Characterization of two Synechococcus sp. PCC7002‐related cyanobacterial strains in relation to 16S rDNA,crtR gene,lipids and pigments

Two Synechococcus strains from the Culture Collection of the Institute for Marine Sciences of Andalusia (Cádiz, Spain), namely Syn01 and Syn02, were found to be closely related to the model strain Synechococcus sp. PCC7002 according to 16S rDNA (99% identity). Pigment and lipid profiles and crtR genes of these strains were ascertained and compared. The sequences of the crtR genes of these strains were constituted by 888 bp, and showed 99% identity between Syn01 and Syn02, and 94% identity of Syn01 and Syn02 to Synechococcus sp. PCC7002. There was coincidence in photosynthetic pigments between the three strains apart from the pigment synechoxanthin, which could be only observed in Synechococcus sp. PCC7002. Species of sulfoquinovosyl‐diacyl‐glycerol (SQDG), phosphatidyl‐glycerol (PG), mono‐ and di‐galactosyl‐diacyl‐glycerol (MGDG and DGDG) were detected by high performance liquid chromatography‐mass spectrometry analysis of lipid extracts. The most abundant species within each lipid class were those containing C18:3 together with C16:0 fatty acyl substituents in the glycerol backbone of the same molecule. From these results it is concluded that these cyanobacterial strains belong to group 2 of the lipid classification of cyanobacteria.     

Engineering of Synechococcus sp. strain PCC 7002 for the photoautotrophic production of light-sensitive riboflavin (vitamin B2)

Due to their capability of photosynthesis and autotrophic growth, cyanobacteria are currently investigated with regard to the sustainable production of a wide variety of chemicals. So far, however, no attempt has been undertaken to engineer cyanobacteria for the biotechnological production of vitamins, which is probably due to the light-sensitivity of many of these compounds. We now describe a photoautotrophic bioprocess to synthesize riboflavin, a vitamin used as a supplement in the feed and food industry. By overexpressing the riboflavin biosynthesis genes ribDGEABHT from Bacillus subtilis in the marine cyanobacterium Synechococcus sp. PCC 7002 riboflavin levels in the supernatant of the corresponding recombinant strain increased 56-fold compared to the wild-type. Introduction of a second promoter region upstream of the heterologous ribAB gene – coding for rate-limiting enzymatic functions in the riboflavin biosynthesis pathway – led to a further increase of riboflavin levels (211-fold compared to the wild-type). Degradation of the light-sensitive product riboflavin was prevented by culturing the genetically engineered Synechococcus sp. PCC 7002 strains in the presence of dichromatic light generated by red light-emitting diodes (λ = 630 and 700 nm). Synechococcus sp. PCC 7002 naturally is resistant to the toxic riboflavin analog roseoflavin. Expression of the flavin transporter pnuX from Corynebacterium glutamicum in Synechococcus sp. PCC 7002 resulted in roseoflavin-sensitive recombinant strains which in turn could be employed to select roseoflavin-resistant, riboflavin-overproducing strains as a chassis for further improvement.     

Roles for heme–copper oxidases in extreme high-light and oxidative stress response in the cyanobacterium Synechococcus sp. PCC 7002

The ctaCIDIEI and ctaCIIDIIEII gene clusters that encode heme–copper cytochrome oxidases have been characterized in the marine cyanobacterium Synechococcus sp. PCC 7002 and the inactivation of ctaDI was shown to affect high-light adaptation. In this study, Synechococcus sp. PCC 7002 wild-type, ctaDI, ctaDII, and ctaDI–ctaDII double mutants were grown under extreme high-light and oxidative stress to further assess the roles of cytochrome oxidases in cyanobacteria. Cells of the ctaDI mutant strain barely grew under extreme high-light illumination of 4.5 mE m⁻² s⁻¹, suggesting that CtaDI is required for high-light acclimation in Synechococcus sp. PCC 7002. The ctaDI–ctaDII double mutant cells unexpectedly tolerated extreme high-light intensity, indicating that the disruption of ctaDII gene suppresses the high-light sensitivity phenotype of the ctaDI single mutant. The ctaDII mutant cells also exhibited higher tolerance to the oxidative stress compound, methyl viologen, in the growth media. The ctaDII mutant and the ctaDI–ctaDII double mutant cells had approximately twofold higher levels of superoxide dismutase (SOD) activity, indicating that the disruption of ctaDII gene increased the capacity to decompose active oxygen species. These results suggest that the CtaII cytochrome oxidase may be involved with the oxidative stress response, including the control of SOD expression.     

