Molecular Cloning and Construction of agp Gene Deletion-mutant in Cyanobacterium Synechocystis sp. PCC 6803

The agp gene encoding the ADP-glucose pyrophosphorylase involved in cyanobacterial glycogen synthesis was amplified by PCR. The resulting agp fragment was cloned in plasmid pUC118 to generate plasmid pUCA. Part of the fragment within the agp DNA was deleted and replaced by an erythromycin resistance cassette to generate plasmid pUCAE, which was used to transform the Synechocystis sp. PCC 6803 wild-type strain and a mutant with resistance to erythromycin was obtained. PCR analysis of the genomic DNA from the resulting mutant indicated that the appropriate deletion and insertion indeed had occurred. The cell growth and Chl a, glycogen content in the mutant showed difference from those in the wild-type strain. The obtained biomass as well as the Chl a content in the mutant strain was higher than that of the wild-type strain, which suggested that the photosynthesis efficiency in the agp(-) strain was higher than that in the wild-type strain. No glycogen was found in the mutant, providing evidence for the correction of the mutant in physiological level.

集胞藻Synechocystis sp. PCC 6803脂质组的分析

以集胞藻Synechocystis sp.PCC 6803为研究对象,研究建立了基于超高效液相色谱耦合串联质谱技术脂质组学分析方法。鸟枪法脂质组学通过电喷雾离子化有效分离油脂粗提物中所含单个脂质分子,在三重四极杆扫描碎片离子,能够利用特征片段离子鉴定光合甘油酯的种类和酰基组成,具有高效、灵敏度高和质量准确度高等优点。对不同光强下生长的Synechocystis sp.PCC 6803细胞的各脂质组分进行了全定量分析,发现单半乳糖甘油二酯(Monogalactosyldiacylglycerol,MGDG)和磷脂酰甘油(Phosphatidyl glycerol,PG)在高光处理的第2小时即显著积累,增长量分别为34.64%和68.49%,其中以含有从头合成、高度饱和的脂肪酸的种类增长最为快速和显著,而后高不饱和度的脂肪酸组成的种类逐渐积累。双半乳糖甘油二脂(Digalactosyldiacylglycerol,DGDG)在各时间点都持续增长,12h后增长量达26.95%,硫代异鼠李糖甘油二酯(Sulfoquinovosyl diacylglycerol,SQDG)的含量则呈现出不断下降的趋势。研究所建立的脂质组学分析方法对进一步研究Synechocystis sp.PCC 6803的脂质代谢及生理功能提供了有力的分析工具。

Effects of Sodium Thiosulfate on the Occurrence of a Novel Glycolipid in Cyanobacterium Synechocystis sp. PCC 6803 Cells Grown in the Presence of Glucose

A novel lipid occurred when cyanobacterium Synechocystis sp. PCC 6803 cells were grown in BG-11 medium with glucose applied. This lipid was determined to be a glycolipid, designated glycolipid-x (Glyco-x), by staining with alpha-naphthol and concentrated sulfuric acid. The occurrence of Glyco-x accompanies the disappearance of other lipids, especially DGDG. Glyco-x can also be observed in cells grown in BG-11 medium with the application of other carbon sources: fructose, maltose and lactose. Sodium thiosulfate, an effective scavenger of reactive oxygen intermediates, showed strong capability to inhibit glucose-induced occurrence of Glyco-x. In the presence of 0.3% sodium thiosulfate, Glyco-x could only be detected in cells grown in BG-11 medium with 100 mmol/L glucose applied in late-exponential phase. These results suggest that reactive oxygen species might be involved in the occurrence of Glyco-x in cyanobacterium Synechocystis sp. PCC 6803 cells grown in the presence of glucose.

Effects of Na2S2O3 and Glucose on the Compositions of Glycerolipids and Their Fatty Acids in Synechocystis sp. PCC 6803 Cells

Compositions of glycerolipids and fatty acid compositions of glycerolipids were compared among Synechocystis sp. PCC 6803 cells grown in the BG-11 medium containing different concentrations of glucose and Na2S2O3 in this study. It was found that Na2S2O3 can effectively increase the percentage of sulphoquinovosyl diacylglycerol (SQDG) and phosphatidylglycerol (PG) to total membrane lipids and the simultaneous application of glucose with Na2S2O3 can counteract the effect of Na2S2O3. In addition, Na2S2O3 can significantly increase the percentage of palmitic acid (C, 16:0) in fatty acid composition of monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) and decrease the fatty acid unsaturation degree accordingly, and these effects can also be eliminated by glucose. These results indicate that Na2S2O3 can take as a reductant to make membrane lipids in a low unsaturated state, and the simultaneous application of glucose can decrease the reducing power of Na2S2O3. In addition, Na2S2O3 can take as a sulfur donor for the synthesis of SQDG.

