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.

1. Introduction

Cyanobacteria are primitive photosynthetic organisms and are considered to be ancestors of higher plant chloroplast [1,2,3,4,5]. Similarly, to the higher plants, the electron transport machinery, photosystem-II (PSII) and -I (PSI), are arranged in the thylakoid membranes. PSII comprises several polypeptides ranging from 3 to 60 KDa. Various proteomic studies and high-resolution electron microscopy [6,7,8,9,10] have helped identify more than 20 proteins associated with different cofactors that participate in redox reactions. Kashino et al. [6] reported the association of five novel proteins, Sll1252, Sll1130, Sll1390, Sll1414, and Sll1638, with highly purified PSII core complexes from the cyanobacterium Synechocystis PCC 6803 (hereafter Synechocystis). Subsequently, it was demonstrated that Sll1130 is not associated with the PSII complex; rather, it is a MazF toxin of a toxin–antitoxin system mediating heat-induced programmed cell death in Synechocystis [11]. Moreover, the Pakrasi group [6,12,13,14] worked with the remaining four novel polypeptides. Sll1638 is found to be a true homolog of higher plant PsbQ [12] along with PsbP, which modulates Ca²⁺ and Cl⁻ required for water splitting [15]. Sll1390 associates with the PSII complex to protect it from photodamage and oxidative stress, accelerating the repair mechanism [13], and Sll1414 is suggested to be involved in PSII biogenesis [14]. Sll1252 seems to be in the downstream of the signal transduction pathway and might sense the redox state of the plastoquinone (PQ) pool for balancing the electron flow between the photosystems [16]. The PQ pool in the thylakoid membrane relays electrons from PSII to PSI via the Cyt b₆/f complex by reduction and re-oxidation. Cyanobacteria are known to coordinate the function of photosystems by sensing different light regimes and regulating the genes that code for proteins of photosynthetic complexes, thereby balancing the electron flow between the photosystems [17,18]. The functional coordination between the photosystems is maintained by regulating the stoichiometry of PSI/PSII [17,18,19]. Whole-transcriptome analysis under high light stress revealed that most of the PSII genes undergo transient downregulation at a short acclimation period except for the D1 protein, while the transcripts accumulate after prolonged exposure. In contrast, the downregulated expression of PSI genes implies a significant inflation of the PSII/PSI ratio during high light stress [19]. Subsequently, a small regulatory RNA PsrR1 has been demonstrated to post-transcriptionally regulate the expression of photosystem genes during high light-stress conditions [20]. The post-transcriptional regulatory mechanism describes a new mode of action to balance functional coordination between photosystems. In addition, mechanisms such as state transitions (due to the mobilization of phycobilisome and energy spillover) for rapid regulation of electron flow and balance between photosystems have also been reported [17,21,22,23,24,25].

The redox state of the PQ pool in turn regulates the expression of many genes of photosynthetic complexes to cope with irregularities and influences the downstream cellular processes [18,26,27,28]. However, the factors that sense the redox state of the PQ pool and balance the electron flow between the photosynthetic complexes are not yet completely understood. In this study, we have elucidated the function of an essential protein, Sll1252, as a regulator of electron transfer between the PQ pool and the Cyt b₆/f complex in the linear photosynthetic electron transport in Synechocystis.

2. Materials and Methods

2.1. Cell and Culture Conditions

A glucose-tolerant strain of Synechocystis (wild-type) was initially obtained from Dr. J.G.K. Williams, Dupont de Nemours [29]. Wild-type cells were grown photo-autotrophically at 34 °C in BG-11 medium buffered with 20 mM HEPES-NaOH (pH 7.5), with a continuous supply of sterile air and illumination from incandescent lamps, as described previously [30]. Light intensities used for growth were 70 µmol photons m⁻² s⁻¹ (optimal light) or 5 µmol photons m⁻² s⁻¹ (low light). Cells were grown under continuous light illumination of 5 µmol photons m⁻² s⁻¹ in BG-11 media supplemented with 5 mM glucose for photo-mixotrophic growth, and 10 µM DCMU along with 5 mM glucose for photo-heterotrophic growth, respectively. Growth of the cultures was monitored by measuring OD at 730 nm using a UV-visible spectrophotometer (Model: UV-VIS 160A Shimadzu Co., Kyoto, Japan).

2.2. Insertional Inactivation of sll1252 by Transposon Mutagenesis (Δsll1252^ins)

Δsll1252^ins mutant was generated by insertional inactivation of the gene, as described by Krishna et al. [31]. A DNA fragment containing the sll1252 ORF with 80 bp upstream and 97 bp downstream flanking regions was PCR-amplified using primers sll1252-F and sll1252-R (

Table A1. List of primers used to generate DNA constructs and sequencing reactions.

