An Evolutionary Fitness Enhancement Conferred by the Circadian System in Cyanobacteria

Abstract

Circadian clocks are found in a wide variety of organisms from cyanobacteria to mammals. Many believe that the circadian clock system evolved as an adaption to the daily cycles in light and temperature driven by the rotation of the earth. Studies on the cyanobacterium, Synechococcus elongatus PCC 7942, have confirmed that the circadian clock in resonance with environmental cycles confers an adaptive advantage to cyanobacterial strains with different clock properties when grown in competition under light-dark cycles. The results thus far suggest that in a cyclic environment, the cyanobacterial strains whose free running periods are closest to the environmental period are the most fit and the strains lacking a functional circadian clock are at a competitive disadvantage relative to strains with a functional clock. In contrast, the circadian system provides little or no advantage to cyanobacteria grown in competition in constant light. To explain the potential mechanism of this clock-mediated enhancement in fitness in cyanobacteria, several models have been proposed; these include the limiting resource model, the diffusible inhibitor model and the cell-to-cell communication model. None of these models have been excluded by the currently available experimental data and the mechanistic basis of clock-mediated fitness enhancement remains elusive.

Figure 1. Competition experiment between S.elongatus strains with different clock phenotypes [3, 11, 12]

The clock phenotypes of two strains, A and B, are shown as luminescence rhythms that report the promoter activity of psbAI. Strains A and B are resistant to different antibiotics such that their fractions in mixed cultures can be tracked by plating on selective media. Pure cultures of A and B were set up under LL, and when they reached log phase, equal numbers of A and B cells were mixed and cultured under different LD cycles or LL condition. Aliquots were taken from the mixed cultures every ~8 generations in LD and every ~16 generations in LL, and they were plated on selective media to count the number of colony-forming units (CFU) of each strain. Meanwhile, the mixed culture was diluted into fresh medium and grown for another ~8 generations (LD) and ~16 generations (LL). This process was repeated for 4 cycles to allow cells to grow for 40-50 generations. The fraction of each strain in the mixed culture was calculated by the number of colonies of each strain growing on selective media. Circadian phenotypes were confirmed by monitoring the luminescence rhythms of colonies of each strain at different sampling times. Figure modified from [3].

Figure 2. Competition of the WT strain with arhythmic strains [11]

A, phenotypes of three strains used in the competition experiments. The WT strain (top) shows circadian rhythms with a ~25 h FRP. CLAb (middle), a clock-disrupted kaiC mutant, is arhythmic. Another kaiC mutant, CLAc (bottom), is also ultimately arhythmic but initially shows a rapidly damped oscillation. B, competitions between the WT strain and arhythmic mutants under LD 12:12 (upper) or LL conditions (lower). Data are plotted as the fraction of the mutant strain in mixed cultures (ordinate) versus the estimated number of generations (abscissa). Figure modified from [3, 11].

Figure 3. Competition of the WT strain with period-altered mutants under LD 11:11 and LD 15:15 cycles [12]

A, circadian phenotypes of the WT strain and period-altered mutants used in these competition experiments. The short period mutants (FRP ~ 22 h) include the kaiB mutant B22a and the kaiC mutant C22a, and the long period mutants (FRP ~ 30 h) include the kaiA mutant A30a and the kaiC mutant C28a. All strains have a luciferase construct that reports the clock-regulated promoter activity of the psbA1 gene by time-dependent luminescence intensity. B, competitions between the WT strain and the period-altered mutants under LD 11:11 cycles (upper) or LD 15:15 cycles (lower). Data are plotted as the fraction of the mutant strain in the mixed culture versus the estimated number of generations. Symbols for each strain are identified under the abscissa. Figure modified from [3, 12].

Figure 4. Growth curves of the WT strain and the clock mutant (CLAb) in pure cultures that were serially diluted four times

A, pure cultures of the WT strain (blue diamond) and CLAb (red squares) were set up under LL conditions. B, pure cultures of the WT strain and CLAb were set up under LD conditions. After the cells reached the stationary phase, they were diluted 1:1000 into fresh BG-11 medium. When the diluted cultures reached the stationary phase, they were diluted again. The cultures were serially diluted four times. Cell density was measured as OD₇₅₀ value.

Figure 5. Test of the Diffusible Inhibitor Model

A, Depiction of the Diffusible Inhibitor Model. Entrained phases of two strains are modeled in terms of their entrained phase relationship to an LD 12:12 cycle. For the optimally-phased cells, the subjective day overlaps with the daytime (white box), and the subjective night phase overlaps with the nighttime (black box). Therefore, their secretion phase (yellow box) coincides with the daytime, and the sensitive phase (red box) coincides with the nighttime. For non-optimally-phased cells, their subjective day starts from middle of the daytime, and their subjective night starts from the middle of the night and ends in the middle of the daytime. Therefore, the secretion phase (yellow box) of the non-optimal-phased cells is only from the middle of the day to the end of the day, while the secretion-competent phase that overlaps with the dark phase (yellow-black box) does not result in secretion because the secretion is postulated to be light-dependent. The sensitive phase (red box) of the non-optimally-phased cells starts in the middle of the night and ends in the middle of the day. B, the semi-co-culture apparatus used to test the existence of a diffusible inhibitor. Two chambers (left and right) were separated by a membrane with 0.2 µm pores. Cells of different strains could be cultured separately in these two chambers, but their media passes freely through this membrane such that the putative inhibitor could diffuse to the other side. The cultures were illuminated by white fluorescent light from the top, and the light intensity was 50 uE*m⁻²*s⁻¹. C, the WT strain and CLAb were semi-co-cultured in this apparatus under LD 12:12 cycles. Cell densities (OD₇₅₀) were measured on the fifth day. Bars which share the same color represent cultures in the same apparatus. Panel A modified from [3, 12].