Computational evaluation of Synechococcus sp. PCC 7002 metabolism for chemical production

Cyanobacteria are ideal metabolic engineering platforms for carbon-neutral biotechnology because they directly convert CO₂ to a range of valuable products. In this study, we present a computational assessment of biochemical production in Synechococcus sp. PCC 7002 (Synechococcus 7002), a fast growing cyanobacterium whose genome has been sequenced, and for which genetic modification methods have been developed. We evaluated the maximum theoretical yields (mol product per mol CO₂ or mol photon) of producing various chemicals under photoautotrophic and dark conditions using a genome-scale metabolic model of Synechococcus 7002. We found that the yields were lower under dark conditions, compared to photoautotrophic conditions, due to the limited amount of energy and reductant generated from glycogen. We also examined the effects of photon and CO₂ limitations on chemical production under photoautotrophic conditions. In addition, using various computational methods such as minimization of metabolic adjustment (MOMA), relative metabolic change (RELATCH), and OptORF, we identified gene-knockout mutants that are predicted to improve chemical production under photoautotrophic and/or dark anoxic conditions. These computational results are useful for metabolic engineering of cyanobacteria to synthesize value-added products.     

An Organic Acid Based Counter Selection System for Cyanobacteria

Cyanobacteria are valuable organisms for studying the physiology of photosynthesis and carbon fixation, as well as metabolic engineering for the production of fuels and chemicals. This work describes a novel counter selection method for the cyanobacterium Synechococcus sp. PCC 7002 based on organic acid toxicity. The organic acids acrylate, 3-hydroxypropionate, and propionate were shown to be inhibitory towards Synechococcus sp. PCC 7002 and other cyanobacteria at low concentrations. Inhibition was overcome by a loss of function mutation in the gene acsA, which is annotated as an acetyl-CoA ligase. Loss of AcsA function was used as a basis for an acrylate counter selection method. DNA fragments of interest were inserted into the acsA locus and strains harboring the insertion were isolated on selective medium containing acrylate. This methodology was also used to introduce DNA fragments into a pseudogene, glpK. Application of this method will allow for more advanced genetics and engineering studies in Synechococcus sp. PCC 7002 including the construction of markerless gene deletions and insertions. The acrylate counter-selection could be applied to other cyanobacterial species where AcsA activity confers acrylate sensitivity (e.g. Synechocystis sp. PCC 6803).     

Nucleotide Sequence of Plasmid pAQ1 of Marine Cyanobacterium Synechococcus sp. PCC7002

We have determined the complete nucleotide sequence of pAQ1,the smallest plasmid of the unicellular marine cyanobacteriumSynechococcus sp. PCC7002. The plasmid consists of 4,809 bpand has at least four open reading frames that potentially encodepolypeptides of 50 or more amino acids. We found that a palindromicelement, the core sequence of which is G(G/A)CGATCGCC, is over-representednot only in plasmid pAQ1 but also in the accumulated cyanobacterialgenomic sequences from Synechococcus sp. PCC6301, PCC7002, PCC7942,vulcanus and Synechocystis sp. PCC6803 within GenBank and EMBLdatabases. It suggests that this sequence might mediate generearrangement, thus increasing genetic diversity, since recombinationevents are frequent in cyanobacteria.     

Identification of a cold-regulated RNA-binding protein from the marine cyanobacteriumSynechococcus sp. PCC7002

TherbpA gene of a marine cyanobacteriumSynechococcus sp. PCC7002 encodes an RNA-binding protein, which exhibited an affinity to poly (G) and poly (U) but not to poly (A) and poly (C). Although there are at least two homologous genes in this cyanobacterium, the RbpA protein was the only RNA-binding protein that was detected in the cell of this strain. This protein was apparently absent in the cells grown at 38 C but was abundant in the cells grown at 25 C.     

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     

The Biosynthetic pathway for synechoxanthin, an aromatic carotenoid synthesized by the euryhaline, unicellular cyanobacterium Synechococcus sp. strain PCC 7002

The euryhaline, unicellular cyanobacterium Synechococcus sp. strain PCC 7002 produces the dicyclic aromatic carotenoid synechoxanthin (χ,χ-caroten-18,18′-dioic acid) as a major pigment (>15% of total carotenoid) and when grown to stationary phase also accumulates small amounts of renierapurpurin (χ,χ-carotene) (J. E. Graham, J. T. J. Lecomte, and D. A. Bryant, J. Nat. Prod. 71:1647-1650, 2008). Two genes that were predicted to encode enzymes involved in the biosynthesis of synechoxanthin were identified by comparative genomics, and these genes were insertionally inactivated in Synechococcus sp. strain PCC 7002 to verify their function. The cruE gene (SYNPCC7002_A1248) encodes β-carotene desaturase/methyltransferase, which converts β-carotene to renierapurpurin. The cruH gene (SYNPCC7002_A2246) encodes an enzyme that is minimally responsible for the hydroxylation/oxidation of the C-18 and C-18′ methyl groups of renierapurpurin. Based on observed and biochemically characterized intermediates, a complete pathway for synechoxanthin biosynthesis is proposed.     