Sl. No	Primer Name	Sequence
1	sll1252-F	5′-AAC CAT AGG CGC ATT GTA GCT CCT TG-3′
2	sll1252-R	5′-GAT ATC AAC ATT ATC CTT TGC CTC GAA CG-3′
3	KAN-2 FP-1	5′-ACC TAC AAC AAA GCT CTC ATC AAC C-3′
4	KAN-2 RP-1	5′-GCA ATG TAA CAT CAG AGA TTT TGA G-3′
5	Supp-F	5′-AGA TCT GCC AAG ATA ATT GAT TG-3′
6	Supp-R	5′-AGA TCT GTC GAC ATT CCA CTG GCA AAA-3′
7	UF	5′-GCA TCA ATT TGT TCT GTC AC-3′
8	UR	5′-AGA TCT CGA GCA ACG GCA TTA GCT-3′
9	DF	5′-CGT TGC TCG AGA TCT CCA GTT TCA AGG CTT GCC-3′
10	DR	5′-CCA CAA TTA AAG TTG TAC TG-3′
11	KAN-2 SalI-F	5′-TCG TCG ACC AAC CAT CAT CGA TGA ATT-3′
12	KAN-2 SalI-R	5′-TCG TCG ACA AAG CCG CCG TCC CGT CAA-3′
13	sll1252-trn-UR	5′-CGA GCA ACG GCA TTA GCT AA-3′
14	sll1252-Ntrn-F	5′-TAA TGC CGT TGC TCG ATG GTG CGT ACT GTA-3′
15	sll1252-Ntrn-R	5′-CCC GGG TTA AAG ATA TCG GGT-3′
16	sll1252-Ntrn-DF	5′-TAT CTT TAA CCC GGG CCA GTT TCA AGG CTT-3′
17	sll1252-trn-DR	5′-CCC GGG TTA CTG GGT TAA ATG AAG ACT-3′
18	sll1252-Ctrn-F	5′-TAA CCC AGT AAC CCG GGC CAG TTT CAA GGC TT-3′

). The resulting 957 bp fragment was ligated to the pT7Blue TA cloning vector using GeNei^TM instant cloning kit (Cat No: 107416, Bangalore Genei Pvt. Ltd., Bengaluru, India). The resultant plasmid, pT-sll1252, was used to inactivate the sll1252 ORF by performing in vitro transposon reaction using the manufacturer’s instructions (EZ::Tn5^TM <KAN-2> Insertion kit, Cat No: EZ1982K, Epicentre, Madison, WI, USA). The plasmid DNA construct in which sll1252 had been disrupted by kanamycin resistance gene cassette (kan^r) was designated as psll1252:: kan^r. This construct was used to transform the wild-type strain. The site of kan^r insertion was located by sequencing psll1252::kan^r using KAN-2 FP-1 primer (Table A1). The mutant strain thus generated was named Δsll1252^ins.

2.3. Screening for Spontaneous Suppressor Mutants of Δsll1252^ins

Suppressor mutant colonies were isolated from Δsll1252^ins and streaked on BG-11 agar plates containing 5 mM glucose and kanamycin (25 µg per mL) under optimal light. After 2 to 3 weeks of incubation, selected colonies were grown in the liquid BG-11 media and genomic DNA was isolated. Specific primers Supp-F and Supp-R (Table A1) located at 146 bp upstream and 49 bp downstream to sll1252, respectively, were used for PCR. The resulting PCR-amplified product was sequenced using the kanamycin-specific primers KAN-2 FP-1 and KAN-2 RP-1 (Table A1).

2.4. Deletional Mutagenesis of the sll1252 Gene (Δsll1252^del)

A 500 bp upstream and a 500 bp downstream flanking region of sll1252 ORF from the genomic DNA were amplified using primer sets UF and UR; DF and DR, respectively. UF and DR primers contained BglII site. The PCR fragments thus generated using UF-UR and DF-DR primer sets were used to set up a fusion PCR with the UF-DR primer set. The fusion PCR-amplified product thus generated was cloned into pMD19 T-Vector (Takara-Bio., Otsu, Japan). The resultant construct was named pT-sll1252^del. kan^r was amplified using the primers containing SalI restriction site: KAN-2 SalI-F and KAN-2 SalI-R (Table A1). SalI-digested Kanamycin gene was cloned onto the BglII site of the pT-sll1252^del by blunting followed by ligation. The mutant was generated by natural transformation in wild-type cells and named Δsll1252^del.

