Transformation and gene replacement in the facultatively chemoheterotrophic,unicellular cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC6714 by electroporation

The unicellular cyanobacterium Synechocystis sp. PCC6714 can grow not only under photoautotrophic conditions, but also under chemoheterotrophic conditions if glucose is added to the medium. This makes it useful for the study of many aspects of bioenergetic mechanisms. In contrast to its closely related strain Synechocystis sp. PCC6803, which cannot grow chemoheterotrophically, Synechocystis PCC6714 is not naturally transformable. To enable gene transfer in this strain, we established a method for the introduction of self-replicating IncQ plasmids and for gene replacement using electroporation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A conjugative plasmid vector for promoter analysis in several cyanobacteria of the genera Synechococcus and Synechocystis

A promoter-probe vector, pSB2A, based on the plasmid RSF1010 and the promoterless chloramphenicol acetyl transferase (cat) reporter gene, has been constructed. pSB2A appeared to be most efficiently transferred by conjugation to the widely used cyanobacteria Synechocystis strains PCC6803 (S.6803) and PCC6714 (S.6714) and Synechococcus strains PCC7942 (S.7942) and PCC6301 (S.6301), where it replicates stably even though it contains no cyanobacterial DNA. Using pSB2A we found that (1) a light-regulated promoter from S.6803 remains controlled by light intensity in S.7942 while it is silent in Escherichia coli, and (2) the E. coli tac promoter behaves as a strong and light-independent promoter in the four cyanobacterial hosts tested.Service de Biochimie et Génétique Moléculaire     

PHOTOTACTIC MOTILITY OF SYNECHOCYSTIS SP. UNIWG (CYANOBACTERIA) FROM BRACKISH ENVIRONMENT1

Although type IV pilus has been implicated in the phototactic motility of some unicellular cyanobacteria, its regulatory mechanism and the effect of environmental factors on motility are still unknown. Equally important is the ability of cyanobacterial cells to anchor themselves to an environment that is conducive for survival. We compared the motility of a newly isolated unicellular brackish cyanobacterium, Synechocystis sp. UNIWG, with the morphologically and phylogenetically similar freshwater cyanobacterium Synechocystis sp. PCC6803 under different environmental conditions. The phototactic motility of Synechocystis sp. UNIWG on semisolid BG‐11 medium with various concentrations of nitrogen source was significantly faster than that of Synechocystis PCC6803. Interestingly, the cell surface of Synechocystis sp. UNIWG showed the presence of rigid spicules when grown in liquid BG‐11, a phenomenon that was absent in Synechocystis PCC6803. Negative staining of Synechocystis sp. UNIWG revealed the presence of two distinct pilus morphotypes, which resembled type IV pili and thin pili of Synechocystis PCC6803. This finding suggested a similar pattern of phototactic motility in both strains. However, the rigid spicules on Synechocystis sp. UNIWG seem to be more of a hindrance during type IV motility. It was determined that the spicules were degraded when the cells moved, such as under prolonged darkness and/or depletion of nitrogen source, indicating that the function of the spicules is to attach the cell to an environment that is conducive for its survival. Thus, Synechocystis sp. UNIWG shows phototaxis regulation that is more complex than Synechocystis PCC6803.     

Identification of conserved domains in the Δ12 desaturases of cyanobacteria

Cyanobacterial genes for enzymes that desaturate fatty acids at the 12 position, designated desA, were isolated from Synechocystis PCC6714, Synechococcus PCC7002 and Anabaena variabilis by crosshybridization with a DNA probe derived from the desA gene of Synechocystis PCC6803. The genes of Synechocystis PCC6714, Synechococcus PCC7002 and A. variabilis encode proteins of 349, 347 and 350 amino acid residues, respectively. The transformation of Synechococcus PCC7942 with the desA genes from Synechocystis PCC6714, Synechococcus PCC7002 and A. variabilis was associated with the ability to introduce a second double bond at the 12 position of fatty acids. The amino acid sequence of the products of the desA genes revealed the presence of four conserved domains. Since one of the conserved domains was also found in the amino acid sequences of 3 desaturases of Brassica napus and mung bean, this domain may play an essential role in the introduction of a double bond into fatty acids bound to membrane lipids.Abbreviations X:Y(Z) fatty acid containing X carbon atoms with Y double bonds in the cis configuration at position Z counted from the carboxyl terminus     

