Effects of H2O2 on M. aeruginosa Circadian Rhythms Figure 4. Effects of H2O2 on the inhibition of M. aeruginosa growth. doi:10.1371/journal.pone.0033347.g004 thesis-related genes responded in the physiological level, such as the retardation of cyanobacterial growth (Figure 8), supporting the opinion again that circadian rhythm might be important for optimal growth [30,31]. The study also analyzed the transcripts of the microcystin- related genes, and demonstrated their circadian patterns under LD conditions. Transcript levels of microcystin biosynthesis genes (mcyA and mcyD) reached their maxima after 4–8 hours of light exposure, while the peak levels of mcyH, the microcystin transport gene, appeared in the dark. These results are similar to those reported by Straub et al. [21], who demonstrated that the biosynthesis of the microcystin genes exhibit circadian rhythms and display their maximum transcript abundance after switching to light exposure. It displayed that microcystin synthesis could be kept in step with photosynthesis, because this process need some substrates related to photosynthesis, while microcystin transport might be taken place in the dark by utilizing ATP produced at degradation of carbohydrate. To verify the rhythm of microcystin at a physiological level, we measured the microcystin content per cell under LD conditions and found that MC content was high during the light cycle and low during the dark cycle, in agreement with the rhythms of the microcystin synthesis-related gene transcripts. These results are similar to the results found by Wiedner et al. [32] in Microcystis strain PCC 7806 and Bittencourt- Oliveira et al. [33] in Microcystis spp. After H2O2 treatment, the circadian of microcystin-related genes transcripts were affected differentially, and MC content and fluctuations were changed, which shows that some physiologies are subjected to the circadian control of circadian genes. It is very common phenomenon that organisms from bacteria to mammals use circadian clock system to adapt to daily environ- mental changes. The signals from the environment affect oscillator circadian rhythm by changing clock genes transcript, which in turn regulates various cellular activities, such as transcription [34,35]. In eukaryotic species, diurnal circadian rhythm has been studied in more detail than in prokaryotic species. Reports have demonstrated that exo-factors (such as food, light) affect circadian rhythms in animals [36,37]. This same phenomenon has also been Figure 5. Morphology of M. aeruginosa cell after H2O2 exposure observed under an optical microscope (61000). A: Control; B: L0 treatment; C: D0 treatment. doi:10.1371/journal.pone.0033347.g005 PLoS ONE | www.plosone.org 5 March 2012 | Volume 7 | Issue 3 | e33347