2.5. Construction of sll1252 N-Terminal Truncated Mutant (Δsll1252-N^trn)

A 500 bp upstream flanking region and 327 bp toward the end of the sll1252 ORF covering the S4-domain were amplified from the genomic DNA using primer sets UF and sll1252-trn-UR; sll1252-N^trn-F and sll1252-N^trn-R, respectively. sll1252-N^trn-F primer was designed with 15 nucleotides complementary to the 5′ region of sll1252-trn-UR primer, followed by ATG. A smaI site was added in both primers. The PCR fragments thus generated using UF, sll1252-trn-UR and sll1252-N^trn-F, sll1252-N^trn-R primer sets were used to set up a fusion PCR with UF and sll1252-N^trn-R primer set. A second fusion PCR was performed using sll1252-N^trn-DF and DR primer sets. The fusion PCR-amplified product thus generated was cloned in pMD19 cloning vector (Takara Inc., Kyoto, Japan). The resultant construct was named pT-sll1252-N^trn. The SalI-digested kan^r, as described in the previous section, was cloned at the smaI site of pT-sll1252-N^trn by blunting followed by ligation. The final construct, in which first 456 bases of the sll1252 ORF had been deleted, was transformed to Synechocystis. The Synechocystis strain thus generated was named Δsll1252-N^trn.

2.6. Construction of sll1252 C-Terminal Truncated Mutant (Δsll1252-C^trn)

A 953 bp DNA fragment including 500 bp upstream flanking region, 453 bp sll1252 ORF, and 500 bp downstream flanking region were amplified from the genomic DNA using primer sets UF, sll1252-trn-DR and sll1252-C^trn-F, DR, respectively. sll1252-trn-DR primer was designed with 14 nucleotides complementary to 5′ region of sll1252-C^trn-F primer along with a stop codon (TAA). smaI site was added to sll1252-trn-DR and sll1252-C^trn-F primers. The PCR fragments generated were used to set up a fusion PCR with UF and DR primer sets. The fusion PCR-amplified product thus generated was cloned into pMD19 T-Vector (Takara-Bio). The resultant construct was named pT-sll1252-C^trn. As mentioned above, kan^r was cloned into smaI site of pT-sll1252-C^trn construct by blunting followed by ligation. Thus, the final construct, in which the 327 bp toward the end of the sll1252 ORF had been deleted, was used to transform wild-type cells of Synechocystis. The Synechocystis strain thus generated was named Δsll1252-C^trn.

2.7. Measurement of Photosynthetic Evolution of Oxygen

Photosynthetic oxygen evolution was measured in 1 mL of cell suspension (OD₇₃₀ of ~1, approximately 5 µg/mL chlorophyll), using an oxygen electrode (Oxygraph plus, Hansatech Instruments Ltd., Norfolk, England). Wild-type and Δsll1252^ins were cultivated photo-autotrophically under optimal or low light with continuous aeration. The rates of whole chain electron transport (WCE) activity and PSII-catalyzed electron transport activity were determined in terms of oxygen evolution in the presence of 2 mM Methyl viologen and 1.0 mM phenyl-p-benzoquinone (PBQ), respectively, under illumination of 1000 µmol photons m⁻² s⁻¹. The activity was determined as a difference between net oxygen evolution in light and oxygen consumption in darkness. The effect of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) at increasing concentrations (0 to 800 nM) was analyzed by measuring the oxygen-evolving activity in the presence of 2 mM Methyl viologen. The rate of PSII-catalyzed electron transport was measured in the presence of increasing concentrations (0 to 600 nM) of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). The chlorophyll concentration of cultures was determined using 80% acetone as a solvent for extraction [32].

2.8. Preparation of cDNA for DNA Microarray Analysis

Wild-type and Δsll1252^ins cells, grown under optimal and low light, were killed instantaneously by the addition of 50 mL ice-cold phenol/ethanol (1:20, w/v) to 50 mL of the cell suspension and then total RNA was extracted, as described previously [30]. The RNA was treated with DNase I (Cat no. 314-08071; Nippon Gene, Tokyo, Japan) to remove contaminating DNA. cDNAs, labeled with fluorescent dyes (Cy3 and Cy5; GE Health Care, Tokyo, Japan), were prepared from 10 μg total RNA with an RNA Fluorescence Labeling Core kit (Cat no. TX807, M-MLV, version 2.0; Takara-Bio) according to the manufacturer’s instructions.

