Characterization of Lysine Monomethylome and Methyltransferase in Model Cyanobacterium Synechocystis sp.PCC 6803

Protein lysine methylation is a prevalent post-translational modification (PTM) and plays critical roles in all domains of life. However, its extent and function in photosynthetic organisms are still largely unknown. Cyanobacteria are a large group of prokaryotes that carry out oxygenic photosynthesis and are applied extensively in studies of photosynthetic mechanisms and environmental adaptation. Here we integrated propionylation of monomethylated proteins, enrichment of the modified peptides, and mass spectrometry (MS) analysis to identify monomethylated proteins in Synechocystis sp. PCC 6803 (Synechocystis). Overall, we identified 376 monomethylation sites in 270 proteins, with numerous monomethylated proteins participating in photosynthesis and carbon metabolism. We subsequently demonstrated that CpcM, a previously identified asparagine methyltransferase in Synechocystis, could catalyze lysine monomethylation of the potential aspartate aminotransferase Sll0480 both in vivo and in vitro and regulate the enzyme activity of Sll0480. The loss of CpcM led to decreases in the maximum quantum yield in primary photosystem II (PSII) and the efficiency of energy transfer during the photosynthetic reaction in Synechocystis. We report the first lysine monomethylome in a photosynthetic organism and present a critical database for functional analyses of monomethylation in cyanobacteria. The large number of monomethylated proteins and the identification of CpcM as the lysine methyltransferase in cyanobacteria suggest that reversible methylation may influence the metabolic process and photosynthesis in both cyanobacteria and plants.     

Proteomic analysis of Synechocystis sp. PCC6803 responses to low-temperature and high light conditions

The global changes in protein expression of Synechocystis sp. PCC6803, a photosynthetic bacterium for the production of secondary metabolites as a green cell factory, were investigated by proteome separation and a subsequent tandem mass spectrometry. Two different proteome separation techniques, strong cation exchange chromatography and off-gel electrophoresis, were applied. The combination of the two proteome separation techniques enabled the comparative analysis of the differential regulation of the Synechocystis proteome in response to two different environmental factors, temperature and light. A total of 1,483 proteins were identified, which represent over 40% of the genes in Synechocystis. Our data showed that fatty acid metabolism was inhibited by (3R)-hydroxymyristol acyl carrier protein dehydrase (Sll1605) under low temperature conditions. The expression of UDP-N-acetylglucosamine acyltransferase (Sll0379) and 3-O-[3-hydroxymyristoyl] glucosamine N-acyltransferase (Slr0776), which is involved in lipopolysaccharide metabolism, was not observed under high light conditions. Under high light exposure, proteins related to iron-sulfur metabolism were detected, which may be responsible for maintaining the redox potential of the photosystem. High light under low temperature caused severe damage to the photosystem. Some of the responses to these stresses were similar to those previously reported for other photosynthetic organisms. Notably, this study revealed the followings: (i) low temperature inhibits fatty acid synthesis; (ii) high light inhibits lipopolysaccharides synthesis and stimulates the expression of iron-sulfur related proteins; and (iii) high light under low temperature induces the photorespiratory cycle. The global proteomic analysis clearly showed that stress conditions such as low temperature and/or high light induce cellular metabolisms related with the protection of their photosystems in the model microalga Synechocystis sp. PCC6803.     

Temperature-dependent hyper-activation of monoglucosyldiacylglycerol synthase is post-translationally regulated in Synechocystis sp. PCC 6803

The mechanism of monoglucosyldiacylglycerol (MGlcDG) increase following heat shock in Synechocystis sp. PCC 6803 was examined by measuring MGlcDG synthase (Sll1377) activity. Temperature-dependent activation of Sll1377 was observed in the membrane fraction of Synechocystis sp. PCC 6803, whereas the Sll1377 protein level remained unchanged, suggesting that the activity is post-translationally regulated without covalent modification of Sll1377 by soluble enzymes. Four individual mutations introduced into recombinant Sll1377 (D147, D200, R329, and R331) significantly reduced the activity and blocked temperature-dependent activation, suggesting that these amino acid residues are essential for Sll1377 activity at both normal growth temperature and the higher temperature.     

