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.     

Putrescine transport in a cyanobacterium Synechocystis sp. PCC 6803

The transport of putrescine into a moderately salt tolerant cyanobacterium Synechocystis sp. PCC 6803 was characterized by measuring the uptake of radioactively-labeled putrescine. Putrescine transport showed saturation kinetics with an apparent K(m) of 92 +/- 10 microM and V(max) of 0.33 +/- 0.05 nmol/min/mg protein. The transport of putrescine was pH-dependent with highest activity at pH 7.0. Strong inhibition of putrescine transport was caused by spermine and spermidine whereas only slight inhibition was observed by the addition of various amino acids. These results suggest that the transport system in Synechocystis sp. PCC 6803 is highly specific for polyamines. Putrescine transport is energy-dependent as evidenced by the inhibition by various metabolic inhibitors and ionophores. Slow growth was observed in cells grown under salt stress. Addition of low concentration of putrescine could restore growth almost to the level observed in the absence of salt stress. Upshift of the external osmolality generated by either NaCl or sorbitol caused an increased putrescine transport with an optimum 2-fold increase at 20 mosmol/kg. The stimulation of putrescine transport mediated by osmotic upshift was abolished in chloramphenicol-treated cells, suggesting possible involvement of an inducible transport system.     

Functional Role of PilA in Iron Acquisition in the Cyanobacterium Synechocystis sp. PCC 6803

Cyanobacteria require large quantities of iron to maintain their photosynthetic machinery; however, in most environments iron is present in the form of insoluble iron oxides. Whether cyanobacteria can utilize these sources of iron, and the potential molecular mechanisms involved remains to be defined. There is increasing evidence that pili can facilitate electron donation to extracellular electron acceptors, like iron oxides in non-photosynthetic bacteria. In these organisms, the donation of electrons to iron oxides is thought to be crucial for maintaining respiration in the absence of oxygen. Our study investigates if PilA1 (major pilin protein) may also provide a mechanism to convert insoluble ferric iron into soluble ferrous iron. Growth experiments supported by spectroscopic data of a strain deficient in pilA1 indicate that the presence of the pilA1 gene enhances the ability to grow on iron oxides. These observations suggest a novel function of PilA1 in cyanobacterial iron acquisition.     

Role of HoxE subunit in Synechocystis PCC6803 hydrogenase

Cyanobacterial NAD(P)(+)-reducing reversible hydrogenases comprise five subunits. Four of them (HoxF, HoxU, HoxY, and HoxH) are also found in the well-described related enzyme from Ralstonia eutropha. The fifth one (HoxE) is not encoded in the R. eutropha genome, but shares homology with the N-terminal part of R. eutropha HoxF. However, in cyanobacteria, HoxE contains a 2Fe-2S cluster-binding motif that is not found in the related R. eutropha sequence. In order to obtain some insights into the role of HoxE in cyanobacteria, we deleted this subunit in Synechocystis PCC6803. Three types of interaction of the cyanobacterial hydrogenase with pyridine nucleotides were tested: (a) reductive activation of the NiFe site, for which NADPH was found to be more efficient than NADH; (b) H(2) production, for which NADH appeared to be a more efficient electron donor than NADPH; and (c) H(2) oxidation, for which NAD(+) was a much better electron acceptor than NADP(+). Upon hoxE deletion, the Synechocystis hydrogenase active site remained functional with artificial electron donors or acceptors, but the enzyme became unable to catalyze H(2) production or uptake with NADH/NAD(+). However, activation of the electron transfer-independent H/D exchange reaction by NADPH was still observed in the absence of HoxE, whereas activation of this reaction by NADH was lost. These data suggest different mechanisms for diaphorase-mediated electron donation and catalytic site activation in cyanobacterial hydrogenase.     

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.     

Salt-dependent protein phosphorylation in the cyanobacterium Synechocystis PCC 6803

Abstract We have isolated a Bradyrhizobium japonicum USDA 438 (serogroup 123) mutant which has the ability to form nodules on serogroup 123 nodulation-restricting plant introduction genotypes and soybeans containing the Rj4 allele. The identity of the mutant was confirmed by using a serocluster 123-specific DNA probe, restriction fragment length polymorphism analysis, and serogroup-specific fluorescent antibodies. While the mutant contains Tn 5 inserted into a cryptic, non nod gene-containing locus, site-directed mutagenesis and complementation studies indicated that the transposon is not responsible for host-range extension. The mutant and the wild-type parent had the same chromatographic profiles of [¹⁴C]acetate-labelled extracellular B. japonicum nod factors.     

