The first protein map of Synechococcus sp. strain PCC 7942

The first protein map was developed of Synechococcus sp. strain PCC 7942, a model organism for studies of photosynthesis, prokaryotic circadian rhythms, cell division, carbon-concentrating mechanisms, and adaptive responses to a variety of stresses. The proteome was analyzed by two-dimensional gel electrophoresis with subsequent MALDI-TOF mass spectroscopy and database analysis. Of the 140 analyzed protein spots, 110 were successfully identified as 62 different proteins, many of which occurred as multiple spots on the gel. The identified proteins participate in the major metabolic and cellular processes in cyanobacterial cells during the exponential growth phase. In addition, 14 proteins which were previously either unknown or considered to be hypothetical were shown to be true gene products in Synechococcus sp. strain PCC 7942. These results may be helpful for the annotation of the recently sequenced genome of this cyanobacterium, as well as for biochemical and physiological studies of Synechococcus.     

Screening of cyanobacterial species for calcification

Species of cyanobacteria in the genera Synechococcus and Synechocystis are known to be the catalysts of a phenomenon called "whitings", which is the formation and precipitation of fine-grained CaCO3 particles. Whitings occur when the cyanobacteria fix atmospheric CO2 through the formation of CaCO3 on their cell surfaces, which leads to precipitation to the ocean floor and subsequent entombment in mud. Whitings represent one potential mechanism for CO2 sequestration. Research was performed to determine the ability of various strains of Synechocystis and Synechococcus to calcify when grown in microcosms amended with 2.5 mM HCO(3-) and 3.4 mM Ca2+. Results indicated that although all strains tested have the ability to calcify, only two Synechococcus species, strains PCC 8806 and PCC 8807, were able to calcify to the extent that a CaCO3 precipitate was formed. Enumeration of the cyanobacterial cultures during testing indicated that cell density did not appear to have a direct effect on calcification. Factors that had the greatest effect on calcification were CO2 removal and subsequent generation of alkaline pH. Whereas cell density was similar for all strains tested, differences in maximum pH were demonstrated. As CO2 was removed, growth medium pH increased and soluble Ca2+ was removed from solution. The largest increases in growth medium pH occurred when CO2 levels dropped below 400 ppmv. Research presented demonstrates that, under the conditions tested, many species of cyanobacteria in the genera Synechocystis and Synechococcus are able to calcify but only two species of Synechococcus were able to calcify to an extent that led to the precipitation of calcium carbonate.     

Effect of heat shock on protein synthesis in the cyanobacterium Synechococcus sp. strain PCC 6301.

The response to heat shock at 47 degrees C was examined in the cyanobacterium (blue-green alga) Synechococcus sp. strain PCC 6301. On heat shock, the growth of the cells decreased and they preferentially synthesized a limited number of polypeptides. The rate of synthesis of these proteins increased markedly in the early period of temperature shift up and gradually decreased afterwards. Among the proteins greatly affected by temperature shift up were those with apparent molecular weights of 91,000 (91K), 79K, 78K, 74K, 65K, 64K, 61K, 49K, 45K, 24K, 22K, 18K, 16K, 14K, 12K, and 11.4K, based on their mobilities in sodium dodecyl sulfate-polyacrylamide gels. From these initial studies on Synechococcus sp. strain PCC 6301 we conclude that in cyanobacteria a heat shock response similar to that known to occur in other eucaryotes and procaryotes might exist.     

The pacL gene of Synechococcus sp. strain PCC 7942 encodes a Ca(2+)-transporting ATPase.

An ATP-dependent Ca2+ uptake activity was identified in plasma membrane vesicles prepared from Synechococcus sp. strain PCC 7942. This activity was insensitive to agents which collapse pH gradients and membrane potentials but sensitive to vanadate, indicating that the activity is catalyzed by a P-type Ca(2+)-ATPase. A gene was cloned from Synechococcus sp. strain PCC 7942 by using a degenerate oligonucleotide based on a sequence conserved among P-type ATPases. This gene (pacL) encodes a product similar in structure to eukaryotic Ca(2+)-ATPases. We have shown that pacL encodes a Ca(2+)-ATPase by demonstrating that a strain in which pacL is disrupted has no Ca(2+)-ATPase activity associated with its plasma membrane. In addition, Ca(2+)-ATPase activity was restored to the delta pacL strain by introducing pacL into a second site in the Synechococcus sp. strain PCC 7942 chromosome.     