Sll1783, a monooxygenase associated with polysaccharide processing in the unicellular cyanobacterium Synechocystis PCC 6803

Cyanobacteria play a pivotal role as the primary producer in many aquatic ecosystems. The knowledge on the interacting processes of cyanobacteria with its environment – abiotic and biotic factors – is still very limited. Many potential exocytoplasmic proteins in the model unicellular cyanobacterium Synechocystis PCC 6803 have unknown functions and their study is essential to improve our understanding of this photosynthetic organism and its potential for biotechnology use. Here we characterize a deletion mutant of Synechocystis PCC 6803, Δsll1783, a strain that showed a remarkably high light resistance which is related with its lower thylakoid membrane formation. Our results suggests Sll1783 to be involved in a mechanism of polysaccharide degradation and uptake and we hypothesize it might function as a sensor for cell density in cyanobacterial cultures.     

The sll1951 Gene Encodes the Surface Layer Protein of Synechocystis sp. Strain PCC 6803

Sll1951 is the surface layer (S-layer) protein of the cyanobacterium Synechocystis sp. strain PCC 6803. This large, hemolysin-like protein was found in the supernatant of a strain that was deficient in S-layer attachment. An sll1951 deletion mutation was introduced into Synechocystis and was easily segregated to homozygosity under laboratory conditions. By thin-section and negative-stain transmission electron microscopy, a ∼30-nm-wide S-layer lattice covering the cell surface was readily visible in wild-type cells but was absent in the Δsll1951 strain. Instead, the Δsll1951 strain displayed a smooth lipopolysaccharide surface as its most peripheral layer. In the presence of chaotropic agents, the wild type released a large (>150-kDa) protein into the medium that was identified as Sll1951 by mass spectrometry of trypsin fragments; this protein was missing in the Δsll1951 strain. In addition, Sll1951 was prominent in crude extracts of the wild type, indicating that it is an abundant protein. The carotenoid composition of the cell wall fraction of the Δsll1951 strain was similar to that of the wild type, suggesting that the S-layer does not contribute to carotenoid binding. Although the photoautotrophic growth rate of the Δsll1951 strain was similar to that of the wild-type strain, the viability of the Δsll1951 strain was reduced upon exposure to lysozyme treatment and hypo-osmotic stress, indicating a contribution of the S-layer to the integrity of the Synechocystis cell wall. This work identifies the S-layer protein in Synechocystis and shows that, at least under laboratory conditions, this very abundant, large protein has a supportive but not a critical role in the function of the cyanobacterium.     

The S-layer biogenesis system of Synechocystis 6803: Role of Sll1180 and Sll1181 (E. coli HlyB and HlyD analogs) as type-I secretion components for Sll1951 export

Multiple secretion pathways are known for export of protein(s) forming the S-layer in bacteria. The unicellular model cyanobacterium Synechocystis sp. strain PCC 6803 (hereafter S. 6803) also possesses a well-defined S-layer composed of Sll1951 protein. However, the mechanism of its secretion is not completely understood. In the present study, the putative T1SS (Type I secretion system) components, Sll1180 and Sll1181 [inner membrane ABC transporter and membrane fusion protein (MFP), respectively] were characterized for their role in Sll1951 secretion. The corresponding ORFs i.e. sll1180 and sll1181 were inactivated by insertion of a spectinomycin resistance gene. The viability of the homozygous mutants of both the genes indicated dispensability of the corresponding proteins under the experimental conditions. Interestingly, the culture supernatants of the mutants i.e. Δsll1180 and Δsll1181, lacked Sll1951 as observed on SDS-PAGE and confirmed by mass spectrometry. Immunofluorescence delineated a distinct outer ring of Sll1951 in S. 6803 cells only that was further iterated by transmission and scanning electron microscopy. The loss of S-layer imparted an aggregative phenotype to both the mutants. Surprisingly, Δsll1181 cells showed increased sensitivity to different antibiotics indicating a role in multidrug efflux. This is the first report establishing Sl1180 and Sll1181 proteins as partners of the previously characterized Slr1270, for Sll1951 secretion and thus S-layer biogenesis in S. 6803. Sll1181 (in conjunction with Slr1270) also acts as MFP in multidrug efflux along with a yet uncharacterized inner membrane protein.     

