Effects of H2O2 on M. aeruginosa Circadian Rhythms Figure 1. The clock gene transcripts in M. aeruginosa under light and dark conditions after H2O2 treatment. The shaded areas correspond to the dark period. Symbols represent mean 6 SEM of triplicate cultures. The mRNA amount of all examined genes is normalized to 16S rDNA. The open circle corresponds to the control treatment; the filled circle corresponds to the L0 treatment; the filled square corresponds to the D0 treatment. A, The effect of H2O2 on the kaiA circadian transcript; B, the effect of H2O2 on the kaiB circadian transcript; C: the effect of H2O2 on the kaiC circadian transcript; D: the effect of H2O2 on the sasA circadian transcript. doi:10.1371/journal.pone.0033347.g001 the cells were bleached (Figure 5). Chlorophyll a (Chl a) excites optimally at 460 nm (blue light) and emits at 685 nm (red light), this fluorescence is directly proportional to the concentration of Chl a. Therefore, we analyzed the fluorescence intensity of Chl a and found that it was stronger in the control than that in the L0 or D0 treatment (Figure 6), indicating that the content of Chl a decreased after H2O2 exposure. We also quantified several pigments content, and found that the content of Chl a, phycocyanobilin (PC), allophycocyanin (APC) and phycoerythrin (PE) decreased after H2O2 exposure at L0 point. The lowest levels of them were 13.9%, 10%, 15.1% and 17.4% of the control, respectively. Similar phenomenon was also displayed after H2O2 exposure at D0 point (Figure 7). We recorded the change of cell numbers in the first 24 h exposure time, and found that M. aeruginosa growth was very rapid under normal conditions, cell numbers increased from 3.76105/ mL to 5.26105/mL; but it slowed or decreased after H2O2 treatment at L0 and D0, cell number was from 3.86105/mL to 3.46105/mL at L0 point, and 3.66105/mL to 3.06105/mL at D0 point (Figure 8). The intracellular microcystin (MC) content fluctuated in the control group, remaining stable (approximately 11.4 pg/102 cells) during the light period, declining shortly after the lights were turned off. The lowest MC content appeared at D4 point, and then returned afterwards (Figure 9). H2O2 treatment at L0 significantly reduced the MC content at D0 and caused the daily rhythm of the MC content to disappear, as determined by the cosine wave analysis. In the D0 treatment, the MC content showed a little increase during the dark period, followed by a decrease in the subsequent light period, and the lowest level was approximately 5.0 pg/102 cells, which is only half of the control. The results of the cosine wave analysis showed that the daily rhythm of the MC content had also disappeared. We had suspected that H2O2 may directly oxidize MC and cause a decrease in MC content. To test this proposal, we added 50 mM H2O2 to a 2 mg/L MC solution and found that the level of MC did not significantly decrease after 24 to 96 h of exposure (Figure 10). Thus, MC is not oxidized by H2O2. Discussion Water blooms are one of the top environmental issues in the world. M. aeruginosa is a common bloom-forming cyanobacteria in freshwater ecosystems and is of primary concern because of its wide distribution and secretion of toxic secondary metabolites, such as microcystins. Cyanobacteria are the only known bacterial species that display circadian rhythms [14], exhibiting self- sustaining oscillations with an approximately 24-h day-night cycle. These expressions are thought to be indicators of a pacemaker that controls various aspects of physiology and metabolism to adapt to daily environmental fluctuations. In cyanobacteria, approximately 30% to 80% genes exhibit robust oscillating expression profiles, and most of these genes are involved in physiological and metabolic processes [23,24]. Using DNA microarrays, Straub et al. [21] demonstrated that more than 25% of the genes involved in many metabolic pathways in M. aeruginosa exhibit significant rhythmic changes in their transcript abundances. Their results revealed that many of the physiological and metabolic activities that occur in cyanobacteria, such as cell division, nitrogen fixation, photosynthesis, carbon uptake and the biosynthesis of secondary metabolites, are controlled by circadian rhythms. Hydrogen peroxide treatment is considered to be more environmentally friendly than the traditional methods for cyanobacterial removal [8,9]. Hydrogen peroxide can inhibit the growth of M. aeruginosa by blocking the transcription of photosynthesis-related genes or destroying photosynthetic pig- ments [10]. However, a clear mechanistic link among the clock genes, cellular physiological and metabolic rhythms and H2O2 (or other xenobiotics) has not been elucidated. Understanding their circadian is essential for determining the relationship between the circadian clock and the observed physiology in M. aeruginosa. Here, PLoS ONE | www.plosone.org 3 March 2012 | Volume 7 | Issue 3 | e33347