Characterization of calcium deposition induced by Synechocystis sp. PCC6803 in BG11 culture medium

Calcium carbonate （CaCO3） crystals in their preferred orientation were obtained in BG11 culture media inoculated with Synechocystis sp. PCC6803 （inoculated BG11）. In this study, the features of calcium carbonate deposition were investigated. Inoculated BGll in different calcium ion concentrations was used for the experimental group, while the BGll culture medium was used for the control group. The surface morphologies of the calcium carbonate deposits in the experimental and control groups were determined by scanning and transmission electron microscopy. The deposits were analyzed by electronic probe micro-analysis, Fourier transform infrared spectrum, X-ray diffraction, thermal gravimetric analysis and differential scanning calorimetry. The results show that the surfaces of the crystals in the experimental group were hexahedral in a scaly pattern. The particle sizes were micrometer-sized and larger than those in the control group. The deposits of the control group contained calcium （Ca）, carbon （C）, oxygen （O）, phosphorus （P）, iron （Fe）, copper （Cu）, zinc （Zn）, and other elements. The deposits in the experimental group contained Ca, C, and O only. The deposits of both groups contained calcite. The thermal decomposition temperature of the deposits in the control group was lower than those in the experimental group. It showed that the CaCO3 deposits of the experimental group had higher thermal stability than those of the control group. This may be due to the secondary metabolites produced by the algae cells, which affect the carbonate crystal structure and result in a close-packed structure. The algae cells that remained after thermal weight loss were heavier in higher calcium concentrations in BGll culture media. There may be more calcium- containing crystals inside and outside of these cells. These results shall be beneficial for understanding the formation mechanism of carbonate minerals.

Effects of heavy metals (Pb^(2+) and Cd^(2+)) on the ultrastructure, growth and pigment contents of the unicellular cyanobacterium Synechocystis sp. PCC 6803

The unicellular cyanobacterium Synechocystis sp. PCC 6803, a model organism known for its unique combination of highly desirable molecular genetic, physiological and morphological characteristics, was employed in the present study. The species was cultured in BG11 liquid medium contained various initial concentrations of Pb^2＋ and Cd〉 （0, 0.5, 1, 2, 4, 6 and 8 mg/L）. The experiment was conducted for six days and the metal induced alterations in the ultrastructure, growth and pigment contents were assessed. Alterations in the ultrastructure of the Synechocystis sp. PCC 6803 cells became evident with the increased （〉4 mg/L Pb^2＋） metal concentration. The photosynthetic apparatus （thylakoid membranes） were found to be the worst affected. Deteriorated or completely destroyed thylakoid membranes have made large empty spaces in the cell interior. In addition, at the highest concentration （8 mg/L pb^2＋）, the polyphosphate granules became more prominent both in size and number. Despite the initial slight stimulations （0.2, 3.8 and 6.5% respectively at 0.5, 1 and 2 mg/L pb^2＋）, both metals inhibited the growth in a dose-dependent manner as incubation progressed. Pigment contents （chlorophyll a, 13 carotene and phycocyanin） were also decreased with increasing metal concentration. Cells exposed to 6 mg/L Pb^2＋, resulted in 36.56, 37.39 and 29.34% reductions of chlorophyll a, 13 carotene and phycocyanin respectively over the control. Corresponding reductions for the same CdZ＋concentrations were 57.83, 48.94 and 56.90%. Lethal concentration （96 h LC50） values （3.47 mg/L Cd^2＋ and 12.11 mg/L Pb^2＋） indicated that Synechocystis sp. PCC 6803 is more vulnerable to Cd^2＋ than Pb^2＋.