Functional Proteomic Discovery of Slr0110 as a Central Regulator of Carbohydrate Metabolism in Synechocystis Species PCC6803

The unicellular photosynthetic model-organism cyanobacterium Synechocystis sp. PCC6803 can grow photoautotrophically using CO₂ or heterotrophically using glucose as the sole carbon source. Several pathways are involved in carbon metabolism in Synechocystis, and the concerted regulation of these pathways by numerous known and unknown genes is critical for the survival and growth of the organism. Here, we report that a hypothetical protein encoded by the open reading frame slr0110 is necessary for heterotrophic growth of Synechocystis. The slr0110-deletion mutant is defective in glucose uptake, heterotrophic growth, and dark viability without detectable defects in autotrophic growth, whereas the level of photosystem II and the rate of oxygen evolution are increased in the mutant. Quantitative proteomic analysis revealed that several proteins in glycolysis and the oxidative pentose phosphate pathway are down-regulated, whereas proteins in photosystem II and phycobilisome are significantly up-regulated, in the mutant. Among the down-regulated proteins are glucose transporter, glucokinase, glucose-6-phosphate isomerase, and glucose-6-phosphate dehydrogenase and its assembly protein OpcA, suggesting that glycolysis, oxidative pentose phosphate, and glycogen synthesis pathways are significantly inhibited in the mutant, which was further confirmed by enzymatic assays and quantification of glycogen content. These findings establish Slr0110 as a novel central regulator of carbon metabolism in Synechocystis, and shed light on an intricate mechanism whereby photosynthesis and carbon metabolism are well concerted to survive the crisis when one or more pathways of the system are impaired.Cyanobacteria are a diverse group of prokaryotes that are capable of oxygenic photosynthesis and are believed to have played a critical role in changing Earth''s atmosphere from ancient anaerobic conditions to the present aerobic conditions (1–3). It is widely accepted that the chloroplasts of higher plants are derived from the endosymbiotic events between cyanobacteria and eukaryotic cells (4). The photosynthetic activity of cyanobacteria is estimated to account for the production of more than half of the biomass on Earth (2). More recently, cyanobacteria have been shown to have great potential as cell factories for the production of clean and renewable biofuels, such as hydrogen (5, 6). Therefore, understanding the physiology and metabolism of cyanobacteria is of great importance not only in basic sciences, but also in biotechnologies dealing with the worldwide crises of energy shortage and environmental pollution.The unicellular cyanobacterium Synechocystis sp. PCC 6803 (hereinafter referred to as Synechocystis) has been widely used as a model system for the study of photosynthesis and other metabolic processes. It is highly transformable, and its genome has been completely sequenced (7), making it an excellent system for studying the functions of unknown proteins that may participate in pathways in central metabolism by means of targeted mutagenesis. The organism can grow under a number of different conditions ranging from photoautotrophic to fully heterotrophic modes, making it a great tool for the study of fundamental processes such as photosynthesis and carbon metabolism (8, 9). Synechocystis contains an outer membrane, a plasma membrane, and large amounts of thylakoid membrane (10), providing an ideal model for functional proteomics aiming at the discovery of novel proteins involved in many fundamental