Characterization and evolution of tetrameric photosystem I from the thermophilic cyanobacterium Chroococcidiopsis sp TS-821

Abstract

Photosystem I (PSI) is a reaction center associated with oxygenic photosynthesis. Unlike the monomeric reaction centers in green and purple bacteria, PSI forms trimeric complexes in most cyanobacteria with a 3-fold rotational symmetry that is primarily stabilized via adjacent PsaL subunits; however, in plants/algae, PSI is monomeric. In this study, we discovered a tetrameric form of PSI in the thermophilic cyanobacterium Chroococcidiopsis sp TS-821 (TS-821). In TS-821, PSI forms tetrameric and dimeric species. We investigated these species by Blue Native PAGE, Suc density gradient centrifugation, 77K fluorescence, circular dichroism, and single-particle analysis. Transmission electron microscopy analysis of native membranes confirms the presence of the tetrameric PSI structure prior to detergent solubilization. To investigate why TS-821 forms tetramers instead of trimers, we cloned and analyzed its psaL gene. Interestingly, this gene product contains a short insert between the second and third predicted transmembrane helices. Phylogenetic analysis based on PsaL protein sequences shows that TS-821 is closely related to heterocyst-forming cyanobacteria, some of which also have a tetrameric form of PSI. These results are discussed in light of chloroplast evolution, and we propose that PSI evolved stepwise from a trimeric form to tetrameric oligomer en route to becoming monomeric in plants/algae.

Figure 1.

Thylakoid Membrane Solubilization and Photosystem Identification. (A) BN-PAGE of solubilized thylakoid membranes from Chroococcidiopsis sp strain TS-821 (CH) and T. elongatus BP-1 (TE). Thylakoid membranes containing 0.2 mg/mL chlorophyll were solubilized in different concentrations of the detergent DDM and loaded on a 4 to 16% BN-PAGE gel. The numbers above each lane indicate the concentration of DDM (w/v %). Soluble protein marker positions are indicated on left. (B) BN-PAGE of 1% DDM solubilized thylakoid membranes (0.2 mg/mL chlorophyll) from TE and CH. Visible bands before staining, as labeled, were cut out and denatured in SDS-PAGE sample solubilization buffer for subsequent SDS-PAGE. (C) SDS-PAGE of proteins from BN-PAGE. Characteristic subunit bands for PSI (asterisks) and PSII (circles) from TE are used to identify photosystems in CH. The identification results are shown underneath each lane with the inference of monomer (Mo), dimer (Di), trimer (Tr), tetramer (Te), and uncertainty (?) indicated. Silver stain was used to visualize SDS-PAGE bands. [See online article for color version of this figure.]

Figure 2.

Isolation of TS-821 PSI Tetramer and Proof of Tetramer presence. (A) PSI tetramer (Tet) isolation from solubilized TS-821 thylakoid membrane using Suc gradient ultracentrifugation. (B) PSI separation in Suc gradient after second ultracentrifugation. Tetramer isolated from thylakoid membranes was dialyzed and loaded on a second Suc gradient to further purify the tetramer. Four bands as labeled were isolated and loaded on BN-PAGE for analysis. (C) BN-PAGE analysis of PSI oligomers isolated from Suc gradient ultracentrifugation. Image was taken after Coomassie blue staining. (D) STEM image of isolated T. elongatus PSI trimer. (E) STEM image of isolated T. elongatus PSI monomer. Arrow heads point to examples of monomeric PSI aggregation. (F) TEM image of isolated TS-821 PSI tetramer. Bars = 50 nm. [See online article for color version of this figure.]

Figure 3.

Final Two-dimensional Maps of TS-821 PSI after Single Particle Averaging with Classification. (A) Close-to nontilted map of tetramers, best 1024 particles out of 5000. (B) Map of frame A, with 2-fold rotational symmetry imposed. (C) A typical tilted map of 2048 summed particles from a homogeneous class of in total 10,000 projections. (D) Map of biochemically isolated PSI dimer, best 2048 particles out of 8000. Bar = 10 nm.

Figure 4.

