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).     

Cyanophycin Production in a Phycoerythrin-Containing Marine Synechococcus Strain of Unusual Phylogenetic Affinity

Thirty-two strains of phycoerythrin-containing marine picocyanobacteria were screened for the capacity to produce cyanophycin, a nitrogen storage compound synthesized by some, but not all, cyanobacteria. We found that one of these strains, Synechococcus sp. strain G2.1 from the Arabian Sea, was able to synthesize cyanophycin. The cyanophycin extracted from the cells was composed of roughly equimolar amounts of arginine and aspartate (29 and 35 mol%, respectively), as well as a small amount of glutamate (15 mol%). Phylogenetic analysis, based on partial 16S ribosomal DNA (rDNA) sequence data, showed that Synechococcus sp. strain G2.1 formed a well-supported clade with several strains of filamentous cyanobacteria. It was not closely related to several other well-studied marine picocyanobacteria, including Synechococcus strains PCC7002, WH7805, and WH8018 and Prochlorococcus sp. strain MIT9312. This is the first report of cyanophycin production in a phycoerythrin-containing strain of marine or halotolerant Synechococcus, and its discovery highlights the diversity of this ecologically important functional group.     

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.     

One-step plasmid construction for generation of knock-out mutants in cyanobacteria: studies of glycogen metabolism in <Emphasis Type="Italic">Synechococcus</Emphasis> sp. PCC 7002

Genome sequences of microorganisms typically contain hundreds of genes with vaguely defined functions. Targeted gene inactivation and phenotypic characterization of the resulting mutant strains is a powerful strategy to investigate the function of these genes. We have adapted the recently reported uracil-specific excision reagent (USER) cloning method for targeted gene inactivation in cyanobacteria and used it to inactivate genes in glycogen metabolism in Synechococcus sp. PCC 7002. Knock-out plasmid constructs were made in a single cloning step, where transformation of E. coli yielded about 90% colonies with the correct construct. The two homologous regions were chosen independently of each other and of restriction sites in the target genome. Mutagenesis of Synechococcus sp. PCC 7002 was tested with four antibiotic resistance selection markers (spectinomycin, erythromycin, kanamycin, and gentamicin), and both single-locus and double-loci mutants were prepared. We found that Synechococcus sp. PCC 7002 contains two glycogen phosphorylases (A0481/glgP and A2139/agpA) and that both need to be genetically inactivated to eliminate glycogen phosphorylase activity in the cells.     

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.     

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.     

<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.     

Introduction of NADH-dependent nitrate assimilation in Synechococcus sp. PCC 7002 improves photosynthetic production of 2-methyl-1-butanol and isobutanol

Cyanobacteria hold promise for renewable chemical production due to their photosynthetic nature, but engineered strains frequently display poor production characteristics. These difficulties likely arise in part due to the distinctive photoautotrophic metabolism of cyanobacteria. In this work, we apply a genome-scale metabolic model of the cyanobacteria Synechococus sp. PCC 7002 to identify strain designs accounting for this unique metabolism that are predicted to improve the production of various biofuel alcohols (e.g. 2-methyl-1-butanol, isobutanol, and 1-butanol) synthesized via an engineered biosynthesis pathway. Using the model, we identify that the introduction of a large, non-native NADH-demand into PCC 7002's metabolic network is predicted to enhance production of these alcohols by promoting NADH-generating reactions upstream of the production pathways. To test this, we construct strains of PCC 7002 that utilize a heterologous, NADH-dependent nitrite reductase in place of the native, ferredoxin-dependent enzyme to create an NADH-demand in the cells when grown on nitrate-containing media. We find that photosynthetic production of both isobutanol and 2-methyl-1-butanol is significantly improved in the engineered strain background relative to that in a wild-type background. We additionally identify that the use of high-nutrient media leads to a substantial prolongment of the production curve in our alcohol production strains. The metabolic engineering strategy identified and tested in this work presents a novel approach to engineer cyanobacterial production strains that takes advantage of a unique aspect of their metabolism and serves as a basis on which to further develop strains with improved production of these alcohols and related products.     

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.     

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.     

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.     

Genome mining of mycosporine-like amino acid (MAA) synthesizing and non-synthesizing cyanobacteria: A bioinformatics study