Osmotic responses of unicellular blue-green algae (cyanobacteria): changes in cell volume and intracellular solute levels in response to hyperosmotic treatment

Abstract Changes in cell volume and solute content upon hyperosmotic shock have been studied for six unicellular blue-green algae (cyanobacteria): Synechococcus PCC 6301, PCC 6311; Synechocystis PCC 6702, PCC 6714, PCC 6803 and PCC 7008. The extent of change in volume was shown to be dependent upon the solute used to establish the osmotic gradient, with cells in NaCl showing a reduced shrinkage when compared to cells in media containing added sorbitol and sucrose. Uptake of extracellular solutes during hyperosmotic shock was observed in Synechocystis PCC 6714, with maximum accumulation of external solutes in NaCl and minimum solute uptake in sucrose solutions. Conversely, solute loss from the cells (K⁺ and amino acids) was greatest in sucrose-containing media and least in NaCl. The results show that these blue-green algae do not behave as ‘ideal osmometers’ in media of high osmotic strength. It is proposed that short-term changes in plasmalemma permeability in these organisms may be due to transient membrane instability resulting from osmotic imbalance between the cell and its surrounding fluid at the onset of hyperosmotic shock.     

Sulfonylurea-resistant mutants and natural tolerance of cyanobacteria

The herbicide sulfometuron methyl (SM) inhibited the growth of the cyanobacterium Synechococcus sp. PCC7942, but not of Synechocystis sp. PCC6714. The inhibitory effect was alleviated by the simultaneous addition of valine, leucine and isoleucine. SM resistant mutants were isolated from Synechococcus 7942, two types of which were further analysed. In these mutants, SM3/20 and SM2/32, the activity of acetolactate synthase (ALS) — a key enzyme in the biosynthesis of branched-chain amino acids —appeared 2600- and 300-fold, respectively, more resistant to SM than that of their wild type. Strain SM2/32 also exhibited a low level of ALS activity. Although the growth of the latter mutant was extremely inhibited by valine, the sensitivity of its ALS activity to feed-back inhibition by the amino acid was unaltered. At high concentrations valine inhibited growth of the wild type strains and of the mutant SM3/20. Isoleucine alleviated the valine-induced growth inhibition. Unlike that of Synechococcus 7942, the ALS activity of Synechocystis was found to tolerate high concentrations (100-fold) of the herbicide. The study confirms that the SM mutations are correlated with a cyanobacterial ilv gene.Abbreviations ALS acetolactate synthase; ile, isoleucine - leu leucine - NTG N-methyl-N-nitro-N-nitrosoguanidine - SM sulfometuron methyl - SM^r sulfometuron methyl resistant - val valine     

Avoidance of protein oxidation correlates with the desiccation and radiation resistance of hot and cold desert strains of the cyanobacterium Chroococcidiopsis

To investigate the relationship between desiccation and the extent of protein oxidation in desert strains of Chroococcidiopsis a selection of 10 isolates from hot and cold deserts and the terrestrial cyanobacterium Chroococcidiopsis thermalis sp. PCC 7203 were exposed to desiccation (air-drying) and analyzed for survival. Strain CCMEE 029 from the Negev desert and the aquatic cyanobacterium Synechocystis sp. PCC 6803 were further investigated for protein oxidation after desiccation (drying over silica gel), treatment with H₂O₂ up to 1 M and exposure to γ-rays up to 25 kGy. Then a selection of desert strains of Chroococcidiopsis with different survival rates after prolonged desiccation, as well as Synechocystis sp. PCC 6803 and Chroococcidiopsis thermalis sp. PCC 7203, were analyzed for protein oxidation after treatment with 10 and 100 mM of H₂O₂. Results suggest that in the investigated strains a tight correlation occurs between desiccation and radiation tolerance and avoidance of protein oxidation.