Studies on Hemolysis of Hemolysin Produced by Synechocystis sp. PCC 6803

Hemolysin produced by various bacteria,may destroy erythrocyte membranes via a pore-forming mechanism,a deter-gent action,or a lipase activity.Previous to this experiment,the mode of action used by cyanobacterial hemolysin had not been re-ported.To characterize the action mode of hemolysin produced by the wild-type strain of Synechocystis sp.PCC6803,hemolysis of erythrocytes originating from human,mouse,sheep,rabbit and goldfish was studied.The erythrocytes of mouse,sheep and rabbit were sensitive,while those of human and fish were resistant,to this hemolysin.Using rabbit erythrocytes,it was shown that hemoly-sis occurred in two steps:a binding step within the first 10 min of treatment and a lytic step after 30 min.Both binding and lysis were highly temperature-dependent.Effects of erythrocyte density on hemolysis suggest that the hemolysin might target erythrocytes via a multiple-hit mechanism.In the osmotic protection experiment,all tested osmotic protectants,with molecular diameters ranging from 0.9 ?5.66 nm,failed to effectively inhibit hemolysis.Scanning electron micrographs showed that the hemolysin caused protuberances or echinocytes in rabbit erythrocytes,and then disrupted and ruptured the erythrocytes.Characteristics of hemolysis showed distinct differences from other pore-forming mechanisms,suggesting that this hemolysin might act through a detergent-like or lipase mecha-nism,rather than a pore-forming mechanism.

Transcriptomic analysis of Synechocystis sp. PCC6803 under low-temperature stress

In this study, cDNA microarrays were developed from 3569 mRNA reads to analyze the expression profiles of the transcriptomes of Synechocystis sp. PCC6803 under low temperature （LT） stress. Among the genes on the cDNA microarrays, 899 LT-affected genes exhibited a 1.5-fold （or greater） difference in expression compared with the genes from normal unstressed Synechocystis sp. PCC6803. Of the differentially expressed genes, 353 were up-regulated and 246 were down-regulated. The results showed that genes involved in photosynthesis were activated at LT （10℃）, including genes for photosystem I, photosystem II, photosynthetic electron transport, and cytochrome b6/f complex. Moreover, desg, one of four genes that encode the fatty acid desaturases, was also induced by LT. However, the LT conditions to some degree enhanced the transcription of some genes. In addition, LT （10℃） may reduce cellular motility by regulating the transcription of spkA （sll1575）, a serine/threonine protein kinase. The results reported in this study may contribute to a better understanding of the responses of the Synechocystis cell to LT, including pathways involved in photosynthesis and repair.

Biotransformation of 6-deoxypseudoanisatin by Synechocystis sp. PCC6803

Objective:To explore the ability of Synechocystis sp.PCC6803 in transforming 6-deoxypseudoanisatin.Methods:The experiment was performed by incubating 6-deoxypseudoanisatin with the freshwater cyanobacterium Synechocystis sp.PCC6803 under continuous white light at 30C for 5 days.The crude converted product was detected using thin-layer chromatography(TLC)and further analyzed using high-performance liquid chromatography(HPLC)as well as HPLC with electron spray ionization mass spectrometry(HPLC-ESI-MS).Results:TLC results showed that 6-deoxypseudoanisatin was converted into a less polar product.HPLC and MS data indicated that the retention time of the converted product increased in comparison with the standard of 6-deoxypseudoanisatin.Conclusion:Thus,the study appears to demonstrate that Synechocystis sp.PCC6803 can transform 6-deoxypseudoanisatin.The polarity of the converted product is less than that of 6-deoxypseudoanisatin.

Production of γ-linolenic acid and stearidonic acid by Synechococcus sp.PCC7002 containing cyanobacterial fatty acid desaturase genes

Genetic modifi cation is useful for improving the nutritional qualities of cyanobacteria. To increase the total unsaturated fatty acid content, along with the ratio of ω-3/ω-6 fatty acids, genetic engineering can be used to modify fatty acid metabolism. S ynechococcus sp. PCC7002, a fast-growing cyanobacterium, does not contain a Δ6 desaturase gene and is therefore unable to synthesize γ-linolenic acid(GLA) and stearidonic acid(SDA), which are important in human health. In this work, we constructed recombinant vectors Syd6 D, Syd15 D and Syd6Dd15 D to express the Δ15 desaturase and Δ6 desaturase genes from Synechocystis PCC6803 in Synechococcus sp. PCC7002, with the aim of expressing polyunsaturated fatty acids. Overexpression of the Δ15 desaturase gene in S ynechococcus resulted in 5.4 times greater accumulation of α-linolenic acid compared with the wild-type while Δ6 desaturase gene expression produced both GLA and SDA. Co-expression of the two genes resulted in low-level accumulation of GLA but much larger amounts of SDA, accounting for as much to 11.64% of the total fatty acid content.

