Complete Genome Structure of the Unicellular Cyanobacterium Synechocystis sp. PCC6803

Cyanobacteria are photoautotrophic organisms capable of oxygen-producingphotosynthesis similar to that in eukaryotic algae and plants,and because of this, they have been used as model organismsfor the study of the mechanism and regulation of oxygen-producingphotosynthesis. To understand the entire genetic system in cyanobacteria,the nucleotide sequence of the entire genome of the unicellularcyanobacterium Synechocystis sp. PCC6803 has been determined.The total length of the circular genome is 3,573,470 bp, witha GC content of 47.7%. A total of 3,168 potential protein codinggenes were assigned. Of these, 145 (4.6%) were identical toreported genes, and 1,259 (39.6%) and 342 (10.8%) showed similarityto reported and hypothetical genes, respectively. The remaining1,422 (45.0%) showed no apparent similarity to any genes registeredin the databases. Classification of the genes by their biologicalfunction and comparison of the gene complement with those ofother organisms have revealed a variety of features of the geneticinformation characteristic of a photoautotrophic organism. Thesequence data, as well as other information on the Synechocystisgenome, is presented in CyanoBase on WWW [http://www.kazusa.or.jp/cyano/]. (Received July 24, 1997; Accepted September 17, 1997)     

A Physical Map of the Genome of a Unicellular Cyanobacterium Synechocystis sp. Strain PCC6803

An accurate physical map of the genome of a cyanobacterium,Synechocystis sp. strain PCC6803, was constructed on the basisof restriction and linking clone analysis. The genome contained6 recognition sites for AscI, 25 sites for MluI, and 31 sitesfor SplI, and the entire genome size was estimated to be 3.6Mb. Sixteen genes or gene clusters, including those involvedin the photosynthetic systems, were localized on the physicalmapof the genome by hybridization. In the course of the above analysis,two extra chromosomal units with approximate sizes of 110 kband 125 kb were identified.     

Genomic Structure of the Cyanobacterium Synechocystis sp. PCC 6803 Strain GT-S

Synechocystis sp. PCC 6803 is the most popular cyanobacterial strain, serving as a standard in the research fields of photosynthesis, stress response, metabolism and so on. A glucose-tolerant (GT) derivative of this strain was used for genome sequencing at Kazusa DNA Research Institute in 1996, which established a hallmark in the study of cyanobacteria. However, apparent differences in sequences deviating from the database have been noticed among different strain stocks. For this reason, we analysed the genomic sequence of another GT strain (GT-S) by 454 and partial Sanger sequencing. We found 22 putative single nucleotide polymorphisms (SNPs) in comparison to the published sequence of the Kazusa strain. However, Sanger sequencing of 36 direct PCR products of the Kazusa strains stored in small aliquots resulted in their identity with the GT-S sequence at 21 of the 22 sites, excluding the possibility of their being SNPs. In addition, we were able to combine five split open reading frames present in the database sequence, and to remove the C-terminus of an ORF. Aside from these, two of the Insertion Sequence elements were not present in the GT-S strain. We have thus become able to provide an accurate genomic sequence of Synechocystis sp. PCC 6803 for future studies on this important cyanobacterial strain.     

Effect of Gravity Changes on the Cyanobacterium Synechocystis sp. PCC 6803

The impact of hypergravity and simulated weightlessness were studied to check whether cyanobacteria perceive changes of gravity as stress. Hypergravity generated by a low-speed centrifuge increased slightly the overall activity of dehydrogenases, but the increase was the same for 90 g and 180 g. The protein pattern did not show qualitative alterations during hypergravity treatment up to 180 g. Cells of Synechocystis PCC 6803 subjected to common stressors like salt, heat, and light clearly accumulated at least four general stress proteins (25, 31, 34, and 63 kDa, respectively). Three of these proteins could also be detected after hypergravity, but in such small amounts that their occurrence could only be taken as a weak indication of stress. Low-molecular-weight stress metabolites were not synthesized in response to hypergravity, indicating that this gravity change was unable to activate the osmotic signal transduction chain. Gravity-dependent alterations were observed only during simulated weightlessness (generated by a fast-rotating clinostat). The glutamate/glutamine ratio was significantly shifted toward a higher glutamine portion. Altogether, the results may indicate that moderate changes of gravity were hardly, if ever, sensed as stress by cyanobacteria. Received: 20 May 1997 / Accepted: 25 June 1997     