     

Metabolic engineering of Synechocystis sp. PCC 6803 for enhanced ethanol production based on flux balance analysis

Synechocystis sp. PCC 6803 is an attractive host for bio-ethanol production due to its ability to directly convert atmospheric carbon dioxide into ethanol using photosystems. To enhance ethanol production in Synechocystis sp. PCC 6803, metabolic engineering was performed based on in silico simulations, using the genome-scale metabolic model. Comprehensive reaction knockout simulations by flux balance analysis predicted that the knockout of NAD(P)H dehydrogenase enhanced ethanol production under photoautotrophic conditions, where ammonium is the nitrogen source. This deletion inhibits the re-oxidation of NAD(P)H, which is generated by ferredoxin-NADP⁺ reductase and imposes re-oxidation in the ethanol synthesis pathway. The effect of deleting the ndhF1 gene, which encodes NADH dehydrogenase subunit 5, on ethanol production was experimentally evaluated using ethanol-producing strains of Synechocystis sp. PCC 6803. The ethanol titer of the ethanol-producing ∆ndhF1 strain increased by 145%, compared with that of the control strain.

     

Isolation and characterization of three types of membranes from the cyanobacterium (blue-green alga) Synechocystis PCC 6714

Three types of membranes were separated from the cyanobacterium Synechocystis PCC 6714 by mechanical disruption and density gradient centrifugation. Orange-colored membranes contained xanthophylls but little -carotene or chlorophyll a, green-colored membranes contained chlorophyll a, -carotene and xanthophylls, and another type of orange-colored membranes contained unknown xanthophylls. These membrane preparations were similar to those from Anacystis nidulans in pigmentation and buoyant density and were identified as purified preparations of the cytoplasmic membranes, thylakoid membranes and cell walls of Synechocystis PCC 6714, respectively.Abbreviations SDS sodium dodecyl sulfate - Tes N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid - TLC thin-layer chromatography     

Functional analysis of the two homologous psbA gene copies in Synechocystis PCC 6714 and PCC 6803

The cyanobacteria Synechocystis 6803 and 6714 contain three genes (psbA) coding for the D₁ protein. This protein is an essential subunit of photosystem II (PSII) and is the target for herbicides. We have used herbicide-resistant mutants to study the role of the two homologous copies of the psbA genes in both strains (the third copy is not expressed). Several herbicide resistance mutations map within the psbAI gene in Synechocystis 6714 (G. Ajlani et al.), Plant Mol. Biol. 13 (1989): (469–479). We have looked for mutations in copy II. Results show that in Synechocystis 6714, only psbAI contains herbicide resistance mutations. Relative expression of psbAI and psbAII has been measured by analysing the proportions of resistant and sensitive D₁ in the thylakoid membranes of the mutants. In normal growth conditions, 95% resistant D₁ and 5% sensitive D₁ were found. In high light conditions, expression of psbAII was enhanced, producing 15% sensitive D₁. This enhancement is specifically due to high light and not to the decrease of D₁ concentration caused by photoinhibition. Copy I of Synechocystis 6714 corresponds to copy 2 of Synechocystis 6803 since it was always psbA2 which was recombined in Synechocystis 6803 transformants. PSII of the transformant strains was found to be 95% resistant to herbicides as in resistant mutants of Synechocystis 6714.     

Multiple rpoD-Related Genes of Cyanobacteria

Genomes of many eubacterial strains have been shown to encode for multiple rpoD-related genes. In this report, we describe the identification of the multiple rpoD-related genes of cyanobacterial strains. DNAs of three cyanobacterial strains, Anabaena sp. PCC7120, Synechococcus sp. PCC7942, and Synechocystis sp. PCC6803, were examined by Southern hybridization, using a synthetic probe designed for detecting rpoD or rpoD-related genes. Four or five hybridization signals were found in each DNA. Four DNA regions of Synechococcus sp. PCC7942 corresponding to the hybridization signals were cloned and partially sequenced. The sequence data indicate the presence of genes, named rpoDl, rpoD2, rpoD3, and rpoD4, whose products are highly similar to the basic structure of the principal σ factors of eubacterial strains. The rpoDl gene showed the greatest similarity to the sigA gene of Anabaena sp. PCC7120.     