Mycosporine-like amino acids (MAAs) are a family of more than 20 compounds having absorption maxima between 310 and 362 nm. These compounds are well known for their UV-absorbing/screening role in various organisms and seem to have evolutionary significance. In the present investigation we tested four cyanobacteria, e.g., Anabaena variabilis PCC 7937, Anabaena sp. PCC 7120, Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 6301, for their ability to synthesize MAA and conducted genomic and phylogenetic analysis to identify the possible set of genes that might be involved in the biosynthesis of these compounds. Out of the four investigated species, only A. variabilis PCC 7937 was able to synthesize MAA. Genome mining identified a combination of genes, YP_324358 (predicted DHQ synthase) and YP_324357 (O-methyltransferase), which were present only in A. variabilis PCC 7937 and missing in the other studied cyanobacteria. Phylogenetic analysis revealed that these two genes are transferred from a cyanobacterial donor to dinoflagellates and finally to metazoa by a lateral gene transfer event. All other cyanobacteria, which have these two genes, also had another copy of the DHQ synthase gene. The predicted protein structure for YP_324358 also suggested that this product is different from the chemically characterized DHQ synthase of Aspergillus nidulans contrary to the YP_324879, which was predicted to be similar to the DHQ synthase. The present study provides a first insight into the genes of cyanobacteria involved in MAA biosynthesis and thus widens the field of research for molecular, bioinformatics and phylogenetic analysis of these evolutionary and industrially important compounds. Based on the results we propose that YP_324358 and YP_324357 gene products are involved in the biosynthesis of the common core (deoxygadusol) of all MAAs.     

Bioinformatics evidence for the transfer of mycosporine-like amino acid core (4-deoxygadusol) synthesizing gene from cyanobacteria to dinoflagellates and an attempt to mutate the same gene (YP_324358) in Anabaena variabilis PCC 7937

We have identified a homologue of 4-deoxygadusol (core of mycosporine-like amino acids) synthesizing gene (ZP_05036788) from Synechococcus sp. PCC 7335 that was found to have additional functionally unknown N-terminal domain similar to homologues from dinoflagellates based on the ClustalW analysis. Phylogenetic analysis revealed that Synechococcus sp. (ZP_05036788) makes a clade together with dinoflagellates and was closest to the Oxyrrhis marina. This study shows for the first time that N-terminal additional sequences that possess upstream plastid targeting sequence in Heterocapsa triquetra and Karlodinium micrum were already evolved in cyanobacteria, and plastid targeting sequence were evolved later in dinoflagellates after divergence from chloroplast lacking Oxyrrhis marina. Thus, MAAs synthesizing genes were transferred from cyanobacteria to dinoflagellates and possibly Synechococcus sp. PCC 7335 acted as a donor during lateral gene transfer event. In addition, we also tried to mutate 4-deoxygadusol synthesizing gene (YP_324358) of Anabaena variabilis PCC 7937 by homologous recombination, however, all approaches to get complete segregation of the mutants from the wild-type were unsuccessful, showing the essentiality of YP_324358 for A. variabilis PCC 7937.     

Characterization of the heme–histidine cross-link in cyanobacterial hemoglobins from<Emphasis Type="Italic"> Synechocystis</Emphasis> sp. PCC 6803 and<Emphasis Type="Italic"> Synechococcus</Emphasis> sp. PCC 7002

The recombinant product of the hemoglobin gene of the cyanobacterium Synechocystis sp. PCC 6803 forms spontaneously a covalent bond linking one of the heme vinyl groups to a histidine located in the C-terminal helix (His117, or H16). The present report describes the ¹H, ¹⁵N, and ¹³C NMR spectroscopy experiments demonstrating that the recombinant hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002, a protein sharing 59% identity with Synechocystis hemoglobin, undergoes the same facile heme adduct formation. The observation that the extraordinary linkage is not unique to Synechocystis hemoglobin suggests that it constitutes a noteworthy feature of hemoglobin in non-N₂-fixing cyanobacteria, along with the previously documented bis-histidine coordination of the heme iron. A qualitative analysis of the hyperfine chemical shifts of the ferric proteins indicated that the cross-link had modest repercussions on axial histidine ligation and heme electronic structure. In Synechocystis hemoglobin, the unreacted His117 imidazole had a normal pK _a whereas the protonation of the modified residue took place at lower pH. Optical experiments revealed that the cross-link stabilized the protein with respect to thermal and acid denaturation. Replacement of His117 with an alanine yielded a species inert to adduct formation, but inspection of the heme chemical shifts and ligand binding properties of the variant identified position 117 as important in seating the cofactor in its site and modifying the dynamic properties of the protein. A role for bis-histidine coordination and covalent adduct formation in heme retention is proposed.Electronic Supplementary Material Supplementary material is available in the online version of this article at Abbreviations DQF-COSY double-quantum-filtered correlated spectroscopy - GlbN cyanoglobin - Hb hemoglobin - hx hexacoordinate - MALDI matrix-assisted laser desorption ionization - NOE nuclear Overhauser effect - NOESY two-dimensional nuclear Overhauser effect spectroscopy - rHb recombinant hemoglobin - rHb-A recombinant hemoglobin with covalently attached heme - rHb-R recombinant heme-reconstituted hemoglobin - S6803 Synechocystis sp. PCC 6803 - S7002 Synechococcus sp. PCC 7002 - TOCSY totally correlated two-dimensional spectroscopy - TPPI time-proportional phase incrementation - trHb truncated hemoglobin - WATERGATE water suppression by gradient-tailored excitation - WEFT water elimination Fourier transform