Assignment of 82 Known Genes and Gene Clusters on the Genome of the Unicellular Cyanobacterium Synechocystis sp. Strain PCC6803

We have previously constructed the physical map of a cyanobacterium,Synechoystis sp. strain PCC6803 on the basis of restrictionand linking clone analysis. Since a total of 82 genes and geneclusters have been isolated from this strain, most of whichare involved in oxygenic photosynthesis, portions of their sequenceswere amplified by the PCR method and assigned on the physicalmap of the genome by hybridization with restriction fragments,ordered clones, which were obtained from cosmid and libraries,and long PCR-products. An exception was the gene psbG2 whichwas mapped on an extra-chromosomal unit of 45 kb. Since geneticmaps of some of genes assigned above, especially those for photosynthesis,have been reported for two other cyanobacterial strains, Anabaenasp. PCC7120 and Synechococcus sp. PCC7002, gene organizationswere compared among the three strains. However, no significantcorrelation was observed, suggesting that rearrangement of genesoccurred in the respective strains during or after establishmentof the species.     

Reduction of Photoautotrophic Productivity in the Cyanobacterium Synechocystis sp. Strain PCC 6803 by Phycobilisome Antenna Truncation

Truncation of the algal light-harvesting antenna is expected to enhance photosynthetic productivity. The wild type and three mutant strains of Synechocystis sp. strain 6803 with a progressively smaller phycobilisome antenna were examined under different light and CO(2) conditions. Surprisingly, such antenna truncation resulted in decreased whole-culture productivity for this cyanobacterium.     

Genomic Responses to Arsenic in the Cyanobacterium Synechocystis sp. PCC 6803

Arsenic is a ubiquitous contaminant and a toxic metalloid which presents two main redox states in nature: arsenite [As^III] and arsenate [As^V]. Arsenic resistance in Synechocystis sp. strain PCC 6803 is mediated by the arsBHC operon and two additional arsenate reductases encoded by the arsI1 and arsI2 genes. Here we describe the genome-wide responses to the presence of arsenate and arsenite in wild type and mutants in the arsenic resistance system. Both forms of arsenic produced similar responses in the wild type strain, including induction of several stress related genes and repression of energy generation processes. These responses were transient in the wild type strain but maintained in time in an arsB mutant strain, which lacks the arsenite transporter. In contrast, the responses observed in a strain lacking all arsenate reductases were somewhat different and included lower induction of genes involved in metal homeostasis and Fe-S cluster biogenesis, suggesting that these two processes are targeted by arsenite in the wild type strain. Finally, analysis of the arsR mutant strain revealed that ArsR seems to only control 5 genes in the genome. Furthermore, the arsR mutant strain exhibited hypersentivity to nickel, copper and cadmium and this phenotype was suppressed by mutation in arsB but not in arsC gene suggesting that overexpression of arsB is detrimental in the presence of these metals in the media.     

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.     

Selection by Anion-Exchange Chromatography of Exopolysaccharide Mutants of the Cyanobacterium Synechocystis Strain PCC 6803

The degree of retention of whole cells of Synechocystis strain PCC 6803 on DEAE-cellulose columns was shown to depend on their content of exopolysaccharides, which are at least in part responsible for the external negative charge of the cells. This feature was used for the isolation of mutants modified in the apparent viscosity caused by these macromolecular constituents. When a wild-type suspension was loaded onto a DE52 column, the cells eluting in the two extreme fractions of a 0 to 5 M NaCl step gradient represented 10⁻⁹ to 10⁻⁷ of the total eluted population. The accuracy of the procedure was established through the analysis of four clones: Suc(0)32 and Suc(0)65 (0 M) and Suc(5)64A and Suc(5)61 (5 M). The decreased viscosity of the exopolymers of the two 0 M clones, which appeared identical, could be related to the production of molecules less charged in uronic acids and more readily liberated from the cells. The two 5 M clones exhibited a lower sedimentation velocity, correlating with either a 60% increase in uronic acid and a doubling of the specific viscosity of the exopolysaccharides [clone Suc(5)64A] or a doubling of the per-cell production of polymers otherwise identical to those from wild-type cells [clone Suc(5)61].     

