 treatment at D0 also significantly decreased the transcript levels and changed the circadian patterns of the photosynthesis- related genes. The peak abundance of psaB decreased to 27.2% of the control treatment, and the circadian rhythm disappeared, as determined by the cosine wave analysis. The amplitude of psbD1 mRNA expression decreased to 75% of the control, but the daily rhythm was maintained (as in the control). The amplitude of rbcL decreased to 62.5% of the control, and its circadian patterns exhibited a 4-h phase afterward from L8 to D0. The effect of H2O2 on the expression of microcystin- related genes in LD-grown cultures The transcript levels of microcystin-related genes (mcyA, mcyD and mcyH) also showed circadian patterns, with peak values at L8, L4, and D8, respectively (Figure 3). H2O2 treatment at L0 did not significantly alter the peak abundances and amplitudes of these three genes with respect to the control; however, H2O2 treatment differentially affected their phases. Although H2O2 treatment at L0 did not change the daily expression patterns of mcyA and mcyH, it immediately suppressed the increase in mcyD mRNA levels and, thus, greatly delayed the peak phase by 20 h (from L4 to L0). H2O2 treatment at D0 also did not affect the peak abundance and phase of mcyA, but it greatly influenced the expression levels of mcyD and mcyH. H2O2 treatment at D0 significantly delayed the peak phases of mcyD and mcyH mRNA expression by 8 h and 12 h, respectively, with respect to the control. The effect of H2O2 on physiological function in LD-grown cultures The growth of M. aeruginosa was significantly inhibited after H2O2 treatment at L0 or D0 point (Figure 4). The percent inhibition was exhibited in a time-dependent manner, which reached 33.2%, 50.5%, 75.3% and 92.7% after 12, 24, 48 and 96 h of H2O2 exposure at L0 point, respectively. Similar inhibitory effects of H2O2 exposure on M. aeruginosa growth at D0 point were also displayed. By observing cellular morphology, cyanobacterial cells were blue in normal cultural condition, but this color became weaker after H2O2 treatment, suggesting that PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e33347