Changes in composition of membrane proteins accompanying the regulation of PS I/PS II stoichiometry observed with Synechocystis PCC 6803

Changes in composition of membrane proteins in Synechocystis PCC 6803 induced by the shift of light regime for photosynthetic growth were studied in relation to the regulation of PS I/PS II stoichiometry. Special attention was paid to the changes in abundance of proteins of PS I and PS II complexes. Composition was examined using a LDS-PAGE and a quantitative enzyme immunoassay. Abundance of PsaA/B polypeptides and the PsaC polypeptide of the PS I complex, on a per cell basis, increased under the light regime exciting preferentially PS II and decreased under the light regime exciting mainly PS I. Similar changes were observed with polypeptides of 18.5, 10 and 8.5 kDa. The abundance of other proteins associated with membranes, including PsbA polypeptide of the PS II complex, was fairly constant irrespective of light regime. These results are consistent with our previous observations with other strains of cyanophytes (Anabaena variabilis M2 and Synechocystis PCC 6714) that PS I is the variable component in changes in PS I/PS II stoichiometry in response to changing light regimes for photosynthesis.Abbreviations CBB Coomassie brilliant blue - Chl chlorophyll - EIA enzyme immunoassay - LDS lithium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - PS photosystem - PVDF polyvinylidene difluoride     

Reconstruction and analysis of genome-scale metabolic model of a photosynthetic bacterium

Background  

Synechocystis sp. PCC6803 is a cyanobacterium considered as a candidate photo-biological production platform - an attractive cell factory capable of using CO₂ and light as carbon and energy source, respectively. In order to enable efficient use of metabolic potential of Synechocystis sp. PCC6803, it is of importance to develop tools for uncovering stoichiometric and regulatory principles in the Synechocystis metabolic network.     

Simultaneous increases in the levels of compatible solutes by cost-effective cultivation of Synechocystis sp. PCC 6803

Synechocystis sp. PCC 6803, a cyanobacterium widely used for basic research, is often cultivated in a synthetic medium, BG-11, in the presence of 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) or 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid buffer. Owing to the high cost of HEPES buffer (96.9% of the total cost of BG-11 medium), the biotechnological application of BG-11 is limited. In this study, we cultured Synechocystis sp. PCC 6803 cells in BG-11 medium without HEPES buffer and examined the effects on the primary metabolism. Synechocystis sp. PCC 6803 cells could grow in BG-11 medium without HEPES buffer after adjusting for nitrogen sources and light intensity; the production rate reached 0.54 g cell dry weight·L⁻¹·day⁻¹, exceeding that of commercial cyanobacteria and Synechocystis sp. PCC 6803 cells cultivated under other conditions. The exclusion of HEPES buffer markedly altered the metabolites in the central carbon metabolism; particularly, the levels of compatible solutes, such as sucrose, glucosylglycerol, and glutamate were increased. Although the accumulation of sucrose and glucosylglycerol under high salt conditions is antagonistic to each other, these metabolites accumulated simultaneously in cells grown in the cost-effective medium. Because these metabolites are used in industrial feedstocks, our results reveal the importance of medium composition for the production of metabolites using cyanobacteria.     

An Evidence Indicating That a Weak Orange Light Absorbed by Phycobilisomes Causes Inactivation of PSII in Cells of the Red Alga Porphyridium cruentum Grown under a Weak Red Light Preferentially Exciting Chl a

Changes in the PSII fluorescence upon shift of light qualitywere studied with the red alga Porphyridium cruentum IAM R-1and supplementarily with P. cruentum ATCC 50161, the cyanophytesSynechocystis spp. PCC6714 and PCC6803 and Synechococcus sp.NIBB1071. When Porphyridium cruentum grown under a weak redlight (PSI light) preferentially absorbed by Chl a was illuminatedwith a weak orange light (PSII light) mainly absorbed by phycobilisomes(PBS), a change of PSII fluorescence at room temperature wasinduced. The ratio of Fvm (Fm— Fo) to Fm was reduced rapidlyaccompanying the increase in Fo (T^1/2 ca. 3 min). The effectsof DCMU and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinoneindicated that the fluorescence change is induced when plastoquinonepool is highly reduced. The fluorescence change after a shortPSII light illumination was reversible; it rapidly recoveredin the dark (T ^1/2 ca. 3 min). The reversibility was graduallyreduced and disappeared after 40 h under PSII light accompanyingdecrease in PSII activity per PBS down to almost 50%. Sincethe pattern of the fluorescence change resembles that observablewhen PSII is photoinactivated, PSII light probably induces thephotoinactivation of PSII, possibly reversibly at first andirreversibly after prolonged illumination. Such a rapid fluorescencechange was insignificant in Synechocystis sp. either PCC6714or PCC6803. Only a slow and small decrease in Fvm/Fm level appearedafter prolonged PSII light illumination (the reduction of PSIIactivity per PBS was around 20%). In Porphyridium, shift fromPSII light to PSI light caused a rapid and chloramphenicol-sensitiveFvm/Fm elevation during the first 10 h while the increase inPSH activity per PBS was only 10% of that before the light shift.Then, a gradual elevation followed up to the level at the steadystate under PSI light. A similar rapid increase in Fvm/Fm wasobserved with Synechocystis PCC6714, in which the synthesisof PSII is not regulated, suggesting that a rapid increase inFvm/Fm does not reflect the acceleration of the synthesis ofPSII. Results were interpreted as that (1) PSII light causesphotoinactivation of PSII. Such a photoinactivation is markedin Prophyridium cells grown under PSI light. (2) In Porphyridium,changes in the abundance of PSII upon shift of light qualityare largely attributed to the photoinactivation of this type. (Received February 19, 1999; Accepted June 14, 1999)     