HSP16.6 Is Involved in the Development of Thermotolerance and Thylakoid Stability in the Unicellular Cyanobacterium, Synechocystis sp. PCC 6803

The low molecular weight (LMW) heat shock protein (HSP), HSP16.6, in the unicellular cyanobacterium, Synechocystis sp. PCC 6803, protects cells from elevated temperatures. A 95% reduction in the survival of mutant cells with an inactivated hsp16.6 was observed after exposure for 1 h at 47°C. Wild-type cell survival was reduced to only 41%. HSP16.6 is also involved in the development of thermotolerance. After a sublethal heat shock at 43°C for 1 h and subsequent challenge exposure at 49°C for 40 min, mutant cells did not survive, while 64% of wild-type cells survived. Ultrastructural changes in the integrity of thylakoid membranes of heat-shocked mutant cells also are discussed. These results demonstrate an important protective role for HSP16.6 in the protection of cells and, in particular, thylakoid membrane against thermal stress. Received: 14 October 1999 / Accepted: 16 November 1999     

Functional Characterization of the slr1944 Gene of Cyanobacterium Synechocystis sp. PCC 6803

The properties of Slr1944 protein encoded by the slr1944 gene and participating in the metabolism of lipophilic compounds in a cyanobacterium Synechocystis were under study. Located in the periplasm, this protein comprises a conserved pentapeptide G-X-S-X-G characteristic of lipases, acetylcholinesterases, and thioesterases. An attempt to delete the gene from the cyanobacterial genome failed; this fact presumes an essential function of Slr1944 protein under the optimum growth conditions. Expression of the slr1944 gene in Escherichia coli cells demonstrated a high affinity of the product for lipophilic compounds. An enhanced slr1944 expression deprived Synechocystis cells of the ability to restore the activity of the photosynthetic electron-transport chain following photoinactivation. The authors believe that Slr1944 participates in the biogenesis of the lipophilic components of photosynthetic complexes.     

Refolding and Enzyme Kinetic Studies on the Ferrochelatase of the Cyanobacterium Synechocystis sp. PCC 6803

Heme is a cofactor for proteins participating in many important cellular processes, including respiration, oxygen metabolism and oxygen binding. The key enzyme in the heme biosynthesis pathway is ferrochelatase (protohaem ferrolyase, EC 4.99.1.1), which catalyzes the insertion of ferrous iron into protoporphyrin IX. In higher plants, the ferrochelatase enzyme is localized not only in mitochondria, but also in chloroplasts. The plastidic type II ferrochelatase contains a C-terminal chlorophyll a/b (CAB) motif, a conserved hydrophobic stretch homologous to the CAB domain of plant light harvesting proteins and light-harvesting like proteins. This type II ferrochelatase, found in all photosynthetic organisms, is presumed to have evolved from the cyanobacterial ferrochelatase. Here we describe a detailed enzymological study on recombinant, refolded and functionally active type II ferrochelatase (FeCh) from the cyanobacterium Synechocystis sp. PCC 6803. A protocol was developed for the functional refolding and purification of the recombinant enzyme from inclusion bodies, without truncation products or soluble aggregates. The refolded FeCh is active in its monomeric form, however, addition of an N-terminal His₆-tag has significant effects on its enzyme kinetics. Strikingly, removal of the C-terminal CAB-domain led to a greatly increased turnover number, k_cat, compared to the full length protein. While pigments isolated from photosynthetic membranes decrease the activity of FeCh, direct pigment binding to the CAB domain of FeCh was not evident.     

集胞藻6803NdhO蛋白多克隆抗体制备及其初步应用

蓝藻NADPH脱氢酶(NDH-1)是一种重要的光合膜蛋白复合体,参与CO2吸收、围绕光系统I的循环电子传递和细胞呼吸.迄今为止,人们在蓝藻细胞中已鉴定出17种NDH-1复合体亚基(NdhA-NdhQ).最近,人们还获得了NdhO亚基的缺失突变株.然而,人们对NdhO亚基的研究还不充份,至今仍不清楚它的功能角色.通过PC...     

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.