2.9. DNA Microarray Analysis

Genome-wide analysis of transcript levels was performed with DNA microarrays, as described previously [30]. In brief, we used a DNA microarray chip (CyanoCHIP; Takara-Bio) that covered 3079 of the 3168 open-reading frames (97% of all genes, except transposon-related genes) of the Synechocystis genome. Hybridization of labeled cDNAs to the DNA microarray probes was allowed to proceed at 65 °C for 16 h. After hybridization, the microarray chips were rinsed with 2x SSC (1x SSC is 150 mM NaCl and 15 mM sodium citrate) at room temperature. They were washed with 2x SSC at 60 °C for 10 min and with 0.2x SSC, 0.1% SDS at 60 °C for 10 min and then rinsed with distilled water at room temperature for 2 min. Moisture was removed with an air spray before analysis with the array scanner (Cat no. GMS418; Affymetrix, Woburn, MA, USA). Each signal was quantified with the ImaGene ver. 6.0 program (BioDiscovery, Los Angeles, CA, USA). The signal from each gene on the microarray was normalized by reference to the total intensity of signals from all genes, with the exception of 159 genes for rRNAs. We then calculated the change in the level of the transcript of each gene relative to the total amount of mRNA.

2.10. Transmission Electron Microscopy

Transmission electron microscopy (TEM) was performed according to Yubuki et al. [33] with few modifications. Cell suspensions were mixed with an equal volume of 1% glutaraldehyde in sodium cacodylate buffer (0.2 M, pH 7.2) and incubated at 4 °C for 2 h. After washing with a cacodylate buffer, the cells were fixed by incubation in 1% osmium tetroxide at 4 °C for 2 h. The cells were dehydrated through a series of ethanol concentrations: 50% ethanol for 1 h, followed by 75, 90, 95, and 99.5% ethanol for 30 min each. Dehydration was followed by treatment of cells with a 1:1 mixture of 99.5% ethanol and propylene oxide twice for 10 min each and then with 99.5% propylene oxide twice for 10 min. The propylene oxide was replaced with AGAR Low viscosity resin (Cat no: R1370, Agar Scientific Ltd., Stansted, England) by treating with a 1:1 mixture of propylene oxide and resin, followed by treatment with the resin. The resin was polymerized at 70 °C for 12 h. Thin sections (50 nm thickness) were cut with an ultramicrotome (EM-Ultracuts, Reichert, Germany) and stained with 2% uranyl acetate and lead citrate. The samples were observed under a transmission electron microscope (H-7650, Hitachi, Tokyo, Japan).

3. Results

3.1. Sll1252 Is a Conserved S4-Domain Containing Protein

Sll1252 protein is shown to have important physiological functions in Synechocystis and is conserved both in cyanobacteria and higher plants. It is associated with photosystem II and is suggested to be involved in regulating photosynthetic electron transport [6,16]. In this study, we generated a mutant of sll1252 by insertional inactivation and subsequently characterized the resultant mutant (Δsll1252^ins) phenotypically to obtain deeper insights into its physiological function. The protein has a calculated mass of 28.4 kDa. Computational domain analysis indicated an S4-like RNA binding domain in the C-terminal region of Sll1252, suggesting it to be a probable RNA binding protein (Figure 1).

3.2. Mutagenesis of sll1252 in Synechocystis

To elucidate the function of Sll1252, the ORF, sll1252, was disrupted by inserting a kanamycin resistance gene by in vitro transposon mutagenesis, described in the Materials and Methods (Figure 2A). The kan^r was inserted within the sll1252 ORF, as shown in the schematic representation (Figure 2A). As Synechocystis contains multiple copies of identical genomes, a mutant without functional Sll1252 can be obtained only after the replacement of all copies of sll1252 by the inactivated sll1252. The extent of replacement of wild-type copies of sll1252 by the mutant copies was analyzed by PCR amplification, which indicated complete segregation of the mutated copy. After several rounds of culturing the mutant cells in increasing concentrations of kanamycin, the genomic DNA was isolated to check the segregation. With the genomic DNA from wild-type cells as a template, a fragment of 957 bp corresponding to the sll1252 ORF and its upstream and downstream flanking regions was amplified by PCR. With the same set of primers, a 2157 bp DNA fragment, corresponding to the wild-type fragment (957 bp) with the inserted kanamycin gene (1200 bp), was amplified when the genomic DNA of Δsll1252^ins cells was used as a template (Figure 2B). These observations suggest that the sll1252 ORF in all genomes was completely disrupted by the kanamycin gene cassette in the Δsll1252^ins strain.

3.3. Insertional Inactivation of sll1252 Influences Growth at Optimal Light

To analyze whether the disruption of sll1252 would influence the physiological activities of the cells, we compared the growth profiles of wild-type and Δsll1252^ins under optimal light. As shown in Figure 3A, the growth of Δsll1252^ins cells was much slower than wild-type cells. Insertional inactivation and slow-growth phenotype of Δsll1252^ins demonstrated that Sll1252 plays an essential role in the physiology of the cells. As Sll1252 seems to be associated with photosystem II [6,16], the mutation in sll1252 might have influenced the photosynthetic efficiency and, thereby, caused slow-growth phenotype. Hence, we compared the growth of wild-type and Δsll1252^ins strains at low light under photo-autotrophic, photo-heterotrophic, and photo-mixotrophic conditions. As shown in Figure 3B, Δsll1252^ins mutant exhibited slow-growth phenotype under photo-autotrophic conditions at low light. Under photo-heterotrophic and -mixotrophic conditions, growth was not detected in Δsll1252^ins culture compared to wild-type (Figure 3B). This suggests that the insertional inactivation of sll1252 leads to a light-sensitive and glucose-sensitive phenotype.