processes, including molecule transport, photosynthesis and respiration, and signal transduction.Photosynthesis and carbon metabolism are two physically and functionally interconnected processes in Synechocystis. The light reaction of photosynthesis provides reductants and energy for the assimilation of inorganic carbon via the Calvin cycle, the net product of which, glyceraldehyde 3-phosphate, can either be further catabolized through the lower energy-conserving phase of glycolysis and the tricarboxylic acid (TCA)¹ cycle, to produce energy, reductants, and precursors for the biosynthesis of other important biomolecules such as amino acids and lipids, or be used as the primary source for the synthesis of glucose and glycogen through gluconeogenesis and glyconeogenesis. Endogenously synthesized or exogenously supplied glucose can also be catabolized through the oxidative pentose phosphate pathway (OPPP) and/or glycolysis to supply carbon and energy for the growth of Synechocystis (11, 12).As the Calvin cycle, OPPP, and glycolysis take place within a single cellular compartment, many reversible reactions, enzymes, regulators, and intermediate metabolites are shared by the three processes. Moreover, the processes are physically and functionally connected with the TCA cycle, nitrogen assimilation, amino acid and protein synthesis, lipid biosynthesis, and many other metabolic processes. A single perturbation of one process may lead to significant effects on the activities and outcomes of the others. For example, the addition of glucose to Synechocystis culture can enhance the activity of OPPP while partly repressing the activity of photosynthesis (13). Similarly, perturbation of any NADPH-utilizing pathways that generate NADP⁺, an allosteric stimulator of the first and the rate-limiting enzyme of the OPPP (i.e. glucose-6-phosphate dehydrogenase (G6PDH)), can significantly affect the activity of the OPPP (14). In this regard, it is of great importance to understand the regulatory mechanism of metabolism at a system level, rather than at the level of a single gene or pathway.The Synechocystis genome contains 3672 putative protein-coding open reading frames (ORFs). Many of these are known to be involved in photosynthesis and carbon metabolism (7). However, nearly 50% of the ORFs encode hypothetical or unknown proteins whose expression and function have not been experimentally determined yet. The lack of functional information on these proteins has seriously hindered the progress toward comprehensive understanding of the mechanism whereby balanced anabolism and catabolism take place in a nonseparated compartment at a system level. Fortunately, well-established and highly time- and cost-effective techniques for generating gene-deletion mutants through insertional mutation allow for the study of functions of hypothetical proteins in a relatively high-throughput way. Moreover, recent progress in proteomics allows the quantitative identification of Synechocystis proteins affected by gene deletion at a system scale (15). In a large-scale screening of the gene-deletion mutants of Synechocystis, we obtained several mutants showing the phenotype defective in heterotrophic or autotrophic growth. Of those, the mutant of the ORF encoding the hypothetical protein Slr0110 (Δslr0110) exhibited completely inhibited heterotrophic growth but did not show any observable phenotype under autotrophic conditions. Here, we describe the details of the phenotype of Δslr0110 and the results obtained from physiological and proteomics studies addressing the functional significance of Slr0110 in the heterotrophic growth of Synechocystis.     