Characterization of the cell wall of the unicellular cyanobacterium Synechocystis PCC 6714

Cell walls free of cytoplasmic- and thylakoid membranes were isolated from Synechocystis PCC 6714 by sucrose density gradient centrifugation and extraction with Triton X-100. The Triton-insoluble cell wall fraction retained the multilayered fine structure. Peptidoglycan, proteins, polysaccharides, lipopolysaccharides, lipids and carotenoids were found as constituents of the cell wall. Polypeptide and lipid patterns of cell walls were completely different from that of the cytoplasmic/thylakoid membrane fraction. The purified cell walls contained about twelve outer membrane proteins. The two major polypeptides (M_r 67,000 and 61,000) were found to be associated with the peptidoglycan by ionic interactions.Myxoxanthophyll (major carotenoid), related carotenoid-glycosides and zeaxanthin were the predominating carotenoids of the cell wall of Synechocystis PCC 6714 over echinenone and -carotene. A polar unknown carotenoid was observed, the absorption spectrum of which resembled that of myxoxanthophyll. It was exclusively found in cell walls, but not in the cytoplasmic/thylakoid membrane fraction.Abbreviations Hep heptose - DGDG digalactosyldiglyceride - MGDG monogalactosyldiglyceride - SL sulfolipid - PC phosphatidylcholin - PG phosphatidylglyceride Dedicated to Prof. Dr. G. Drews on the occasion of his 60th birthday     

Identification and bioinformatic analysis of the membrane proteins of synechocystis sp. PCC 6803

Background  

The membranes of Synechocystis sp. PCC 6803 play a central role in photosynthesis, respiration and other important metabolic pathways. Comprehensive identification of the membrane proteins is of importance for a better understanding of the diverse functions of its unique membrane structures. Up to date, approximately 900 known or predicted membrane proteins, consisting 24.5% of Synechocystis sp. PCC 6803 proteome, have been indentified by large-scale proteomic studies.     

Effects of fatty acid activation on photosynthetic production of fatty acid-based biofuels in Synechocystis sp. PCC6803

Background

Direct conversion of solar energy and carbon dioxide to drop in fuel molecules in a single biological system can be achieved from fatty acid-based biofuels such as fatty alcohols and alkanes. These molecules have similar properties to fossil fuels but can be produced by photosynthetic cyanobacteria.

Results

Synechocystis sp. PCC6803 mutant strains containing either overexpression or deletion of the slr1609 gene, which encodes an acyl-ACP synthetase (AAS), have been constructed. The complete segregation and deletion in all mutant strains was confirmed by PCR analysis. Blocking fatty acid activation by deleting slr1609 gene in wild-type Synechocystis sp. PCC6803 led to a doubling of the amount of free fatty acids and a decrease of alkane production by up to 90 percent. Overexpression of slr1609 gene in the wild-type Synechocystis sp. PCC6803 had no effect on the production of either free fatty acids or alkanes. Overexpression or deletion of slr1609 gene in the Synechocystis sp. PCC6803 mutant strain with the capability of making fatty alcohols by genetically introducing fatty acyl-CoA reductase respectively enhanced or reduced fatty alcohol production by 60 percent.

Conclusions

Fatty acid activation functionalized by the slr1609 gene is metabolically crucial for biosynthesis of fatty acid derivatives in Synechocystis sp. PCC6803. It is necessary but not sufficient for efficient production of alkanes. Fatty alcohol production can be significantly improved by the overexpression of slr1609 gene.