3.4. Insertional Inactivation of sll1252 Alters the Rate of Whole Chain Electron Transport

The rate of PSII-catalyzed electron transport and WCE activity was compared in the wild-type and Δsll1252^ins to elucidate the effect of inactivation of sll1252 on photosynthesis. As shown in

Table 1. Comparison of photosynthetic activity and growth of Δsll1252 strains with the wild-type. Photosystem II and whole chain electron transport activities of wild-type and Δsll1252 strains, which were grown at 34 °C and bubbled with filter-sterilized air and 70 µmol photon m⁻² s⁻¹. Data represent the average of three independent experiments with standard deviations.

Strain	Mean Generation Time (h)	PSII Activity (H2O to PBQ)	WCE Activity (H2O to MV)
Wild-type	8.3 ± 0.3	1 ± 0.11	1 ± 0.13
Δsll1252ins	Not detected	0.7 ± 0.11	0.55 ± 0.06
Δsll1252del	9.8 ± 0.2	0.78 ± 0.23	0.82 ± 0.06
Δsll1252inv-Rev	8.1 ± 0.2	0.89 ± 0.16	0.91 ± 0.09
Δsll1252-Ntrn	22.0 ± 2.5	0.75 ± 0.04	0.55 ± 0.06
Δsll1252-Ctrn	11.5 ± 0.7	0.59 ± 0.03	0.91 ± 0.06

, the rate of WCE in Δsll1252^ins cells was reduced by 45% compared to wild-type cells.

3.5. The Global Gene Expression Profiles of DBMIB-Treated Wild-Type and Δsll1252^ins Cells Grown under Optimal Light Are Similar

Since the photosynthetic transfer of electrons and/or the redox status of electron-transport components are the primary signals for the expression of few genes, we investigated the effect of sll1252 insertional inactivation on the genome-wide expression of genes in Δsll1252^ins and wild-type grown under low light (Figure 4A). It was observed that the insertional inactivation did not affect the global gene expression profile compared to wild-type at low light (Figure 4A). However, we observed that the expression of 37 genes was upregulated more than 2.5 times in Δsll1252^ins compared to wild-type, when grown under optimal light (Figure 4B). Genes that were upregulated in Δsll1252^ins are listed in

Table 2. Genes whose expression were significantly affected due to the mutation in Δsll1252^ins..