Visualization of Tetrameric PSI Complexes in TS-821 Thylakoid Membrane. (A) Overview of a part of a double thylakoid membrane depicted in a thick layer of negative stain (2% uranyl acetate). Some visible tetrameric PSI particles are marked by boxes. (B) A gallery of 24 particles, selected from a series of membranes. (C) An average of the best 47 projections (out of 150 selected), presented on the same scale as the membrane. (D) Same average as in (C), magnified 2×. Bar = 100 nm. [See online article for color version of this figure.]

Figure 5.

Purified PSI Tetramer Dissociation upon Detergent Treatment. (A) BN-PAGE analysis of purified PSI tetramer treated using different amounts of the detergent DDM. The final concentrations of DDM (w/v %) are shown on top of each lane. The final concentration of chlorophyll for each lane is 0.15 mg/mL. (B) Percentages of PSI oligomers under different DDM concentrations. The percentages of TS-821 PSI monomer (filled circle), dimer (filled triangle), trimer (open square), and tetramer (open circle) under different concentrations of DDM are shown.

Figure 6.

Properties of TS-821 PSI Tetramer. (A) Low-temperature fluorescence of TS-821 PSI monomer (M), dimer (D), and tetramer (Tet), compared with T. elongatus (TE) PSI trimer and monomer. (B) Low-temperature fluorescence of TS-821 whole cell and isolated PSI compared with those from T. elongatus. (C) AUC analysis of TS-821 PSI tetramer. The dashed line denotes previously published S value of the PSI trimer from T. elongatus (Iwuchukwu et al., 2010). (D) Thermostability of PSI tetramer inferred from the CD spectra. Triangles indicate the maximum absorbance around 515 nm and circles show those values around 705 nm. (E) PSI tetramer reduction using T. elongatus cyt c₆. Different concentrations of cytochrome were used. Circle, square, triangle, and upside down triangle show the cytochrome c to PSI ratio of 0, 10, 20, and 40, respectively. (F) Photooxidation of TS-821 PSI monomer, dimer, and tetramer with different light intensities. The same graph with linear light intensity axis is shown in the embedded panel, which shows the saturation of the oxidation. [See online article for color version of this figure.]

Figure 7.

Cloning and Genomic Organization of psaL Genes. (A) Cloning of the TS-821 psaL gene was achieved through several steps. First, partial psaL sequence was amplified using primers (1 and 2) derived from conserved consensus in heterocyst-forming cyanobacteria psaL genes (see Methods for details). With known sequences inside the TS-821 psaL gene, primer 3 and primer 4 were designed, both of which independently amplified a flanking region of psaL. Primer 5 and primer 6 were designed to confirm the presence of a single psaL. The genes were identified using translated DNA sequences. (B) Cyanobacterial psaL genomic locus comparison. Most cyanobacterial psaL genes locate either adjacent to psaF, psaJ, and gmk (heterocyst-forming cyanobacteria or related) or adjacent to psaI (other groups). psaF, pasJ, psaL, psaI, and gmk genes are shown in orange, yellow, green, red, and blue, respectively. Other genes are shown in gray. Rulers are shown separately for (A) and (B), in units of base pairs.

Figure 8.

Alignment of Selected PsaL Sequences and Phylogenetic Tree Based on PsaL Protein Sequences. (A) Alignment of TS-821 PsaL (TS-821, sequence acquired in this study) with other PsaL sequences from T. elongatus BP-1 (TE), Synechocystis, Chlamydomonas, and Arabidopsis. Putative transmembrane helices are highlighted according to the available structure in PDB, 1JB0. The major differences of PsaL sequences among different species are underlined. (B) Phylogenetic tree of cyanobacteria and plastids based on PsaL protein sequences. Heterocyst-forming cyanobacteria are denoted with filled diamonds, while other filamentous cyanobacteria are labeled with open diamonds. Known PSI trimers (triangles) and tetramers (squares) are labeled at the end of the species’ names. Bootstrap values of maximum likelihood and neighbor joining methods are shown at each branch point, upper and lower, respectively. Viridiplantae and Rhodoplantae are denoted as V and R in circles, respectively. Three major cyanobacterial clades are numbered as I, II, and III in circles as well.