Depletion of Vipp1 results in a Synechocystis strain with less thylakoid membranes. A, Representative electron micrographs of Synechocystis wild-type (wt) and Vipp1 depleted cells are shown. B, Under the presented growth conditions, the wild-type cells (black bars) had on average four to six layers of thylakoid membranes, whereas the mutant strain (gray bars) had only two to three thylakoid layers. Synechocystis cells were grown at 20 μmol/(m² s) as described in “Materials and Methods.”

Absorbance spectra of Synechocystis wild-type and vipp1 merodiploid cells. Cell suspensions were diluted to 3 × 10⁵ cells/mL (for details see the text), and spectra were recorded in the range between 350 to 750 nm. Solid line, Wild-type cells; dashed line, vipp1 merodiploid cells.

Oxygen evolution of wild-type (light bars) and vipp1 merodiploid (gray bars) Synechocystis cells. Cells were grown at different light intensities and oxygen evolution was measured using either PPBQ (top) or HCO₃⁻ (bottom) as electron acceptor. The statistical significance of the observed difference in oxygen evolution was calculated by a one-tailed t test. For all the observed oxygen evolution rates, the differences between the means were due to chance (P > 0.05) and thus statistically not relevant.

Determination of the PSI activity. PSI activity of Synechocystis cells grown at different light intensities was measured as oxygen consumption due to the Mehler reaction (for details, see “Materials and Methods”). Oxygen consumption of wild-type (white bars) and vipp1 merodiploid (gray bars) cells was normalized to 10⁵ cells. The statistical significance of the observed difference in oxygen consumption was calculated by a one-tailed t test. For the observed oxygen consumption rates at 40, 80, and 120 μmol photons/(m² s), the differences were statistically relevant at a 1% level (P < 0.01). Due to the relatively high scattering of the measured rates at 20 μmol photons/(m² s), the observed difference between the means were statistically not relevant (P > 0.05).

77K fluorescence emission spectra of Synechocystis wild-type (solid line) and Δvipp1 cells (dotted line) after excitation of phycobilisomes at 580 nm. Cells were grown at 120 μmol/(m² s). The spectra were normalized at 650 nm. a. u., Arbitrary units.

Separation of membrane protein complexes from wild-type and Vipp1 depleted Synechocystis cells. A, Membrane proteins from Synechocystis wild-type (wt) and Vipp1 depleted cells were solubilized by β-DM and membrane protein complexes were separated on a 10% to 30% Suc density gradient. Three colored bands (I–III) were clearly resolved. In wild-type membranes, band II of the gradient contained about 3.5 times more chlorophyll than band III, whereas in the depletion strain, this ratio was lowered to about 1.8. B, Individual fractions of the gradient from wild-type membranes were analyzed by immunoblot analysis. The individual membrane protein complexes were identified using antibodies directed against PSI (PsaA, B, D) and PSII (PsbA, B) subunits. Fraction 1 corresponds to the top of the Suc gradient.

Coomassie-stained BN-PAGE gel of membrane protein complexes isolated from wild-type (wt) and Vipp1 depleted (Δ) Synechocystis cells. For identification of the photosystems, the individual membrane protein complexes were identified with antibodies directed against PSI and PSII subunits (see Fig. 9 legend) after separation in a second dimension by SDS-PAGE (data not shown). Densities of the PSI monomer and trimer bands in BN-PAGE gels were determined using the program Scion Image (Scion Corp.). Based on these measurements, the wild type contained at least 2.5 times more trimeric over monomeric PSI, whereas in the depletion strain, this ratio was shifted to approximately 0.5. [See online article for color version of this figure.]

Disruption of vipp1 in Synechocystis sp. PCC 6803. A, Strategy for disruption of vipp1 in the genome of Synechocystis sp. PCC 6803. The aphII gene confers resistance to kanamycin (Km^R). Arrows indicate the direction of the aphII and the vipp1 gene. Numbered arrows indicate primers, which were used for segregation control (compare B). B, The vipp1 gene cannot be deleted completely in Synechocystis. Amplification of the vipp1 gene from wild-type (wt) and Δvipp1 Synechocystis genomic DNA resulted only in an amplification of the wild-type fragment (800 bp) and no fragment, which corresponds in size to the interrupted vipp1 gene (approximately 2,000 bp) was observed. When a primer pair was used, one primer of which anneals to the 5′-end of the vipp1 gene and the other anneals to the aphII gene (compare A), a fragment of about 1,200 bp was amplified, which demonstrates that the aphII gene is interrupted in some genomic vipp1 copies. C, Immunoblot analysis of total cellular extracts from Synechocystis wild type and the Vipp1 depletion strain grown at 120 μmol s⁻¹ m⁻². Five micrograms protein have been separated by SDS-PAGE and subsequently analyzed by immunoblot analysis using an α-Vipp1 antibody. For comparison, for the wild-type cell extract, 3.75, 2.5, 1.25, and 0.5 μg protein have also been analyzed.

Chlorophyll content in Synechocystis wild-type and Vipp1 depleted cells. Chlorophyll content of Synechocystis wild-type (light bars) and Vipp1 depleted cells (gray bars) was determined as described in “Materials and Methods” after growing the cells at different light intensities [20, 40, 80, 120 μmol/(m² s)]. Chl is shown for 10⁵ cells.

Light-dependent regulation of the PSI-to-PSII ratio in wild-type and vipp1 merodiploid cells. A and B, 77K fluorescence emission spectra of Synechocystis wild-type (A) and vipp1 merodiploid (B) cells after excitation of chlorophylls at 435 nm. Increase of the growth light intensity [20, 40, 80, 120 μmol/(m² s)] results in a decreased PSI-to-PSII ratio in both the wild-type and Vipp1 depletion strain. At each line, the light intensity, at which the cells were grown, is indicated. Under all tested light intensities, the PSI-to-PSII ratio was decreased when compared to wild-type cells grown under identical growth conditions. For better comparability, the spectra were normalized at 695 nm and the fluorescence emission at 695 nm was set at 1.