Sll1252 Coordinates Electron Transport between Plastoquinone and Cytochrome b6/f Complex in Synechocystis PCC 6803

Abstract

A mutant, Δsll1252^ins, was generated to functionally characterize Sll1252. Δsll1252^ins exhibited a slow-growth phenotype at 70 µmol photons m^-2 s^-1 and glucose sensitivity. In Δsll1252^ins, the rate of PSII activity was not affected, whereas the whole chain electron transport activity was reduced by 45%. The inactivation of sll1252 led to the upregulation of genes, which were earlier reported to be induced in DBMIB-treated wild-type, suggesting that Sll1252 may be involved in electron transfer from the reduced-PQ pool to Cyt b₆/f. The inhibitory effect of DCMU on PSII activity was similar in both wild-type and Δsll1252^ins. However, the concentration of DBMIB for 50% inhibition of whole chain electron transport activity was 140 nM for Δsll1252^ins and 300 nM for wild-type, confirming the site of action of Sll1252. Moreover, the elevated level of the reduced-PQ pool in Δsll1252^ins supports that Sll1252 functions between the PQ pool and Cyt b₆/f. Interestingly, we noticed that Δsll1252^ins reverted to wild-type phenotype by insertion of natural transposon, ISY523, at the disruption site. Δsll1252-N^trn, expressing only the C-terminal region of Sll1252, exhibited a slow-growth phenotype and disorganized thylakoid structure compared to wild-type and Δsll1252-C^trn (expressing only the N-terminal region). Collectively, our data suggest that Sll1252 regulates electron transfer between the PQ pool and the Cyt b₆/f complex in the linear photosynthetic electron transport chain via coordinated function of both the N- and C-terminal regions of Sll1252.

Keywords: PQ pool; Sll1252; photosynthetic electron transport; suppressor mutant.

Figure 1

Multiple sequence alignment of the Sll1252 from Synechocystis sp. PCC 6803 and its homologous proteins in other cyanobacterial genomes, algae and higher plants. The sequences were aligned by the ClustalW algorithm. Identical amino acids are shown in black and conservative substitutions are in gray. * indicates algae and higher plants. NP_001044235.2, Oryza sativa; ACG39001.1, Zea mays; AT1G53120.1, Arabidopsis thaliana; XP_001690978.1, Chlamydomonas reinhardtii; Synpcc7942_1503, Synechococcus elongatus PCC7942; syc2488_d, Synechococcus elongatus PCC6301; alr2890, Anabaena sp.PCC7120; Tery_4198, Trichodesmium erythraeum IMS101; MAE24350, Microcystis aeruginosa NIES-843; sll1252, Synechocystis sp. PCC6803; gll0391, Gloeobacter violaceus PCC7421; Syncc9902_0525, Synechococcus sp. CC9902; Syncc9605_2152, Synechococcus sp. CC9605; PMT1433, Prochlorococcus marinus MIT9313; SynWH7803_1983, Synechococcus sp. WH 7803. The S4-domain is labeled. The site of kan^r insertion in Δsll1252^ins is indicated with an open arrow.

Figure 2

Strategy for disruption of sll1252 gene in the genome of Synechocystis sp. PCC 6803. The wild-type copy of the sll1252 gene was completely replaced by ∆sll1252^ins. (A) A 957 bp DNA fragment corresponding to sll1252 was insertionally inactivated kan^r (1200 bp). (B) Schematic representation of the genotype of ∆sll1252^ins. The sll1252 and kan^r cassette are shown in light gray and dark gray arrows, respectively. Thick arrows indicate sll1252-F (FP) and sll1252-R (RP) primers used for PCR amplification of the wild-type copy of sll1252 gene and that of the kan^r cassette. M represents 1 kb DNA ladder; WT, PCR product with wild-type DNA as template; 1252^ins, PCR product with Δsll1252^ins DNA as template.

Figure 3

Effect of light intensity on growth profiles of wild-type and Δsll1252^ins. (A) Photo-autotrophic growth of wild-type (open circles) and Δsll1252^ins (closed circles) at optimal light. (B) Photo-autotrophic growth of wild-type (open circles) and Δsll1252^ins (closed circles) at low light. Photo-heterotrophic growth in the presence of 5 mM glucose and 10 µM of DCMU of wild-type (open triangles) and Δsll1252^ins (closed triangles). Growth under mixotrophic conditions (5 mM glucose) of wild-type (open squares) and Δsll1252^ins (closed squares). Standard deviations are shown in vertical bars. h, hours.