ORF and Description	(Δsll1252ins/Wild-Type)		Effect of Inhibitor on Expression a
	5 µmol/m−2 s−1	70 µmol/m−2 s−1	DBMIB	DCMU
sll0330, sepiapterine reductase	1.3 ± 0.4	18.7 ± 5.5
slr1544	1.1 ± 0.1	17.8 ± 1.7	I
slr1704	1.3 ± 0.1	11.9 ± 0.2
slr1674	1.1 ± 0.1	8.3 ± 0.6	I
sll0528	1.1 ± 0.1	7.6 ± 0.2	I
sll1483, periplasmic protein	1.1 ± 0.0	7.2 ± 0.7	I	I
sll1652	1.2 ± 0.1	6.6 ± 3.6
sll0306, sigB, rpoD, RNA polymerase group 2 sigma factor	1.5 ± 0.1	6.5 ± 0.4	I
sll1862	1.2 ± 0.1	5.8 ± 0.8
sll1515, gifB, glutamine synthetase inactivating factor IF17	1.2 ± 0.1	5.6 ± 1.0	I	I
sll1653 menG, probable phylloquinone biosynthesis methlytransferase	0.9 ± 0.1	5.5 ± 0.9
sll1514, hspA, hsp1, 16.6 kDa small heat shock protein	1.1 ± 0.1	5.5 ± 1.3	I
ssr2194	1.1 ± 0.1	5.2 ± 0.1
slr1516, sodB, superoxide dismutase	0.8 ± 0.1	4.9 ± 0.4
sll1236	1.0 ± 0.1	4.9 ± 0.0
slr1738	1.1 ± 0.1	4.7 ± 0.2	I
slr0581	1.0 ± 0.1	4.5 ± 0.5	I	I
ssr2016	1.1 ± 0.0	4.1 ± 0.2
slr2075, groES, 10 kDa chaperonin, GroES protein	0.7 ± 0.0	3.9 ± 0.5	I
ssr2595, hilB, scpD, high light-inducible protein	1.1 ± 0.1	3.9 ± 0.2	I	I
sll1598, mntC, ABC-type manganese transport system substrate-binding protein	1.1 ± 0.2	3.7 ± 0.8
slr1675, hypA1, putative hydrogenase expression/formation protein HypA1	0.7 ± 0.1	3.7 ± 0.5	I
slr2076, groEL1, cpn60, 60 kDa chaperonin 1, GroEL1, molecular chaperone	0.9 ± 0.0	3.7 ± 0.1	I	R
slr0967	0.8 ± 0.2	3.6 ± 0.4	I	I
ssl3044, hydrogenase component	1.0 ± 0.1	3.5 ± 0.4
sl0416, groEL2, cpn 60, 60 kDa chaperonin 2, GroEL2,	1.1 ± 0.0	3.5 ± 1.1		R
sll0846	1.0 ± 0.0	3.2 ± 0.3	I
sll1863	1.0 ± 0.1	3.2 ± 1.3
slr0093, dnaJ, DnaJ protein, heat shock protein 40, molecular chaperone	1.1 ± 0.0	3.1 ± 0.2	I
ssl1911, gifA, glutamine synthetase inactivating factor IF7	1.3 ± 0.1	2.9 ± 0.5	I	I
slr1687	1.1 ± 0.2	2.8 ± 0.1	I	I
sll1620	1.2 ± 0.0	2.8 ± 0.6	I
slr1204, htrA, serine protease Htra	1.1 ± 0.2	2.8 ± 1.2
sll1621, membrane protein	0.8 ± 0.1	2.7 ± 0.4	I
slr1963, water-soluble carotenoid protein	1.1 ± 0.1	2.6 ± 0.8	I	R
ssl3364, cp12	1.3 ± 0.0	2.6 ± 0.7
ssl2542, hliA, scpC, high light-inducible protein	0.8 ± 0.1	2.6 ± 0.5	I

Wild-type and Δsll1252^ins cells were grown at 34 °C for 48 h, at an illumination of 70 µmol photons m⁻² s⁻¹ or at 5 µmol photons m⁻² s⁻¹. Each value indicates the ratio of levels of the mRNA from Δsll1252^ins cells to those from wild-type cells. The values shown are the means ± range of two independent experiments. The numbering of ORFs corresponds to that of Kaneko et al. [34]. ^a DBMIB, the effect of DBMIB, and DCMU, the effect of DCMU, on the expression of genes, according to Hihara et al. [18]. “I” denotes induction of the gene and “R” denotes repression of the gene.

. It was observed that 7 out 37 upregulated genes in Δsll1252^ins were also reported to be commonly upregulated in DBMIB- or DCMU-treated wild-type cells. Fifteen upregulated genes were reported to be exclusively induced in the wild-type cells with DBMIB, but none of the upregulated genes were reported to be exclusively induced by DCMU [18] (Table 2). Thus, the effect of sll1252 insertional inactivation is similar to the inhibitory effect of DBMIB on global gene expression profile. This indicates that Sll1252 is involved in the electron transfer from the reduced-PQ pool to the Cyt b₆/f complex. With this clue, to establish the involvement of Sll1252 in the regulation of electron transport, we analyzed the relative sensitivity of wild-type and Δsll1252^ins to DCMU, which act as an inhibitor of electron transfer between PSII and Q_B. Similarly, the relative sensitivity to DBMIB, which inhibits electron transfer between the PQ pool and Cyt b₆/f, was also analyzed.

3.6. Δsll1252^ins Cells Are More Sensitive to Inhibition by DBMIB Than Wild-Type Cells

Changes in the DNA microarray gene expression profile and the decrease in the WCE activity of ∆sll1252^ins suggest that Sll1252 is required for electron transfer from the PQ pool to the Cyt b₆/f complex. Differential sensitivity of wild-type and Δsll1252^ins cells to specific inhibitors, DCMU and DBMIB, was examined by measuring photosynthetic electron transport rates at increasing concentrations of the inhibitors. The oxygen-evolving activity with PBQ as an electron acceptor under the rate-saturating irradiance showed a gradual decrease in PSII activity with an increase in the concentration of DCMU in both wild-type and Δsll1252^ins (Figure 5A). The ratios of uninhibited (V₀)/inhibited (V_i) PBQ-mediated PSII activities versus DCMU concentration were calculated. The DCMU required for 50% inhibition of the original activity was almost the same (80 nM) in both wild-type and Δsll1252^ins cells. These results suggest that the mutation in sll1252 had no effect on the site of DCMU action and confirm that the Sll1252 is not involved in the PSII-catalyzed electron transport activity. We analyzed the effect of increasing concentrations of DBMIB on WCE activity in the wild-type and Δsll1252^ins cells with Methyl viologen as an electron acceptor (Figure 5B). The inhibitor required for 50% inhibition of the WCE activity was 300 nM in the wild-type and 140 nM in Δsll1252^ins cells. Moreover, Δsll1252^ins had elevated levels of reduced-PQ pool compared to wild-type. The reduced-PQ content in Δsll1252^ins is almost similar to DBMIB (an inhibitor which blocks electron transport between the PQ pool and the Cyt b₆/f complex)-treated wild-type cells (Figure S1), which suggests that the site of action of Sll1252 is between the PQ pool and Cyt b₆/f. This suggests that the Δsll1252^ins cells are more sensitive to DBMIB and confirms that Sll1252 is required for electron transport from the PQ pool to the Cyt b₆/f complex.