Expression of holo-proteorhodopsin in Synechocystis sp. PCC 6803

Retinal-based photosynthesis may contribute to the free energy conversion needed for growth of an organism carrying out oxygenic photosynthesis, like a cyanobacterium. After optimization, this may even enhance the overall efficiency of phototrophic growth of such organisms in sustainability applications. As a first step towards this, we here report on functional expression of the archetype proteorhodopsin in Synechocystis sp. PCC 6803. Upon use of the moderate-strength psbA2 promoter, holo-proteorhodopsin is expressed in this cyanobacterium, at a level of up to 10⁵ molecules per cell, presumably in a hexameric quaternary structure, and with approximately equal distribution (on a protein-content basis) over the thylakoid and the cytoplasmic membrane fraction. These results also demonstrate that Synechocystis sp. PCC 6803 has the capacity to synthesize all-trans-retinal. Expressing a substantial amount of a heterologous opsin membrane protein causes a substantial growth retardation Synechocystis, as is clear from a strain expressing PROPS, a non-pumping mutant derivative of proteorhodopsin. Relative to this latter strain, proteorhodopsin expression, however, measurably stimulates its growth.     

Coregulated Genes Link Sulfide:Quinone Oxidoreductase and Arsenic Metabolism in Synechocystis sp. Strain PCC6803

Although the biogeochemistry of the two environmentally hazardous compounds arsenic and sulfide has been extensively investigated, the biological interference of these two toxic but potentially energy-rich compounds has only been hypothesized and indirectly proven. Here we provide direct evidence for the first time that in the photosynthetic model organism Synechocystis sp. strain PCC6803 the two metabolic pathways are linked by coregulated genes that are involved in arsenic transport, sulfide oxidation, and probably in sulfide-based alternative photosynthesis. Although Synechocystis sp. strain PCC6803 is an obligate photoautotrophic cyanobacterium that grows via oxygenic photosynthesis, we discovered that specific genes are activated in the presence of sulfide or arsenite to exploit the energy potentials of these chemicals. These genes form an operon that we termed suoRSCT, located on a transposable element of type IS4 on the plasmid pSYSM of the cyanobacterium. suoS (sll5036) encodes a light-dependent, type I sulfide:quinone oxidoreductase. The suoR (sll5035) gene downstream of suoS encodes a regulatory protein that belongs to the ArsR-type repressors that are normally involved in arsenic resistance. We found that this repressor has dual specificity, resulting in 200-fold induction of the operon upon either arsenite or sulfide exposure. The suoT gene encodes a transmembrane protein similar to chromate transporters but in fact functioning as an arsenite importer at permissive concentrations. We propose that the proteins encoded by the suoRSCT operon might have played an important role under anaerobic, reducing conditions on primordial Earth and that the operon was acquired by the cyanobacterium via horizontal gene transfer.     

Chorismate Pyruvate-Lyase and 4-Hydroxy-3-solanesylbenzoate Decarboxylase Are Required for Plastoquinone Biosynthesis in the Cyanobacterium Synechocystis sp. PCC6803

Plastoquinone is a redox active lipid that serves as electron transporter in the bifunctional photosynthetic-respiratory transport chain of cyanobacteria. To examine the role of genes potentially involved in cyanobacterial plastoquinone biosynthesis, we have focused on three Synechocystis sp. PCC 6803 genes likely encoding a chorismate pyruvate-lyase (sll1797) and two 4-hydroxy-3-solanesylbenzoate decarboxylases (slr1099 and sll0936). The functions of the encoded proteins were investigated by complementation experiments with Escherichia coli mutants, by the in vitro enzyme assays with the recombinant proteins, and by the development of Synechocystis sp. single-gene knock-out mutants. Our results demonstrate that sll1797 encodes a chorismate pyruvate-lyase. In the respective knock-out mutant, plastoquinone was hardly detectable, and the mutant required 4-hydroxybenzoate for growth underlining the importance of chorismate pyruvate-lyase to initiate plastoquinone biosynthesis in cyanobacteria. The recombinant Slr1099 protein displayed decarboxylase activity and catalyzed in vitro the decarboxylation of 4-hydroxy-3-prenylbenzoate with different prenyl side chain lengths. In contrast to Slr1099, the recombinant Sll0936 protein did not show decarboxylase activity regardless of the conditions used. Inactivation of the sll0936 gene in Synechocystis sp., however, caused a drastic reduction in the plastoquinone content to levels very similar to those determined in the slr1099 knock-out mutant. This proves that not only slr1099 but also sll0936 is required for plastoquinone synthesis in the cyanobacterium. In summary, our data demonstrate that cyanobacteria produce plastoquinone exclusively via a pathway that is in the first reaction steps almost identical to ubiquinone biosynthesis in E. coli with conversion of chorismate to 4-hydroxybenzoate, which is then prenylated and decarboxylated.     

Microevolution in Cyanobacteria: Re-sequencing a Motile Substrain of Synechocystis sp. PCC 6803

Synechocystis sp. PCC 6803 is a widely used model cyanobacterium for studying photosynthesis, phototaxis, the production of biofuels and many other aspects. Here we present a re-sequencing study of the genome and seven plasmids of one of the most widely used Synechocystis sp. PCC 6803 substrains, the glucose tolerant and motile Moscow or ‘PCC-M’ strain, revealing considerable evidence for recent microevolution. Seven single nucleotide polymorphisms (SNPs) specifically shared between ‘PCC-M’ and the ‘PCC-N and PCC-P’ substrains indicate that ‘PCC-M’ belongs to the ‘PCC’ group of motile strains. The identified indels and SNPs in ‘PCC-M’ are likely to affect glucose tolerance, motility, phage resistance, certain stress responses as well as functions in the primary metabolism, potentially relevant for the synthesis of alkanes. Three SNPs in intergenic regions could affect the promoter activities of two protein-coding genes and one cis-antisense RNA. Two deletions in ‘PCC-M’ affect parts of clustered regularly interspaced short palindrome repeats-associated spacer-repeat regions on plasmid pSYSA, in one case by an unusual recombination between spacer sequences.     