Dynam,c Changes of IsiA-Containing Complexes during Long-Term Iron Deficiency in Synechocystis sp, PCC 6803

Iron stress-induced protein A （IsiA）, a major chlorophyll-binding protein in the thylakoid membrane, is significantly induced under iron deficiency conditions. Using immunoblot analysis and 77 K fluorescence spectroscopy combined with sucrose gradient fractionation, we monitored dynamic changes of IsiA- containing complexes in Synechocystis sp. PCC 6803 during exposure to long-term iron deficiency. Within 3 days of exposure to iron deficiency conditions, the initially induced free IsiA proteins preferentially con- jugated to PSI trimer to form IsiA18-PS I trimers, which serve as light energy collectors for efficiently trans- mitting energy to PS h With prolonged iron deficiency, IsiA proteins assembled either into IsiA aggregates or into two other types of IsiA-PS I supercomplexes, namely IsiA-PS I high fluorescence supercomplex （IHFS） and IsiA-PS I low fluorescence supercomplex （ILFS）. Further analysis revealed a role for IsiA as an energy dissipater in the IHFS and as an energy collector in the ILFS. The trimeric structure of PS I mediated by PsaL was found to be indispensable for the formation of IHFS/ILFS. Dynamic changes in IsiA-containing complexes in cyanobacteria during long-term iron deficiency may represent an adaptation to iron limitation stress for flexible light energy distribution, which balances electron transfer between PS I and PS II, thus minimizing photooxidative damage.

Contributions of DPOR at Low Light Intensity to Chlorophyll Biosynthesis and Growth in the Synechocystis sp. PCC 6803

The chlL gene encoding one component of light-independent (dark) protochlorophyllide oxido reductase (DPOR) was deleted in cyanobacterium Synechocystis sp. PCC 6803 (S.6803). The resulting chlL- mutant lost DPOR activity. No significant differences of chlorophyll (Chl) content and growth rate were observed between the wild and the mutant strains grown at 50 mE·m2·s1 light intensity for photomixtrophic and photoautotrophic growth. However, differences were observed at 1 mE·m2·s1 light intensity. For photomixtrophic growth, the mutant Chl content was 50% of the wild content with continuous light and 35.7% of the wild content with a 10 h light/ 14 h dark cycle. For photoautotriphic growth, the mutant Chl level was 76.3% of the wild content with continuous light and 63.2% with a 10 h light/ 14 h dark cycle. The results indicate that DPOR contributes to Chl synthesis and increases the growth rate in cyanobacteria phototrophically cultured at 1mE·m2·s1 light intensity. In contrast, the photosynthetic capacity on a per-cell basis of the mutant is 5% higher than that of the wild strain with continuous light and 27% higher than that of the wild strain with a 10 h light/14 h dark cycle at 1 mE·m2·s1 light intensity for photoautotrophic growth. With the low Chl content, the cyanobacteria have the ability to improve their photosynthetic capacity by decreasing the ratio of PSI to PSII by unknown morphological or physiological means.

Sodium Acetate Stimulates PHB Biosynthesis in Synechocystis sp. PCC 6803

Synechocystis sp. PCC 6803 cell growth and poly β hydroxybutyrate (PHB) biosynthesis were studied in the presence of sodium acetate (NaAc). For nitrogen sufficient conditions, 15 mmol/L NaAc improved the PHB content up to 9.9% (w/w) while for nitrogen starved conditions, the PHB content was up to 15.2% (w/w). NaAc at levels below 20 mmol/L promoted cell growth in the first six days, but the growth slowed on the seventh day when the NaAc concentration exceeded 15 mmol/L. The PHB content in the final biomass reached 11.0% of the dry cellular weight in the presence of 20 mmol/L NaAc. Two adjacent open reading frames (ORFs) in the genome of Synechocystis sp. PCC 6803, slr1993 and slr1994, were assigned to phbA and phbB, respectively, while the phbC gene was found to be far from these genes. This may account for the low expression of PHB in cyanobacteria.