Characterization of a zwf mutant of Synechococcus sp. strain PCC 7942.

A mutant of the cyanobacterium Synechococcus sp. strain PCC 7942 carrying a disrupted gene encoding glucose-6-phosphate dehydrogenase (zwf) produced no detectable glucose-6-phosphate dehydrogenase as assessed by enzyme assay and Western blot (immunoblot) analysis. This mutant exhibited significantly impaired dark viability.     

Characterization of a Synechococcus sp. strain PCC 7002 spontaneous mutant strain defective in accumulation of photosystem II core chlorophyll-protein complexes.

Two photosystem II-associated chlorophyll-protein complexes of Synechococcus sp. strain PCC 7002 were identified. Their polypeptide compositions were similar to those of chlorophyll-containing antenna complexes of other cyanobacteria. Strain GT8B did not possess the complex responsible for 695-nm fluorescence and was unable to grow photoautotrophically; hence, this complex is necessary for photosystem II function in vivo.     

Physical genome map of the unicellular cyanobacterium Synechococcus sp. strain PCC 7002.

A physical restriction map of the genome of the cyanobacterium Synechococcus sp. strain PCC 7002 was assembled from AscI, NotI, SalI, and SfiI digests of intact genomic DNA separated on a contour-clamped homogeneous electric field pulsed-field gel electrophoresis system. An average genome size of 2.7 x 10(6) bp was calculated from 21 NotI, 37 SalI, or 27 SfiI fragments obtained by the digestions. The genomic map was assembled by using three different strategies: linking clone analysis, pulsed-field fragment hybridization, and individual clone hybridization to singly and doubly restriction-digested large DNA fragments. The relative positions of 21 genes or operons were determined, and these data suggest that the gene order is not highly conserved between Synechococcus sp. strain PCC 7002 and Anabaena sp. strain PCC 7120.     

Induction of the heat shock protein ClpB affects cold acclimation in the cyanobacterium Synechococcus sp. strain PCC 7942.

The heat shock protein ClpB is essential for acquired thermotolerance in cyanobacteria and eukaryotes and belongs to a diverse group of polypeptides which function as molecular chaperones. In this study we show that ClpB is also strongly induced during moderate cold stress in the unicellular cyanobacterium Synechococcus sp. strain PCC 7942. A fivefold increase in ClpB (92 kDa) content occurred when cells were acclimated to 25 degrees C over 24 h after being shifted from the optimal growth temperature of 37 degrees C. A corresponding increase occurred for the smaller ClpB' (78 kDa), which arises from a second translational start within the clpB gene of prokaryotes. Shifts to more extreme cold (i.e., 20 and 15 degrees C) progressively decreased the level of ClpB induction, presumably due to retardation of protein synthesis within this relatively cold-sensitive strain. Inactivation of clpB in Synechococcus sp. increased the extent of inhibition of photosynthesis upon the shift to 25 degrees C and markedly reduced the mutant's ability to acclimate to the new temperature regime, with a threefold drop in growth rate. Furthermore, around 30% fewer delta clpB cells survived the shift to 25 degrees C after 24 h compared to the wild type, and more of the mutant cells were also arrested during cell division at 25 degrees C, remaining attached after septum formation. Development of a cold thermotolerance assay based on cell survival clearly demonstrated that wild-type cells could acquire substantial resistance to the nonpermissive temperature of 15 degrees C by being pre-exposed to 25 degrees C. The same level of cold thermotolerance, however, occurred in the delta clpB strain, indicating ClpB induction is not necessary for this form of thermal resistance in Synechococcus spp. Overall, our results demonstrate that the induction of ClpB contributes significantly to the acclimation process of cyanobacteria to permissive low temperatures.     