Structural and Mutational Analysis of Band 7 Proteins in the Cyanobacterium Synechocystis sp. Strain PCC 6803

Band 7 proteins, which encompass members of the stomatin, prohibitin, flotillin, and HflK/C protein families, are integral membrane proteins that play important physiological roles in eukaryotes but are poorly characterized in bacteria. We have studied the band 7 proteins encoded by the cyanobacterium Synechocystis sp. strain PCC 6803, with emphasis on their structure and proposed role in the assembly and maintenance of the photosynthetic apparatus. Mutagenesis revealed that none of the five band 7 proteins (Slr1106, Slr1128, Slr1768, Sll0815, and Sll1021) was essential for growth under a range of conditions (including high light, salt, oxidative, and temperature stresses), although motility was compromised in an Slr1768 inactivation mutant. Accumulation of the major photosynthetic complexes in the thylakoid membrane and repair of the photosystem II complex following light damage were similar in the wild type and a quadruple mutant. Cellular fractionation experiments indicated that three of the band 7 proteins (Slr1106, Slr1768, and Slr1128) were associated with the cytoplasmic membrane, whereas Slr1106, a prohibitin homologue, was also found in the thylakoid membrane fraction. Blue native gel electrophoresis indicated that these three proteins, plus Sll0815, formed large (>669-kDa) independent complexes. Slr1128, a stomatin homologue, has a ring-like structure with an approximate diameter of 16 nm when visualized by negative stain electron microscopy. No evidence for band 7/FtsH supercomplexes was found. Overall, our results indicate that the band 7 proteins form large homo-oligomeric complexes but do not play a crucial role in the biogenesis of the photosynthetic apparatus in Synechocystis sp. strain PCC 6803.Members of the band 7 superfamily of proteins are found throughout nature and are defined by a characteristic sequence motif, termed the SPFH domain, after the initials of the various subfamilies: the stomatins, the prohibitins, the flotillins (also known as “reggies”), and the HflK/C proteins (12, 49). The stomatins and prohibitins and to a lesser extent flotillins are highly conserved protein families and are found in a variety of organisms ranging from prokaryotes to higher eukaryotes (29, 34, 49), whereas HflK and HflC homologues are only present in bacteria.In eukaryotes band 7 proteins are linked with a variety of disease states consistent with important cellular functions (6). In general the eukaryotic band 7 proteins tend to be oligomeric and are involved in membrane-associated processes: for example, prohibitins are involved in modulating the activity of a membrane-bound FtsH protease (17, 46) and the assembly of mitochondrial respiratory complexes (30), stomatins are involved in ion channel function (47), and flotillins are involved in signal transduction and vesicle trafficking (25).In the case of prokaryotes, most work so far has focused on the roles of the HflK/C and YbbK (also known as QmcA, a stomatin homologue) band 7 proteins of Escherichia coli (7, 16, 17, 36) and the structure of a stomatin homologue in the archaeon Pyrococcus horikoshii (57). Much less is known about the structure, function, and physiological importance of band 7 proteins in other prokaryotes, especially the cyanobacteria (12).The unicellular cyanobacterium Synechocystis sp. strain PCC 6803 is a widely used model organism for studying various aspects of cyanobacterial physiology and, in particular, oxygenic photosynthesis. One of the main areas of our research is to understand the mechanism by which the oxygen-evolving photosystem II (PSII) complex found in the thylakoid membrane of Synechocystis sp. strain PCC 6803 is repaired following light damage. Recent work has identified an important role for FtsH proteases in PSII repair (19, 41). Given that FtsH is known to form large supercomplexes with HflK/C in E. coli (36) and with prohibitins in Saccharomyces cerevisiae mitochondria (46), we hypothesized that one or more band 7 proteins might interact with FtsH in cyanobacteria and play a role in the selective turnover of the D1 reaction center polypeptide during PSII repair and so provide resistance to high light stress (40). This idea was given early support by the detection of both FtsH and Slr1106, a prohibitin homologue, in a His-tagged PSII preparation isolated from Synechocystis sp. strain PCC 6803 (40) and the detection of Slr1128 (a stomatin homologue), Sll1021 (a possible flotillin homologue), and FtsH in a His-tagged preparation of ScpD, a small chlorophyll a/b-like-binding protein that associates with PSII (56). Recent mutagenesis experiments have also suggested a role for Slr1128 in maintaining growth at high light intensities (53).In this paper we have used targeted gene disruption mutagenesis and various biochemical approaches to investigate the structure and function of band 7 proteins in Synechocystis sp. strain PCC 6803, with particular emphasis on PSII function. We provide evidence that four predicted band 7 proteins in Synechocystis sp. strain PCC 6803 (Slr1106, Slr1768, Slr1128, and Sll8015) form large independent complexes, which in the case of Slr1128 forms a ring-like structure. No evidence was found for the formation of supercomplexes with FtsH. Importantly, single and multiple insertion mutants lacking up to four of the five band 7 proteins are able to grow as well as the wild type (WT) under a range of growth conditions, including high light stress. Our results suggest that band 7 proteins are not essential in Synechocystis sp. strain PCC 6803 and are not required for efficient PSII repair. Possible functions of the cyanobacterial band 7 proteins are discussed in the light of recent results from other systems.     