3.7. Suppressor Mutants of Δsll1252^ins (Δsll1252^ins-Rev)

Few Δsll1252^ins colonies grew faster than others with different colony morphology in the BG-11 agar plates containing kanamycin. Hence, we analyzed the kanamycin insertion site of the phenotypically different colonies by PCR amplification and DNA sequencing. Surprisingly, we observed the presence of a naturally occurring cyanobacterial transposable element (ISY523) along with kan^r used for the inactivation of the sll1252 at the insertion site (Figure 6A). The appearance of transposable elements at the inactivation site caused the cells to revert to the wild-type phenotype, labeled as Δsll1252^ins-Rev. Reversion of the mutant to the wild-type phenotype suggests that the insertional inactivation of sll1252 might be detrimental to the cells.

3.8. Truncated and Deletional Mutants of sll1252 (Δsll1252N^trn, Δsll1252C^trn, and Δsll1252^del)

The mean generation time calculated for Δsll1252^del (Figure 6B) is 9.8 h with no effect on PSII and WCE activities compared to wild-type (Table 1). We assumed that in Δsll1252^ins, the insertion of kan^r might have resulted in the expression of either the N-terminal or C-terminal region of Sll1252 (Sll1252-N or Sll1252-C), leading to the severe slow-growth phenotype. The natural transposon has been placed between the kanamycin and C-terminal encoding region of sll1252, probably to inhibit the expression of either Sll1252-N or Sll1252-C, thereby suppressing the mutation effect. Thus, the expression of either Sll1252-N or Sll1252-C may be detrimental to the cells. To test this hypothesis, two truncated mutants were generated: Δsll1252-N^trn expressing the N-terminal region consisting of 453 bp from start codon and Δsll1252C^trn expressing the C-terminal region covering 454 bp till the end of the sll1252 ORF (Figure 6C,D). The constructs thus generated were transformed into wild-type cells. Complete replacement of the wild-type copies of sll1252, by Δsll1252-N^trn or Δsll1252-C^trn, were confirmed by PCR amplification using UF and DR primer (Figure 6C,D, Table A1).

3.9. Expression of Only C-Terminal Region of sll1252 Influences Growth of the Cells

As expected, we observed no significant decrease in the photosynthetic oxygen evolution in Δsll1252^ins-Rev, Δsll1252^del, and Δsll1252C^trn. In Δsll1252N^trn cells, there was a 50% decrease in the WCE rate (Table 1). A slight decline in PSII-catalyzed oxygen evolution was observed in all strains of sll1252 mutants, except for Δsll1252C^trn, in which approximately 40% loss in the PSII activity was observed (Table 1). The mean generation time calculated for wild-type cells was ~8 h and for Δsll1252^del it was 9.8 h. As mentioned before, Δsll1252^ins exhibited a severe slow-growth phenotype. The mean generation time of Δsll1252-N^trn was 22 h. Since only Δsll1252-N^trn exhibited slow-growth phenotype like Δsll1252^ins and reduction in whole chain electron transport, it suggests that expression of the C-terminal region of Sll1252 is deteriorative to the cells (Table 1). Hence, we hypothesize that both the C-terminal and N-terminal regions of Sll1252 regulate the electron transport between the plastoquinone pool and the Cyt b₆/f complex.