Figure 4

DNA microarray analysis to compare the gene expression changes in ∆sll1252^ins and wild-type cells. RNA extracted from wild-type and ∆sll1252^ins cells was used to synthesize Cy3- and Cy-5-labeled cDNAs, respectively. (A) Cy5/Cy3 ratio of almost all the genes was between 2.0 and 0.5 (indicated by dashed lines), implying no alteration in the gene expression profile at low light. (B) Cy5/Cy3 ratio of several genes were altered in Δsll1252^ins when grown at optimal light, suggesting that the insertional inactivation of sll1252 affected the gene expression. Similar results were obtained in two independent experiments, and the figure represents one of the experiments.

Figure 5

Effect of varying concentrations of DCMU and DBMIB on photosystem II and whole chain electron transport, respectively. Rates of uninhibited/inhibited (V₀/V_i) activities plotted as a function of DCMU (A) and DBMIB (B) concentrations. Three independent experiments were performed, and the data are represented as mean ± SD: wild-type cells (filled circles) and Δsll1252^ins cells (open circles). Standard deviations are shown as vertical bars.

Figure 6

(A) Schematic representation of the genotype of ∆sll1252^ins-Rev. Insertional inactivation of sll1252 was suppressed by spontaneous insertion of naturally occurring transposon, ISY523. An open arrow indicates ISY523. Supp-F (SF) and Supp-R (SR) primers are used to amplify DNA fragments covering the sll1252 ORF. KAN-2 FP-1 (FP1) and KAN-2 RP-1 (RP1) kanamycin-specific primers were used for sequencing to locate the position of spontaneous insertion of ISY523. (B) Schematic representation of the genotype of Δsll1252^del cells. A 1780 bp DNA fragment corresponding to sll1252 ORF was replaced with kan^r (1200 bp). PCR analysis with the primers as indicated in the construct map; M represents 1 kb plus DNA ladder; WT, PCR product with wild-type DNA as template; 1252^del, PCR product with Δsll1252^del DNA as template. (C) Schematic representation of the genotype of Δsll1252-N^trn. A 453 bp DNA fragment corresponding to 5′ region of sll1252 was deleted using fusion PCR, as described in Materials and Methods. PCR analysis with the primers as indicated in the construct map; WT, PCR product with wild-type DNA as template; N^trn, PCR product with Δsll1252-N^trn DNA as template. (D) Schematic representation of the genotype of the Δsll1252-C^trn mutant. A 327 bp DNA fragment corresponding to 3′ region of the sll1252 gene was deleted using fusion PCR. PCR analysis with the primers as indicated in the construct map; WT, PCR product with wild-type DNA as template; C^trn, PCR product with Δsll1252-C^trn DNA as template. Thick arrows in panels B, C, and D indicate UF and DR primers used for PCR amplification of the wild-type and mutant copy including kan^r cassette. sll1252 and kan^r are shown in light gray and dark gray arrows, respectively. The deleted part of sll1252 is shown with dotted lines in Δsll1252-N^trn and Δsll1252-C^trn construct maps.

Figure 7

Changes in cell morphology due to truncation of 5′ and 3′ regions of the sll1252 ORF. Representative electron micrograph of (A) wild-type cells, (B) Δsll1252-N^trn, and (C) Δsll1252-C^trn cells. The cells were grown at optimal light for 24 h and then fixed for transmission electron microscopic observations. Carboxysomes (C), thylakoid membrane (T), and polyphosphate bodies (P) are indicated by arrows. Black spherical bodies (S) are seen only in the Δsll1252-N^trn mutant. Scale bar = 500 nm.

Figure 8

The model presented summarizes the function of Sll1252. (A) As the PQ pool is a common electron carrier and receives electrons from the NDH complex and PSII, the size of the PQH₂ is represented with a bigger oval shape than the oxidized PQ. Sll1252 is probably associated with the Cyt b₆/f complex. Electron transfer is regulated by the Sll1252 N- and C-terminal regions depending on the efficiency of the electron transport chain, thus balancing the functional coordination between PSII and PSI. For simplification, cyclic electron transport, terminal oxidases, and ATP synthase complexes are not shown. (B) Inhibition of electron transfer from the PQ pool to the Cyt b₆/f complex due to the expression of the C-terminal region of Sll1252. The expression of the C-terminal leads to a further increase in the reduced-PQ pool. (C) Expression of the N-terminal region of Sll1252 has no effect on electron transfer between the PQ pool and the Cyt b₆/f complex.