Photophysiological and Photosynthetic Complex Changes during Iron Starvation in Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942

Iron is an essential component in many protein complexes involved in photosynthesis, but environmental iron availability is often low as oxidized forms of iron are insoluble in water. To adjust to low environmental iron levels, cyanobacteria undergo numerous changes to balance their iron budget and mitigate the physiological effects of iron depletion. We investigated changes in key protein abundances and photophysiological parameters in the model cyanobacteria Synechococcus PCC 7942 and Synechocystis PCC 6803 over a 120 hour time course of iron deprivation. The iron stress induced protein (IsiA) accumulated to high levels within 48 h of the onset of iron deprivation, reaching a molar ratio of ∼42 IsiA : Photosystem I in Synechococcus PCC 7942 and ∼12 IsiA : Photosystem I in Synechocystis PCC 6803. Concomitantly the iron-rich complexes Cytochrome b₆f and Photosystem I declined in abundance, leading to a decrease in the Photosystem I : Photosystem II ratio. Chlorophyll fluorescence analyses showed a drop in electron transport per Photosystem II in Synechococcus, but not in Synechocystis after iron depletion. We found no evidence that the accumulated IsiA contributes to light capture by Photosystem II complexes.     

Accumulation of poly-beta-hydroxybutyrate in cyanobacterium Synechocystis sp. PCC6803

Accumulation of poly-beta-hydroxybutyrate (PHB) by photoautotrophic microorganisms makes it possible to reduce the production cost of PHB. The Synechocystis sp. PCC6803 cells grown in BG11 medium under balanced, nitrogen-starved or phosphorus-starved conditions were observed by transmission electron microscope. Many electron-transparent granules in the nitrogen-starved cells had a diameter up to 0.8 micron. In contrast, the number of granules in the normally cultured cells decreased obviously and only zero to three much smaller granules were in each cell. These granules were similar to those in bacteria capable of synthesizing PHB. They were proved to be PHB by gas chromatography after subjecting the cells to methanolysis. Effects of glucose as carbon source and light intensity on PHB accumulation in Synechocystis sp. PCC6803 under nitrogen-starved cultivation were further studied. Glucose and illumination promoted cell growth but did not favor PHB synthesis. After 7 days of growth under nitrogen-starved photoautotrophic conditions, the intracellular level of PHB was up to 4.1% of cellular dry weight and the PHB concentration in the culture broth was 27 mg/l.     

Mutants of Synechocystis PCC6803 Defective in Inorganic Carbon Transport

Eighty mutants of Synechocystis PCC6803 that require high CO₂ for growth were examined with a mass spectrometer for their ability to take up CO₂ in the light. Two of these mutants (type A) did not show any CO₂ uptake while the rest of the mutants (type B) took up CO₂ actively. Type A mutants (RKa and RKb) and one type B mutant (RK11) were partially characterized. At 3% CO₂, growth rates of the mutants and the wild type (WT) were similar. Under air levels of CO₂, growth of RKa and RKb was very slow, and RK11 did not grow at all. The photosynthetic affinities for inorganic carbon (C_i) in these three mutants were about 100 times lower than the affinity in WT. The following characteristics of type A mutants indicated that the mutants have a defect in their CO₂-transport system: (a) the activity of ¹³C¹⁸O₂ uptake in RKa and RKb in the light was less than 5% the activity in WT, and (b) each mutant had only a low level of activity of ¹⁴CO₂ uptake as measured by the method of silicone oil-filtering centrifugation. The HCO₃⁻-transport system was also impaired in these mutants. The activity of H¹⁴CO₃⁻ uptake was negligibly low in RKb and was one-third the activity of WT in RKa. On the other hand, the type B mutant, RK11, transported CO₂ and HCO₃⁻ into the intracellular C_i pool as actively as WT but was unable to utilize it for photosynthesis. Complementation analysis of type A mutants indicated that RKa and RKb have mutations in different regions of the genome. These results suggested that at least two kinds of proteins are involved in the C_i-transport system.     

Characterisation of acyltransferases from Synechocystis sp. PCC6803

As phylogenetic ancestors of plant chloroplasts cyanobacteria resemble plastids with respect to lipid and fatty acid composition. These membrane lipids show the typical prokaryotic fatty acid pattern in which the sn-2 position is exclusively esterified by C(16) acyl groups. In the course of de novo glycerolipid biosynthesis this prokaryotic fatty acid pattern is established by the sequential acylation of glycerol-3-phosphate with acyl-ACPs by the activity of different acyltransferases. In silico approaches allowed the identification of putative Synechocystis acyltransferases involved in glycerolipid metabolism. Functional expression studies in Escherichia coli showed that sll1848 codes for a lysophosphatidic acid acyltransferase with a high specificity for 16:0-ACP, whereas slr2060 encodes a lysophospholipid acyltransferase, with a broad acyl-ACP specificity but a strong preference for lysophosphatidyglycerol especially its sn-2 acyl isomer as acyl-acceptor. The generation and analysis of the corresponding Synechocystis knockout mutants revealed that lysophosphatidic acid acyltransferase unlike the lysophospholipid acyltransferase is essential for the vital functions of the cells.     