Paradigm of Monoterpene (β-phellandrene) Hydrocarbons Production via Photosynthesis in Cyanobacteria

A direct “photosynthesis-to-fuels” approach envisions application of a single organism, absorbing sunlight, photosynthesizing, and converting the primary products of photosynthesis into ready-made fuel. The work reported here applied this concept for the photosynthetic generation of monoterpene (β-phellandrene) hydrocarbons in the unicellular cyanobacteria Synechocystis sp. PCC 6803. Heterologous expression of a codon-optimized Lavandula angustifolia β-phellandrene synthase (β-PHLS) gene in Synechocystis enabled photosynthetic generation of β-phellandrene in these microorganisms. β-phellandrene accumulation occurred constitutively and in tandem with biomass accumulation, generated from sunlight, CO₂, and H₂O. Results showed that β-phellandrene diffused through the plasma membrane and cell wall of the cyanobacteria and accumulated on the surface of the liquid culture. Spontaneous β-phellandrene separation from the biomass and its removal from the liquid phase alleviated product inhibition of cellular metabolism and enabled a continuous production process. The work showed that oxygenic photosynthesis can be directed to generate monoterpene hydrocarbons, while consuming CO₂, without a prior requirement for the harvesting, dewatering, and processing of the respective biomass.     

Observation of the solid-state photo-CIDNP effect in entire cells of cyanobacteria Synechocystis

Cyanobacteria are widely used as model organism of oxygenic photosynthesis due to being the simplest photosynthetic organisms containing both photosystem I and II (PSI and PSII). Photochemically induced dynamic nuclear polarization (photo-CIDNP) ¹³C magic-angle spinning (MAS) NMR is a powerful tool in understanding the photosynthesis machinery down to atomic level. Combined with selective isotope enrichment this technique has now opened the door to study primary charge separation in whole living cells. Here, we present the first photo-CIDNP observed in whole cells of the cyanobacterium Synechocystis.     