Substrate specificities and availability of fucosyltransferase and beta-carotene hydroxylase for myxol 2'-fucoside synthesis in Anabaena sp. strain PCC 7120 compared with Synechocystis sp. strain PCC 6803

To elucidate the biosynthetic pathways of carotenoids, especially myxol 2'-glycosides, in cyanobacteria, Anabaena sp. strain PCC 7120 (also known as Nostoc sp. strain PCC 7120) and Synechocystis sp. strain PCC 6803 deletion mutants lacking selected proposed carotenoid biosynthesis enzymes and GDP-fucose synthase (WcaG), which is required for myxol 2'-fucoside production, were analyzed. The carotenoids in these mutants were identified using high-performance liquid chromatography, field desorption mass spectrometry, and (1)H nuclear magnetic resonance. The wcaG (all4826) deletion mutant of Anabaena sp. strain PCC 7120 produced myxol 2'-rhamnoside and 4-ketomyxol 2'-rhamnoside as polar carotenoids instead of the myxol 2'-fucoside and 4-ketomyxol 2'-fucoside produced by the wild type. Deletion of the corresponding gene in Synechocystis sp. strain PCC 6803 (sll1213; 79% amino acid sequence identity with the Anabaena sp. strain PCC 7120 gene product) produced free myxol instead of the myxol 2'-dimethyl-fucoside produced by the wild type. Free myxol might correspond to the unknown component observed previously in the same mutant (H. E. Mohamed, A. M. L. van de Meene, R. W. Roberson, and W. F. J. Vermaas, J. Bacteriol. 187:6883-6892, 2005). These results indicate that in Anabaena sp. strain PCC 7120, but not in Synechocystis sp. strain PCC 6803, rhamnose can be substituted for fucose in myxol glycoside. The beta-carotene hydroxylase orthologue (CrtR, Alr4009) of Anabaena sp. strain PCC 7120 catalyzed the transformation of deoxymyxol and deoxymyxol 2'-fucoside to myxol and myxol 2'-fucoside, respectively, but not the beta-carotene-to-zeaxanthin reaction, whereas CrtR from Synechocystis sp. strain PCC 6803 catalyzed both reactions. Thus, the substrate specificities or substrate availabilities of both fucosyltransferase and CrtR were different in these species. The biosynthetic pathways of carotenoids in Anabaena sp. strain PCC 7120 are discussed.     

Molecular cloning and characterization of the recA gene from the cyanobacterium Synechococcus sp. strain PCC 7002.

The recA gene of Synechococcus sp. strain PCC 7002 was detected and cloned from a lambda gtwes genomic library by heterologous hybridization by using a gene-internal fragment of the Escherichia coli recA gene as the probe. The gene encodes a 38-kilodalton polypeptide which is antigenically related to the RecA protein of E. coli. The nucleotide sequence of a portion of the gene was determined. The translation of this region was 55% homologous to the E. coli protein; allowances for conservative amino acid replacements yield a homology value of about 74%. The cyanobacterial recA gene product was proficient in restoring homologous recombination and partial resistance to UV irradiation to recA mutants of E. coli. Heterologous hybridization experiments, in which the Synechococcus sp. strain PCC 7002 recA gene was used as the probe, indicate that a homologous gene is probably present in all cyanobacterial strains.     

Carbon supply and 2-oxoglutarate effects on expression of nitrate reductase and nitrogen-regulated genes in Synechococcus sp. strain PCC 7942

Synthesis of nitrate reductase in the unicellular cyanobacterium Synechococcus sp. strain PCC 7942 took place at a slow rate when the cells were incubated without a supply of inorganic carbon, but addition to these cells of CO(2)/bicarbonate or, in a Synechococcus strain transformed with a gene encoding a 2-oxoglutarate permease, 2-oxoglutarate stimulated expression of the enzyme. Induction by 2-oxoglutarate was also observed at the mRNA level for two nitrogen-regulated genes, nir and amt1, but not for the photosystem II D1 protein-encoding gene psbA1. Our results are consistent with a role of 2-oxoglutarate in nitrogen control in cyanobacteria.     

Insertional inactivation of genes to isolate mutants of Synechococcus sp. strain PCC 7942: isolation of filamentous strains.