FutA2 is a ferric binding protein from Synechocystis PCC 6803

Synechocystis PCC 6803 has a high demand for iron (10 times greater than Escherichia coli) to sustain photosynthesis and is unusual in possessing at least two putative iron-binding proteins of a type normally associated with ATP-binding cassette-type importers. It has been suggested that one of these, FutA2, binds ferrous iron, but herein we clearly demonstrate that this protein avidly binds Fe(III), the oxidation state preference of periplasmic iron-binding proteins. Structures of apo-FutA2 and Fe-FutA2 have been determined at 1.7 and 2.7A, respectively. The metal ion is bound in a distorted trigonal bipyramidal arrangement with no exogenous anions as ligands. The metal-binding environment, including the second coordination sphere and charge properties, is consistent with a preference for Fe(III). Atypically, FutA2 has a Tat signal peptide, and its inability to coordinate divalent cations may be crucial to prevent metals from binding to the folded protein prior to export from the cytosol. A loop containing the His(43) ligand undergoes considerable movement in apo-versus Fe-FutA2 and may control metal release to the importer. Although these data are consistent with FutA2 being the periplasmic component involved in iron uptake, deletion of another putative ferric binding protein, FutA1, has a greater effect on the accumulation of iron and is more analogous to a DeltafutA1DeltafutA2 double mutant than DeltafutA2. Here, we also discover that there is a reduced level of ferric FutA2 in the periplasm of the DeltafutA1 mutant providing an explanation for its severe iron-uptake phenotype.     

Proteomic analysis of heterotrophy in Synechocystis sp. PCC 6803

To provide an insight into the heterotrophic metabolism of cyanobacteria, a proteomic approach has been employed with the model organism Synechocystis sp. PCC 6803. The soluble proteins from Synechocystis grown under photoautotrophic and light-activated heterotrophic conditions were separated by 2-DE and identified by MALDI-MS or LC-MS/MS analysis. 2-DE gels made using narrow- and micro-range IPG strips allowed quantitative comparison of more than 900 spots. Out of 67 abundant protein spots identified, 13 spots were increased and 9 decreased under heterotrophy, representing all the major fold changes. Proteomic alterations and activity levels of selected enzymes indicate a shift in the central carbon metabolism in response to trophic change. The significant reduction in light-saturated rate of photosynthesis as well as in the expression levels of rubisco and CO(2)-concentrating mechanism proteins under heterotrophy indicates the down-regulation of the photosynthetic machinery. Alterations in the expression level of proteins involved in carbon utilization pathways refer to enhanced glycolysis, oxidative pentose phosphate pathway as well as tricarboxylic acid cycle under heterotrophy. Proteomic evidences also suggest an enhanced biosynthesis of amino acids such as histidine and serine during heterotrophic growth.     

Precipitation of calcite induced by Synechocystis sp. PCC6803

Calcite with laminate structure was successfully prepared by culturing Synechocystis sp. PCC6803 with different concentrations of calcium chloride (CaCl₂) in BG11 media. S. PCC6803 was examined by scanning electron microscopy (SEM), transmission electron microscopy (TEM), laser confocal scanning microscope (LCSM) and energy dispersive spectroscopy (EDS). The effects of Ca²⁺ concentrations and pH values on calcification were investigated and the micro morphs of the CaCO₃ crystals were observed by means of SEM. These results showed that CaCO₃ crystals could be more easily formed with increasing the concentration of CaCl₂ in S. PCC6803 culture solution. S. PCC6803 could largely bind calcium ions, most of which were present in extracellular polymeric substances and on the cell wall. Inside the cells there were a lot of circular areas rich in calcium ions without the crystallization of calcium. Some cells produced a thicker gelatinous sheath outside of the translucent organic thin layer. And the cells inside also produced major changes that the original chloroplasts were almost transformed into starch grains whose sizes were from 0.5 to 1 μm with relatively uniform in sizes. At the same time the cell sizes significantly reduced to only about 8–9 μm almost changing to half of its original diameters. The calcite crystals with a highly preferred orientation induced by S. PCC6803 were observed with X-ray diffraction (XRD). A critical implication was that S. PCC6803 could induce bio-calcification and then mediate the further growth of CaCO₃ crystals in the biological system.