3.10. Thylakoid Membrane Is Disorganized in Δsll1252N^trn

Since we observed slow-growth phenotype in Δsll1252-N^trn, but not in Δsll1252-C^trn, we further examined the ultrastructural changes caused in cells due to expression of the truncated forms of Sll1252. The wild-type and Δsll1252-C^trn looked alike, as can be seen in Figure 7. Well-organized thylakoid membranes are visible when examined under transmission electron microscope. Layers of thylakoid membranes were arranged in rows. In contrast to wild-type and Δsll1252-C^trn cells, thylakoid membranes were disorganized in Δsll1252-N^trn. Only a few polyphosphate granules were observed in wild-type and Δsll1252-C^trn cells, indicating actively growing and dividing cells in these cultures, which might be using the stored phosphate for their active growth. The presence of more polyphosphates in Δsll1252-N^trn might be a reason for its slow-growth phenotype. The microscopic image revealed spherical dense bodies in Δsll1252-N^trn, probably containing the degraded thylakoid membrane lipids. Thus, it is clear from the transmission electron microscopic observations that expression of only the C-terminal region of Sll1252 leads to ultrastructural changes, especially the disorganization of thylakoid membranes. The observations thus support the essential physiological role of Sll1252 in photosynthetic electron transport.

4. Discussion

In the present study, using DNA microarray gene expression profiling and comparative transcriptome analysis, we can obtain clues about the function of Sll1252. Sll1252, associated with the thylakoid membrane, is now proven to be an essential protein in photosynthetic electron transport, assisting election transfer between the PQ pool and the Cyt b₆/f complex. The genes upregulated due to insertional inactivation of sll1252 (Table 1) are reportedly induced by DBMIB, which inhibits electron transfer from the PQ pool to the Cyt b₆/f complex. Physiologically, ∆sll1252^ins had a slow-growth phenotype and decreased WCE activity (Figure 3, Table 1). The elevated level of the reduced-PQ pool in ∆sll1252^ins suggests that Sll1252 might facilitate electron transfer from the PQ pool to the Cyt b₆/f complex (Figure S1). This is consistent with the observation by Kashino et al. [16]. The insertionally disrupted mutant is observed to be reverted by mobilization of a naturally occurring transposable element (ISY523) by an unknown mechanism. Interestingly, Synechocystis cells use a natural transposable element to suppress the mutations that occur in genes, if they are lethal to the cells [35]. The mechanism by which these cells place the transposable element at the insertion site to suppress the mutation is yet to be elucidated. It is also interesting to study whether the placement of the transposon for suppressing the lethal effect of the mutation is by selective insertion or random transposition.

Further, Δsll1252^ins also exhibited glucose sensitivity. Earlier studies, using comparative genome analysis of Synechocystis, revealed that frameshift/substitution mutations in several genes, like glcP encoding glucose transporter and the genes encoding protein components of NDH-complexes involved in respiratory electron transport, result in glucose sensitivity [36,37]. Moreover, inactivation of pmgA, which is known to be essential for photo-mixotrophic growth and involved in the regulation of photosystem stoichiometry and high light response, also results in glucose sensitivity [38,39]. The involvement of Sll1252 in the electron transport from the PQ pool to the Cyt b₆/f complex, like NDH complexes, might be the reason for the glucose sensitivity of Δsll1252^ins. However, the exact mechanism by which glucose exerted slow-growth phenotype under photo-mixotrophic and photo-heterotrophic conditions needs to be studied in detail.

We have seen that the strain expressing only the C-terminal region of Sll1252 is detrimental to the cell, unlike the deletional mutant, in which Sll1252 is not expressed or the wild-type in which Sll1252 is expressed. The C-terminal region of Sll1252, consisting of the S4-domain, may act as an inhibitor of electron flow. The observations suggest that only N-terminal or full-length Sll1252 expression is not lethal to the cell. The lethality may have neutralized when both terminal proteins are expressed together. Thus, we propose that the interaction of the N-terminal region with the C-terminal region probably masks the inhibitory effect of the C-terminal region, thereby regulating the electron flow. Depending on the requirement, the electron transport rate may be regulated by Sll1252 using both its N-terminal and C-terminal regions. However, a detailed study is needed on the mechanism by which Sll1252 is involved in such a regulation. We present a model that shows the various experimental results as the basis for drawing a conclusion about the function of Sll1252 in the thylakoid membrane (Figure 8). Being associated with purified PSII complexes [6], this protein is assumed to have a transmembrane domain. However, computational analysis of the protein indicated that it does not possess any predicted transmembrane domains (Figure 1). Sll1252 may be associated with the thylakoid membrane in close vicinity of the PSII complex and may not be a direct member, but it is involved in efficient electron transfer from the reduced-PQ pool to the Cyt b₆/f complex (Figure 8A). Expression of only the C-terminal region of Sll1252 leads to inhibition of electron transfer between the PQ pool and the Cyt b₆/f complex (Figure 8B). Expression of only the N-terminal region of Sll1252 has no effect on electron transfer (Figure 8C). From the data obtained using sll1252 truncated mutants, we suggest that the C-terminal region of Sll1252 down-regulates the electron flow. A kind of dynamic conformational change in Sll1252 probably leads to the specific interaction of the C-terminal region with the Cyt b₆/f complex and is the key to the regulation of electron transport.