The cloning and sequencing of Synechococcus sp. PCC 7002 petCA operon: Implications for the cytochrome c-553 binding domain of cytochrome f

The genes encoding the Rieske iron-sulfur protein and cytochrome f from a unicellular, naturally transformable, photoheterotrophic cyanobacterium, Synechococcus sp. PCC 7002, formerly Agmenellum quadruplicatum, have been isolated and sequenced. The two genes were found to be on a single operon, petCA.The Synechococcus sp. PCC 7002 iron-sulfur protein contains 181 amino acids, the conserved putative iron-binding domains CTHLGCV, residues 108–114, and CPCHGS, residues 128–133, no presequence and has a 73% sequence identity to the Nostoc PCC 7906 iron-sulfur protein. The 325 amino acid apocytochrome f sequence contains a 42 amino acid presequence, a CANCH heme binding domain, residues 20–24 from the presumed start of the mature protein, and a predicted hydrophobic membrane-spanning domain, residues 250–269. The mature cytochrome f sequence has a 71.5% sequence identity with Nostoc PCC 7906 cytochrome f and possesses a large (-14) negative charge and low calculated pI of 4.47 compared to higher plant chloroplast sequences. Nine separate domains showing differences in charged residues among cyanobacteria and plants have been identified and the possibility that these domains are involved in the ionic interactions with plastocyanin or cytochrome c-553 is discussed.The sequences reported in this paper have been deposited in the EMBL/Genbank data base (IntelliGenetics, Mountain View, CA, and Eur. Mol. Biol. Lab., Heidelberg) (accession no. M74514).     

<Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 CruA (sll0147) encodes lycopene cyclase and requires bound chlorophyll <Emphasis Type="Italic">a</Emphasis> for activity

The genome of the model cyanobacterium, Synechococcus sp. PCC 7002, encodes two paralogs of CruA-type lycopene cyclases, SynPCC7002_A2153 and SynPCC7002_A0043, which are denoted cruA and cruP, respectively. Unlike the wild-type strain, a cruA deletion mutant is light-sensitive, grows slowly, and accumulates lycopene, γ-carotene, and 1-OH-lycopene; however, this strain still produces β-carotene and other carotenoids derived from it. Expression of cruA from Synechocystis sp. PCC 6803 (cruA ₆₈₀₃) in Escherichia coli strains that synthesize either lycopene or γ-carotene did not lead to the synthesis of either γ-carotene or β-carotene, respectively. However, expression of this orthologous cruA ₆₈₀₃ gene (sll0147) in the Synechococcus sp. PCC 7002 cruA deletion mutant produced strains with phenotypic properties identical to the wild type. CruA₆₈₀₃ was purified from Synechococcus sp. PCC 7002 by affinity chromatography, and the purified protein was pale yellow-green due to the presence of bound chlorophyll (Chl) a and β-carotene. Native polyacrylamide gel electrophoresis of the partly purified protein in the presence of lithium dodecylsulfate at 4 °C confirmed that the protein was yellow-green in color. When purified CruA₆₈₀₃ was assayed in vitro with either lycopene or γ-carotene as substrate, β-carotene was synthesized. These data establish that CruA₆₈₀₃ is a lycopene cyclase and that it requires a bound Chl a molecule for activity. Possible binding sites for Chl a and the potential regulatory role of the Chl a in coordination of Chl and carotenoid biosynthesis are discussed.     

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.     

Respiratory terminal oxidases alleviate photo-oxidative damage in photosystem I during repetitive short-pulse illumination in <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

Oxygenic phototrophs are vulnerable to damage by reactive oxygen species (ROS) that are produced in photosystem I (PSI) by excess photon energy over the demand of photosynthetic CO₂ assimilation. In plant leaves, repetitive short-pulse (rSP) illumination produces ROS to inactivate PSI. The production of ROS is alleviated by oxidation of the reaction center chlorophyll in PSI, P700, during the illumination with the short-pulse light, which is supported by flavodiiron protein (FLV). In this study, we found that in the cyanobacterium Synechocystis sp. PCC 6803 P700 was oxidized and PSI was not inactivated during rSP illumination even in the absence of FLV. Conversely, the mutant deficient in respiratory terminal oxidases was impaired in P700 oxidation during the illumination with the short-pulse light to suffer from photo-oxidative damage in PSI. Interestingly, the other cyanobacterium Synechococcus sp. PCC 7002 could not oxidize P700 without FLV during rSP illumination. These data indicate that respiratory terminal oxidases are critical to protect PSI from ROS damage during rSP illumination in Synechocystis sp. PCC 6803 but not Synechococcus sp. PCC 7002.     