Dissecting the Photoprotective Mechanism Encoded by the flv4‐2 Operon: a Distinct Contribution of Sll0218 in Photosystem II Stabilization

In Synechocystis sp. PCC 6803, the flv4‐2 operon encodes the flavodiiron proteins Flv2 and Flv4 together with a small protein, Sll0218, providing photoprotection for Photosystem II (PSII). Here, the distinct roles of Flv2/Flv4 and Sll0218 were addressed, using a number of flv4‐2 operon mutants. In the ?sll0218 mutant, the presence of Flv2/Flv4 rescued PSII functionality as compared with ?sll0218‐flv2, where neither Sll0218 nor the Flv2/Flv4 heterodimer are expressed. Nevertheless, both the ?sll0218 and ?sll0218‐flv2 mutants demonstrated deficiency in accumulation of PSII proteins suggesting a role for Sll0218 in PSII stabilization, which was further supported by photoinhibition experiments. Moreover, the accumulation of PSII assembly intermediates occurred in Sll0218‐lacking mutants. The YFP‐tagged Sll0218 protein localized in a few spots per cell at the external side of the thylakoid membrane, and biochemical membrane fractionation revealed clear enrichment of Sll0218 in the PratA‐defined membranes, where the early biogenesis steps of PSII occur. Further, the characteristic antenna uncoupling feature of the ?flv4‐2 operon mutants is shown to be related to PSII destabilization in the absence of Sll0218. It is concluded that the Flv2/Flv4 heterodimer supports PSII functionality, while the Sll0218 protein assists PSII assembly and stabilization, including optimization of light harvesting.     

The alternative sigma factor SigF is a key player in the control of secretion mechanisms in Synechocystis sp. PCC 6803

Cyanobacterial alternative sigma factors are crucial players in environmental adaptation processes, which may involve bacterial responses related to maintenance of cell envelope and control of secretion pathways. Here, we show that the Group 3 alternative sigma factor F (SigF) plays a pleiotropic role in Synechocystis sp. PCC 6803 physiology, with a major impact on growth and secretion mechanisms, such as the production of extracellular polysaccharides, vesiculation and protein secretion. Although ΔsigF growth was significantly impaired, the production of released polysaccharides (RPS) increased threefold to fourfold compared with the wild-type. ΔsigF exhibits also impairment in formation of outer-membrane vesicles (OMVs) and pili, as well as several other cell envelope alterations. Similarly, the exoproteome composition of ΔsigF differs from the wild-type both in amount and type of proteins identified. Quantitative proteomics (iTRAQ) and an in silico analysis of SigF binding motifs revealed possible targets/pathways under SigF control. Besides changes in protein levels involved in secretion mechanisms, our results indicated that photosynthesis, central carbon metabolism and protein folding/degradation mechanisms are altered in ΔsigF. Overall, this work provided new evidences about the role of SigF on Synechocystis physiology and associates this regulatory element with classical and non-classical secretion pathways.     

Cyanobacterial hybrid kinase Sll0043 regulates phototaxis by suppressing pilin and twitching motility protein

The unicellular cyanobacterium Synechocystis sp. PCC 6803 glides toward a light source through the interplay of positive phototaxis genes and proteins. In genetic analysis, the complete disruption of the hybrid sensory kinase sll0043 produced negative phototaxis. Furthermore, Sll0043 was found to be a hub protein by in silico prediction of protein-protein interaction, in which Sll0043 was predominantly linked to seven two-component proteins with high confidence. To understand the regulation and networking of positive phototaxis proteins, the proteomic profile of the sll0043 mutant was compared to that of wild-type. In the sll0043 mutant, 18 spots corresponding to 15 unique proteins were altered by 1.3 to 59 fold; the spots were identified by 2-DE/MALDI-MS analysis. Down-regulated proteins in the sll0043 null-mutant included chaperonins, superoxide dismutase, and phycocyanin beta-subunit. In contrast, nine proteins involved in photosynthesis, translation, regulatory function, and other functions were up-regulated. In particular, a twitching motility protein (PilT1) was induced over 2-fold in sll0043 mutant. Moreover, semi-quantitative and quantitative RT-PCR analysis revealed that pilin (pilA1), pili motor (pilT1), and pili switch gene (pilT2) were significantly increased in sll0043 mutant. These results suggest that the hybrid kinase Sll0043 regulates positive phototaxis by suppressing the expression of pili biosynthesis and regulatory genes and through the interplay with positive phototaxis/motility two-component proteins.