Involvement of the SppA1 peptidase in acclimation to saturating light intensities in Synechocystis sp. strain PCC 6803

Abstract

The sll1703 gene, encoding an Arabidopsis homologue of the thylakoid membrane-associated SppA peptidase, was inactivated by interposon mutagenesis in Synechocystis sp. strain PCC 6803. Upon acclimation from a light intensity of 50 to 150 microE m(-2) s(-1), the mutant preserved most of its phycobilisome content, whereas the wild-type strain developed a bleaching phenotype due to the loss of about 40% of its phycobiliproteins. Using in vivo and in vitro experiments, we demonstrate that the DeltasppA1 strain does not undergo the cleavage of the L(R)(33) and L(CM)(99) linker proteins that develops in the wild type exposed to increasing light intensities. We conclude that a major contribution to light acclimation under a moderate light regime in cyanobacteria originates from an SppA1-mediated cleavage of phycobilisome linker proteins. Together with changes in gene expression of the major phycobiliproteins, it contributes an additional mechanism aimed at reducing the content in phycobilisome antennae upon acclimation to a higher light intensity.

FIG. 1.

Growth phenotype (A) and absorbance spectra (B) of the wild type (WT) and the ΔsppA1 mutant during acclimation to the ML regime and nitrogen deprivation. Synechocystis cultures were grown under LL to an OD₇₅₀ of 1.0. Cells were then transferred to ML or kept at LL for the next 3 days. For nitrogen limitations, cells were grown in BG11 medium and then transferred to nitrogen-free BG11 (-N) medium for 3 days as described in Materials and Methods. The spectra were measured on whole cyanobacterial cells. Bold line, wild type; dashed line, ΔsppA1 mutant.

FIG. 2.

Cell growth and pigment analysis of the wild type and the ΔsppA1 mutant strain under the ML regime. (A) Synechocystis cells were grown first under LL till exponential phase, diluted with BG11 medium, and transferred to ML for 72 h. The cell growth (A) and Chl (B) and PC (C) concentrations were measured as percentages per OD₇₅₀. Bold line, wild type; dashed line, ΔsppA1 mutant.

FIG. 3.

77K excitation spectra of the wild type and the ΔsppA1 strain with LL and ML regimes. Synechocystis cells of the wild type and the ΔsppA1 mutant were grown in LL and adapted to ML for 36 h. The excitation spectra were recorded for PSII emission at 695 nm. Bold line, wild type; dashed line, ΔsppA1 mutant.

FIG. 4.

Biochemical analysis of major photosynthetic complexes. Thylakoid membrane proteins isolated from cyanobacterial cells adapted for 3 days to LL and ML were separated by 12% PAGE. The major photosynthetic proteins were visualized by immunodetection with antisera raised against the β subunit of ATP synthase, PsaA/B reaction center proteins of PSI, the D1 protein of PSII, the Rieske FeS protein of cytochrome b/f complex, and major bilin-containing proteins of PBS antennae. The proteins were normalized to the content in the β subunit of ATP synthase. WT, wild type.

FIG. 5.

Evolution of whole-cell content in PC^α in vivo. Wild-type (WT) and ΔsppA1 strains were grown in LL till mid-log phase and then diluted with BG11 to an OD₇₅₀ of 0.5 (point 0) and transferred to ML or kept in LL for 36 h. Cells were taken after 12, 24, or 36 h in ML and after 36 h in LL. Total proteins were extracted from cells at the same OD₇₅₀ and separated by 12% SDS-PAGE. Proteins were visualized by Coomassie blue staining. Coomassie-stained cell proteins from the wild type (A) and visualization of PC^α and PC^β bands during acclimation to ML (B) are shown. The upper part of the panel with α- and β-ATPase subunits was used as a loading control.

FIG. 6.

Analysis of protein translation rate in the ΔsppA1 mutant and the wild type under various light regimes by pulse labeling with l-[³⁵S]methionine. Cells were adapted to LL and ML for 12, 24, or 36 h. Cells were labeled with l-[³⁵S]methionine as described in Materials and Methods, after immediate transfer to various light regimes for 30 min (lane 0.5) and after each time point of incubation under ML. The whole-cell proteins were separated by 12% SDS-PAGE, and the gel was fluorographed in a Fuji phosphorimager.

FIG. 7.

Degradation of linker polypeptides of PBS antennae. (A) Wild-type and ΔsppA1 mutant cells were grown under LL and then transferred to the ML regime for the next 72 h. Thylakoid membrane proteins were separated, transferred onto nitrocellulose membranes, and immunodetected with antisera against the various linker proteins: membrane linker L_CM⁹⁹ and rod linkers L_R³⁵ and L_R³³. Protein loading was normalized to the β-ATPase subunit as shown in Fig. 4. (B) Gene expression of sppA1 under LL and ML. RNA was extracted from the wild-type and mutant strains grown under LL and then acclimated to ML for 36 h. SppA1 transcripts were identified by hybridization analysis with a gene-specific probe. (C) PBS were isolated from wild-type, pVZsppA1-complemented, and ΔsppA1 cells grown in LL. Isolated PBS were incubated at 4°C in the dark (lanes 1, 3, and 5) and at 37°C under ML (lanes 2, 4, and 6) for 3 h. The reaction was stopped by placing the samples on ice. Proteins were separated by 12% SDS-PAGE. For protein visualization, the gel was silver stained.