Evaluation of quenching methods for metabolite recovery in photoautotrophic Synechococcus sp. PCC 7002

The first step of many metabolomics studies is quenching, a technique vital for rapidly halting metabolism and ensuring that the metabolite profile remains unchanging during sample processing. The most widely used approach is to plunge the sample into prechilled cold methanol; however, this led to significant metabolite loss in Synecheococcus sp. PCC 7002. Here we describe our analysis of the impacts of cold methanol quenching on the model marine cyanobacterium Synechococcus sp. PCC 7002, as well as our brief investigation of alternative quenching methods. We tested several methods including cold methanol, cold saline, and two filtration approaches. Targeted central metabolites were extracted and metabolomic profiles were generated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results indicate that cold methanol quenching induces dramatic metabolite leakage in Synechococcus, resulting in a majority of central metabolites being lost prior to extraction. Alternatively, usage of a chilled saline quenching solution mitigates metabolite leakage and improves sample recovery without sacrificing rapid quenching of cellular metabolism. Finally, we illustrate that metabolite leakage can be assessed, and subsequently accounted for, in order to determine absolute metabolite pool sizes; however, our results show that metabolite leakage is inconsistent across various metabolite pools and therefore must be determined for each individually measured metabolite.     

Chemical reactivity of <Emphasis Type="Italic">Synechococcus</Emphasis> sp. PCC 7002 and <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 hemoglobins: covalent heme attachment and bishistidine coordination

In the absence of an exogenous ligand, the hemoglobins from the cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 coordinate the heme group with two axial histidines (His46 and His70). These globins also form a covalent linkage between the heme 2-vinyl substituent and His117. The in vitro mechanism of heme attachment to His117 was examined with a combination of site-directed mutagenesis, NMR spectroscopy, and optical spectroscopy. The results supported an electrophilic addition with vinyl protonation being the rate-determining step. Replacement of His117 with a cysteine demonstrated that the reaction could occur with an alternative nucleophile. His46 (distal histidine) was implicated in the specificity of the reaction for the 2-vinyl group as well as protection of the protein from oxidative damage caused by exposure to exogenous H₂O₂.     

PsbU, a Protein Associated with Photosystem II, Is Required for the Acquisition of Cellular Thermotolerance in Synechococcus species PCC 7002

PsbU is an extrinsic protein of the photosystem II complex of cyanobacteria and red algae. Our previous in vitro studies (Y. Nishiyama, D.A. Los, H. Hayashi, N. Murata [1997] Plant Physiol 115: 1473–1480) revealed that PsbU stabilizes the oxygen-evolving machinery of the photosystem II complex against heat-induced inactivation in the cyanobacterium Synechococcus sp. PCC 7002. To elucidate the role of PsbU in vivo, we inactivated the psbU gene in Synechococcus sp. PCC 7002 by targeted mutagenesis. Inactivation of the psbU gene resulted in marked changes in the acclimative responses of cells to high temperature: Mutated cells were unable to increase the thermal stability of their oxygen-evolving machinery when grown at moderately high temperatures. Moreover, the cellular thermotolerance of the mutated cells failed to increase upon acclimation of cells to high temperature. The heat-shock response, as assessed in terms of the levels of homologs of the heat-shock proteins Hsp60, Hsp70, and Hsp17, was unaffected by the mutation in psbU, suggesting that heat-shock proteins were not involved in the changes in the acclimative responses. Our observations indicate that PsbU is involved in the mechanism that underlies the enhancement of the thermal stability of the oxygen-evolving machinery and that the stabilization of the oxygen-evolving machinery is crucial for the acquisition of cellular thermotolerance.     

A simple method for isolation and construction of markerless cyanobacterial mutants defective in acyl-acyl carrier protein synthetase

Cyanobacterial mutants defective in acyl-acyl carrier protein synthetase (Aas) secrete free fatty acids (FFAs) into the external medium and hence have been used for the studies aimed at photosynthetic production of biofuels. While the wild-type strain of Synechocystis sp. PCC 6803 is highly sensitive to exogenously added linolenic acid, mutants defective in the aas gene are known to be resistant to the externally provided fatty acid. In this study, the wild-type Synechocystis cells were shown to be sensitive to lauric, oleic, and linoleic acids as well, and the resistance to these fatty acids was shown to be enhanced by inactivation of the aas gene. On the basis of these observations, we developed an efficient method to isolate aas-deficient mutants from cultures of Synechocystis cells by counter selection using linoleic acid or linolenic acid as the selective agent. A variety of aas mutations were found in about 70 % of the FFA-resistant mutants thus selected. Various aas mutants were isolated also from Synechococcus sp. PCC 7002, using lauric acid as a selective agent. Selection using FFAs was useful also for construction of markerless aas knockout mutants from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002. Thus, genetic engineering of FFA-producing cyanobacterial strains would be greatly facilitated by the use of the FFAs for counter selection.