Two-Component Signal Transduction Systems in the Cyanobacterium Synechocystis sp. PCC 6803

Two-component systems are signal transduction systems which enable bacteria to regulate cellular functions in response to changing environmental conditions. The unicellular Synechocystis sp. PCC 6803 has become a model organism for a range of biochemical and molecular biology studies aiming at investigating environmental stress response. The publication of the complete genome sequence of the cyanobacterium Synechocystis sp. PCC 6803 provided a tremendous stimulus for research in this field, and at least 80 open reading frames were identified as members of the two-component signal transduction systems in this single species of cyanobacteria. To date, functional roles have been determined for only a limited number of such proteins. This review summarizes our current knowledge about the two-component signal transduction systems in Synechocystis sp. PCC 6803 and describes recent achievements in elucidating the functional roles of these systems.

Construction and application of the Synechocystis sp.PCC6803-ftnA in microbial contamination control in a coupled cultivation and wastewater treatment

Inspired by iron fertilization experiments in HNLC（high-nitrate, low-chlorophyll） sea areas,we proposed the use of iron-rich engineered microalgae for microbial contaminant control in iron-free culture media. Based on the genome sequence and natural transformation system of Synechocystis sp. PCC6803, ftn A（encoding ferritin） was selected as our target gene and was cloned into wild-type Synechocystis sp. PCC6803. Tests at the molecular level confirmed the successful construction of the engineered Synechocystis sp. PCC6803-ftn A. After Fe3＋-EDTA pulsing, the intracellular iron content of Synechocystis sp. PCC6803-ftn A was significantly enhanced, and the algae was used in the microbial contamination control system. In the coupled Synechocystis sp. PCC6803-ftn A production and municipal wastewater（MW, including Scenedesmus obliquus and Bacillus） treatment, Synechocystis sp. PCC6803-ftn A accounted for all of the microbial activity and significantly increased from 70% of the microbial community to 95%.These results revealed that while the stored iron in the Synechocystis sp. PCC6803-ftn A cells was used for growth and reproduction of this microalga in the MW, the growth of other microbes was inhibited because of the iron limitation, and these results provide a new method for microbial contamination control during a coupling process.

Expression and activity of heterologous hydroxyisocaproate dehydrogenases in Synechocystis sp.PCC 6803ΔhoxYH

Exploiting light to drive redox reactions is currently a hot topic since light is considered as an environmentally friendly source of energy.Consequently,cyanobacteria,which can use light e.g.,for generating NADPH,are in the focus of research.Previously,it has been shown that various heterologous redox enzymes could be expressed in these microorganisms.Here we demonstrated the successful inducer-free expression of𝛼-keto-acid dehydroge-nases(L-HicDH and D-HicDH)from Lactobacillus confusus DSM 20196 and Lactobacillus paracasei DSM 20008 in Synechocystis sp.PCC 6803ΔhoxYH mutant using replicative plasmids.While the L-HicDH showed poor activity limited by the amount of expressed enzyme,the D-HicDH was applied both in vivo and in vitro,transforming the selected𝛼-keto acids to the corresponding optically pure(R)-𝛼-hydroxy acids(ee>99%)in up to 53%and 90%conversion,respectively.

藻类生物钙化研究进展

藻类钙化是生物钙化的主要形式之一,也是钙化生产力的重要组成部分。微藻与大型藻中均包括有钙化现象的种类,且二者的钙化过程都包括四个主要步骤:CO_(2)的浓缩,Ca^(2+)的聚集,CO_(2)^(3-)浓度上升,Ca^(2+)与CO_(2)^(3-)结合生成CaCO_(3)。然而,微藻和大型藻由于形态和生理上的差异,钙化过程各具特点。本文以钙化模式种集胞藻(Synechocystis sp.PCC6803)为例介绍了藻类钙化的四个主要步骤,并对关键的钙化机理进行了解析。通过比较分析,分别以珊瑚藻和颗石藻为例,介绍了大型藻和微藻在钙化过程和钙化机理上的异同。基于上述机制和特点的深化研究发现:钙化藻类在碳汇渔业、蓝碳领域和纳米材料方向具有较大的发展潜力。