A novel plasmid recombination mechanism of the marine cyanobacterium Synechococcus sp. PCC7002.

We describe a novel mechanism of site-specific recombination in the unicellular marine cyanobacterium Synechococcus sp. PCC7002. The specific recombination sites on the smallest plasmid pAQ1 were localized by studying the properties of pAQ1-derived shuttle-vectors. We found that a palindromic element, the core sequence of which is G(G/A)CGATCGCC, functions as a resolution site for site-specific plasmid recombination. Furthermore, site-directed mutagenesis analysis of the element show that the site-specific recombination in the cyanobacterium requires sequence specificity, symmetry in the core sequence and, in part, the spacing between the elements. Interestingly, this element is over-represented not only in pAQ1 and in the genome of the cyanobacterium, but also in the accumulated cyanobacterial sequences from Synechococcus sp. PCC6301, PCC7942, vulcanus and Synechocystis sp. PCC6803 within GenBank and EMBL databases. Thus, these findings strongly suggest that the site-specific recombination mechanism based on the palindromic element should be common in these 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.     

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.     

A high temperature-sensitive mutant of Synechococcus sp. PCC 7002 with modifications in the endogenous plasmid, pAQ1

To study thermal adaptations in the cyanobacterium, Synechococcus sp. PCC 7002, we screened about 3,000 mutants for their tolerance to high temperature, and found one, SHT1, that is sensitive to high-temperature stress. The mutant had a modified gene construct in the endogenous plasmid, pAQ1. One of the four ORFs, ORF93, was duplicated, and its mRNA level was higher than in the wild type. At 38 degrees C, the growth of SHT1 was retarded as compared with the wild type, and above 38 degrees C, almost all the cells of SHT1 died. This temperature is much lower than that required for induction of heat shock proteins. Interestingly, in both the wild type and SHT1, the thermal stability of oxygen-evolving machinery increased upon acclimation to high temperatures. These findings indicate that the lack of thermal tolerance in the SHT1 strain is likely independent of the adaptation of the PSII complex and heat shock responses, whereas there are essential contributions of genes in the endogenous plasmid to the adaptation to high temperature. Thus, understanding the role of pAQ1 in the adaptation of Synechococcus sp. PCC 7002 to high-temperature environments is the first step in elucidating the function of this plasmid.     

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.     

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.     

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).     

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.     

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.     

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).     

Donation of Electrons from Cytosolic Components to the Intersystem Chain in the Cyanobacterium Synechococcus sp. PCC 7002 as Determined by the Reduction of P700+

Cells, of Synechococcus sp. PCC 7002 showed a low oxidationlevel of P700 under a far-red light at 6 W m^–2 which inducednearly complete oxidation of P700 in spinach leaves, and a strongerfar-red light was required to observe the oxidation of P700.DCMU did not affect the level of P700⁺² but 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinoneinduced the oxidation of P700 under far-red light, indicatingthat the low oxidation level of P700 was due to the donationof electrons to P700⁺² from the cytosolic respiratory donorsthrough the intersystem chain at the plastoquinone pool. Theelectron transfer from the cytosolic donors to the intersystemchain was inhibited by HgCl₂ but not by antimycin A. The reductionof P700⁺ in Synechococcus cells, after illumination by strongfar-red light was mostly accounted for by the electron flowto the inter system chain from the respiratory donors (t     

<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.     

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 Novel Expression Vector for the Cyanobacterium, Synechococcus PCC 6301

A cyanobacterial expression vector was constructed using ribulose-1,5-bisphosphatecarboxylase/oxygenase (RuBisCO) promoter and terminator sequencesderived from Synechococcus PCC 6301. The recombinant plasmid,designated pARUB19, has an ampicillin-resistant (Ap^R) gene asa selectable marker and four unique restriction sites to allowthe insertion of foreign genes. Using this vector, the luciferasegene from the firefly, Photinus pyralis, was introduced intoSynechococcus PCC 6301 cells. The luciferase expression vectorcould be maintained stably in the host cells. Light productionof luciferin/luciferase was detected in the transformants. Luciferaseamounted to 1.2% of the total soluble protein. This plasmidmay facilitate higher levels of foreign gene expression in SynechococcusPCC 6301.     

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.     

Identification of replication and stability functions in the complete nucleotide sequence of plasmid pUH24 from the cyanobacterium Synechococcus sp. PCC 7942

The complete nucleotide sequence is presented for pUH24, the small plasmid of Synechococcus sp. PCC 7942. pUH24 consists of 7835bp and has a G+C content of 59%. The distribution of translation start and stop codons in the sequence allows 36 open reading frames that potentially encode polypeptides of 50 or more amino acids. We postulate that eight of these open reading frames are actual coding sequences. A region has been identified, by experiment, that contains two functions, designated pmaA and pmaB, involved in the segregational stability of the plasmid. The minimal region of pUH24 fully capable of supporting autonomous replication consists of a 3.6kb DNA fragment, which is almost entirely occupied by two overlapping genes most likely coding for essential replication proteins (repA and repB).     

Genetic analysis of two new mutations resulting in herbicide resistance in the cyanobacterium Synechcoccus sp. PCC 7002

Two herbicide-resistant strains of the cyanobacterium Synechococcus sp. PCC 7002 are compared to the wild-type with respect to the DNA changes which result in herbicide resstance. The mutations have previously been mapped to a region of the cyanobacterial genome which encodes oneof three copies of psbA, the gene which encodes the 32 kDa Q_b-binding protein also known as D1 (Buzby et al. 1987). The DNA sequence of the wild-type gene was first determined and used as a comparison to that of the mutant alleles. A point mutation at codon 211 in the psbA1 coding locus (TTC) to TCC) results in an amino acid change from phenylalanine to serine in the D1 protein. This mutation confers resistance to atrazine and diuron at seven times and at two times the minimal inhibitory concentration (MIC) for the wild-type, respectively. A mutation at codon 211 resulting in herbicide resistance has not previously been described in the literature. A second point mutation at codon 219 in the psbA1 coding locus (GTA to ATA) results in an amino acid change from valine to isoleucine in the D1 protein. This mutation confers resistance to diuron and atrazine at ten times and at two times the MIC for the wild-type, respectively. An identical codon change conferring similar herbicide resistance patterns has previously been described in Chlamydomonas reinhardtii. The atrazine-resistance phenotype in Synechococcus sp. PCC 7002 was shown to be dominant by plasmid segregation analysis.Abbreviations At ^r atrazine resistance - Du ^r diuron resistance - Km ^r kanamycin resistance - Ap ^r ampicillin resistance - MIC Minimum inhibitory concentration