We have developed a simple procedure for generating mutants of the cyanobacterium Synechococcus sp. strain PCC 7942 in which the site of the lesion can be readily identified. This procedure involves transforming Synechococcus sp. strain PCC 7942 with a library of its own DNA that was fully digested with Sau3A and ligated into the plasmid vector pUC8. The homologous integration of the recombinant plasmid into the genome will often result in the disruption of a gene and the loss of gene function. We have used this method to generate many mutants of Synechococcus sp. strain PCC 7942 which grow as multicellular filaments rather than as unicells. Since the gene harboring the lesion was tagged with pUC8, it was easily isolated. In this paper, we discuss the usefulness of this procedure for the generation of mutants, and we characterize one mutant in which the lesion may be in an operon involved in the assembly of lipopolysaccharides.     

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.     

转小鼠金属硫蛋白-Ⅰ基因聚球藻7002的生长潜力分析

以野生聚球藻7002为对照,从室温吸收光谱、光合放氧速率、生长动力学参数以及气升式光生物反应器中的生长特性阐述了转小鼠金属硫蛋白-Ⅰ基因聚球藻7002的生长优势和培养潜力.结果表明:转MT聚球藻室温可见光光谱吸收峰比野生藻略高;最大净光合速率和饱和光强比野生藻高,呼吸速率和补偿光强比野生藻低;转MT聚球藻摇瓶培养的最大细胞浓度为野生藻的1.74倍,具有较高的细胞生长速率;气升式光生物反应器有利于转MT基因聚球藻生长潜力的发挥.     

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.     

Relationship between DNA cycle and growth rate in Synechococcus sp. strain PCC 6301.

Flow cytometry was used to examine cell cycle regulation in Synechococcus sp. strain PCC 6301 under a variety of growth conditions. The DNA frequency distributions of exponentially growing and dark-blocked populations confirmed that this cyanobacterium contains multiple chromosome copies even at very slow growth rates. Furthermore, the presence of major peaks corresponding to other than 2" chromosome copies strongly suggests that DNA replication is initiated asynchronously. Although this suggestion is at odds with the standard formulation of the procaryotic cell cycle model, it is similar to recent observations of asynchrony in Escherichia coli replication mutants.     

Molecular structure and enzymatic function of lycopene cyclase from the cyanobacterium Synechococcus sp strain PCC7942.

A gene encoding the enzyme lycopene cyclase in the cyanobacterium Synechococcus sp strain PCC7942 was mapped by genetic complementation, cloned, and sequenced. This gene, which we have named crtL, was expressed in strains of Escherichia coli that were genetically engineered to accumulate the carotenoid precursors lycopene, neurosporene, and zeta-carotene. The crtL gene product converts the acyclic hydrocarbon lycopene into the bicyclic beta-carotene, an essential component of the photosynthetic apparatus in oxygen-evolving organisms and a source of vitamin A in human and animal nutrition. The enzyme also converts neurosporene to the monocyclic beta-zeacarotene but does not cyclize zeta-carotene, indicating that desaturation of the 7-8 or 7'-8' carbon-carbon bond is required for cyclization. The bleaching herbicide 2-(4-methylphenoxy)triethylamine hydrochloride (MPTA) effectively inhibits both cyclization reactions. A mutation that confers resistance to MPTA in Synechococcus sp PCC7942 was identified as a point mutation in the promoter region of crtL. The deduced amino acid sequence of lycopene cyclase specifies a polypeptide of 411 amino acids with a molecular weight of 46,125 and a pI of 6.0. An amino acid sequence motif indicative of FAD utilization is located at the N terminus of the polypeptide. DNA gel blot hybridization analysis indicated a single copy of crtL in Synechococcus sp PCC7942. Other than the FAD binding motif, the predicted amino acid sequence of the cyanobacterial lycopene cyclase bears little resemblance to the two known lycopene cyclase enzymes from nonphotosynthetic bacteria. Preliminary results from DNA gel blot hybridization experiments suggest that, like two earlier genes in the pathway, the Synechococcus gene encoding lycopene cyclase is homologous to plant and algal genes encoding this enzyme.