Características del testamento

Physiological characterization of the ∆sppA1 mutant did not show any significant difference in the growth under LL when compare to wild-type (see Section 3.1). To analyze the phenotypical modifications of wild-type, ∆sppA1 and pVZsppA1 due to different light intensities the cell batches grown at LL to the end of the exponential phase were diluted to an A750 of0.5 and then transferred to ML and HL or kept at LL for further three days. In contrast to the LL ∆sppA1 mutant behaved differently from the wild-type when the cells were grown at ML and HL. The ∆sppA1 mutant exhibited non-bleaching after the shift to ML and bleached faster under HL relative to the wild-type cells (Fig. 18).

Typical absorption spectra of the wild-type cells display a broad absorption peak with several shoulders that correspond to the Soret region of the chlorophyll a absorption spectrum (440

nm) and to the absorption of various carotenoid bands (above 450 nm). The red region displays two distinct peaks, one centered around 620 nm due to phycobilin-containing proteins, phycocyanin (PC) and allophycocyanin (APC), the other around 680 nm corresponding to chlorophyll a. The absorption spectra of thylakoids extracted from wild-type and mutant strains grown at various light conditions showed that the amounts of phycobilisomes as well as of chlorophyll a and carotenoid decreased within 3 – 4 days after transfer of Synechocystis cells from LL light to HL (Fig. 19). Under ML and HL the wild-type and pVZsppA1 strains revealed lower absorbance of the PC peak than at LL. The ∆sppA1 strain behaved in a similar way at LL and HL. However, although a decrease in chlorophyll a absorption relative to that of carotenoids was observed, the PC/APC absorption peak at 620 nm remained higher in ∆sppA1 under ML. Consequently, the ratio between the chlorophyll a and PC/APC absorption peaks in mutant cells grown under ML remained similar to that grown under LL. These observations suggest that the wild-type adapts to ML by losing more of its PC/APC-containing phycobiliproteins than ∆sppA1.

Figure 18. Phenotypical characterization of wild-type, ∆sppA1 and pVZsppA1 strains upon acclimation to various light intensities. Wild-type, ∆sppA1 and pVZsppA1 were incubated under LL to an A750 of 0.6 and then transferred to LL, ML or HL for 3 days. The phenotypical changes were monitored after 3 days of light acclimation.

Table 8 shows the generation time of wild-type and ∆sppA1 mutant at three light intensities, DL, LL and ML. It has been observed that cell division was light-limited in the 20 - 50 µE m-2 s-1 range. The latter intensity was close to saturation since a further increase in light intensity by a factor of three produced only a moderate increase in growth rates for the two strains. That these growth conditions were not limited by CO2 availability is demonstrated by similar generation times observed, independently whether cultures were bubbled with CO2or not.

Figure 19. Absorbance spectra of thylakoids of the wild-type, ∆sppA1 and pVZsppA1

during acclimation to different light regimes. Synechocystis cells were grown at LL to an A₇₅₀ of 0.5. Cells were then transferred to ML or HL, or kept at LL for the next three days. Thylakoid membranes were isolated from cultures and adjusted to the same A₇₅₀ before determining the spectra.

TABLE 8. Doubling time of wild-type and ∆sppA1 mutant at different light intensities

Strain and light intensity Doubling time (h) Cell concentration _{(number/ml) at T72}a

wild-type (DL; 20 µE m-2 _s-1_{) 36.3} ± 3.0 8.0 ± 0.3 x 108 ∆sppA1 (DL; 20 µE m-2 _s-1_{) 35.6} ± 1.5 8.4 ± 0.4 x 108 wild-type (LL; 50 µE m-2 _s-1_{) 11.4} ± 0.9 26.0 ± 0.2 x 108 ∆sppA1 (LL; 50 µE m-2 _s-1_{) 10.5 ± 1.8 29.5 ± 0.3 x 10}8 wild-type (ML; 150 µE m-2 _s-1_{) 10.0} ± 1.7 40.1 ± 0.1 x 108 ∆sppA1 (ML; 150 µE m-2 _s-1_{) 9.8} ± 0.8 44.5 ± 0.3 x 108 wild-type (HL; 350 µE m-2 _s-1_{) 40.0} ± 0.7 17.6 ± 0.8 x 108 ∆sppA1 (HL; 350 µE m-2 _s-1_{) 36.8 ± 0.2 24.5 ± 0.2 x 10}8 wild-type (3% CO2; 50 µE m-2 s-1) 12.0 ± 0.3 21.7 ± 0.4 x 108 ∆sppA1 (3% CO2; 50 µE m-2 s-1) 11.1 ± 0.8 26.1 ± 0.7 x108 wild-type (3% CO2; 150 µE m-2 s-1 ) 10.3 ± 1.2 38.7 ± 0.2 x 108 ∆sppA1 (3% CO2; 150 µE m-2 s-1) 9.1 ± 1.1 44.0 ± 0.1 x 108 a _T

For the analysis of pigment contents in wild-type and ∆sppA1 strains during adaptation to different light regimes cyanobacterial cells were acclimated to LL, ML and HL for 3 days and the concentration of phycocyanin and chlorophyll a was measured for 3 days every 12 h. Measurement of the pigment concentrations per optical density of cells (A750 = 0.4) showed that the PC pool in the wild-type decreases when cells are adapted to ML and HL, in the

∆sppA1 strain its amount remained constant under ML as under LL. Under HL both strains, wild-type and ∆sppA1, contained almost equal amount of PC (Fig. 20).

Figure 20. Contents of chlorophyll and phycobiliproteins in wild-type and ∆sppA1 cells under different light regimes. Synechocystis cells were grown at LL till log-phase, diluted with BG-11 medium and transferred to LL, ML and HL for 3 days. The measurements were taken from the beginning of acclimation. Chlorophyll (A) and phycocyanin (B) concentrations at A750 of 0.4 were measured.

The loss of phycobiliproteins in Synechocystis wild-type is well documented under nitrogen deprivation. To check whether SppA1 inactivation also resulted in the preservation of PBS under these conditions, wild-type and ∆sppA1 were grown in nitrogen-depleted medium for 3 days (Fig. 21). The loss of PBS was visualized by the loss of the PC/APC absorbance peak in the 620 nm region of the spectrum. The same decrease in PC/APC absorbance was observed in both strains showing that SppA1 plays no role in the PBS loss under nitrogen starvation.

Figure 21. Phenotypical modifications and absorption spectra of wild-type and ∆sppA1

mutant during nitrogen starvation. Wild-type cells and ∆sppA1 mutant were incubated under LL till A750 of 0.6 and then transferred to BG-11 medium depleted in nitrogen. The phenotypical changes and spectra were monitored after 3 days of deprivation.