Glutaminase is widely distributed among microorganisms and mammals with important functions. Lit-tle is known regarding the biochemical properties and functions of the deamidating enzyme glutami-nase in cyanobacteria....Glutaminase is widely distributed among microorganisms and mammals with important functions. Lit-tle is known regarding the biochemical properties and functions of the deamidating enzyme glutami-nase in cyanobacteria. In this study a putative glutaminase encoded by gene slr2079 in Synechocystis sp. PCC 6803 was investigated. The slr2079 was expressed as histidine-tagged fusion protein in Es-cherichia coli. The purified protein possessed glutaminase activity, validating the functional assign-ment of the genomic annotation. The apparent Km value of the recombinant protein for glutamine was 26.6 ± 0.9 mmol/L, which was comparable to that for some of other microbial glutaminases. Analysis of the purified protein revealed a two-fold increase in catalytic activity in the presence of 1 mol/L Na+. Moreover, the Km value was decreased to 12.2 ± 1.9 mmol/L in the presence of Na+. These data demon-strate that the recombinant protein Slr2079 is a glutaminase which is regulated by Na+ through in-creasing its affinity for substrate glutamine. The slr2079 gene was successfully disrupted in Synecho-cystis by targeted mutagenesis and the △slr2079 mutant strain was analyzed. No differences in cell growth and oxygen evolution rate were observed between △slr2079 and the wild type under standard growth conditions, demonstrating slr2079 is not essential in Synechocystis. Under high salt stress condition, however, △slr2079 cells grew 1.25-fold faster than wild-type cells. Moreover, the photosyn-thetic oxygen evolution rate of △slr2079 cells was higher than that of the wild-type. To further charac-terize this phenotype, a number of salt stress-related genes were analyzed by semi-quantitative RT-PCR. Expression of gdhB and prc was enhanced and expression of desD and guaA was repressed in △slr2079 compared to the wild type. In addition, expression of two key enzymes of ammonium assimi-lation in cyanobacteria, glutamine synthetase (GS) and glutamate synthase (GOGAT) was examined by semi-quantitative RT-PCR. Expression of GOGAT 展开更多
The electron-transport machinery in photosynthetic membranes is known to be very sensitive to heat. In this study, the rate of electron transport (ETR) driven by photosystem I (PSI) and photosystem II (PSII) during he...The electron-transport machinery in photosynthetic membranes is known to be very sensitive to heat. In this study, the rate of electron transport (ETR) driven by photosystem I (PSI) and photosystem II (PSII) during heat stress in the wild-type Synechocystis sp. strain PCC 6803 (WT) and its ndh gene inactiva-tion mutants △ndhB (M55) and △ndhD1/ndhD2 (D1/D2) was simultaneously assessed by using the novel Dual-PAM-100 measuring system. The rate of electron transport driven by the photosystems (ETRPSs) in the WT, M55, and D1/D2 cells incubated at 30℃ and at 55℃ for 10 min was compared. Incubation at 55 ℃ for 10 min significantly inhibited PSII-driven ETR (ETRPSII) in the WT, M55 and D1/D2 cells, and the ex-tent of inhibition in both the M55 and D1/D2 cells was greater than that in the WT cells. Further, PSI-driven ETR (ETRPSI) was stimulated in both the WT and D1/D2 cells, and this rate was increased to a greater extent in the D1/D2 than in the WT cells. However, ETRPSI was considerably inhibited in the M55 cells. Analysis of the effect of heat stress on ETRPSs with regard to the alterations in the 2 active NDH-1 complexes in the WT, M55, and D1/D2 cells indicated that the active NDH-1 supercomplex and medi-umcomplex are essential for alleviating the heat-induced inhibition of ETRPSII and for accelerating the heat-induced stimulation of ETRPSI, respectively. Further, it is believed that these effects are most likely brought about by the electron transport mediated by each of these 2 active NDH-1 complexes.展开更多
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 d...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.展开更多
Heterologous expression of the isoprene synthase gene in the cyanobacterium Synechocystis PCC 6803 conferred upon these microorganisms the property of photosynthetic isoprene （C5H8） hYdrocarbons production. Continuo...Heterologous expression of the isoprene synthase gene in the cyanobacterium Synechocystis PCC 6803 conferred upon these microorganisms the property of photosynthetic isoprene （C5H8） hYdrocarbons production. Continuous production of isoprene from CO2 and H2O was achieved in the light, occurring via the endogenous methylerythritolphosphate （MEP） pathway, in tandem with the growth of Synechocystis. This work addressed the issue of photosynthetic carbon partitioning between isoprene and biomass in Synechocystis. Evidence is presented to show heterologous genomic integration and cellular expression of the mevalonic acid （MVA） pathway genes in Synechocystis endowing a non-native pathway for carbon flux amplification to isopentenyl-diphosphate （IPP） and dimethylallyl-diphosphate （DMAPP） precursors of isoprene. Heterologous expression of the isoprene synthase in combination with the MVA pathway enzymes resulted in photosynthetic isoprene yield improvement by approximately 2.5-fold, compared with that measured in cyanobacteria transformed with the isoprene synthase gene only. These results suggest that the MVA pathway introduces a bypass in the flux of endogenous cellular substrate in Synechocystis to IPP and DMAPP, overcoming flux limitations of the native MEP pathway. The work employed a novel chromosomal integration and expression of synthetic gene operons in Synechocystis, comprising up to four genes under the control of a single promoter, and expressing three operons simultaneously. This is the first time an entire biosynthetic pathway with seven recombinant enzymes has been heterologously expressed in a photosynthetic microorganism. It constitutes contribution to the genetic engineering toolkit of photosynthetic microorganisms and a paradigm in the pursuit of photosynthetic approaches for the renewable generation of high-impact products.展开更多
Biogenesis of photosynthetic pigment/protein complexes is a highly regulated process that requires various assisting factors. Here, we report on the molecular analysis of the Pitt gene （sir1644） from the cyanobacter...Biogenesis of photosynthetic pigment/protein complexes is a highly regulated process that requires various assisting factors. Here, we report on the molecular analysis of the Pitt gene （sir1644） from the cyanobacterium Synechocystis sp. PCC 6803 （Synechocystis 6803） that encodes a membrane-bound tetratricopeptide repeat （TPR） protein of formerly unknown function. Targeted inactivation of Pitt affected photosynthetic performance and light-dependent chlorophyll synthesis. Yeast two-hybrid analyses and native PAGE strongly suggest a complex formation between Pitt and the light-dependent protochlorophyllide oxidoreductase （POR）. Consistently, POR levels are approximately threefold reduced in the pitt insertion mutant. The membrane sublocalization of Pitt was found to be dependent on the presence of the periplasmic photosystem Ⅱ （PSⅡ） biogenesis factor PratA, supporting the idea that Pitt is involved in the early steps of photosynthetic pigment/protein complex formation.展开更多
Formation of the multi-subunit oxygen-evolving photosystem II （PSII） complex involves a number of auxiliary protein factors. In this study we compared the localization and possible function of two homolo- gous PSU a...Formation of the multi-subunit oxygen-evolving photosystem II （PSII） complex involves a number of auxiliary protein factors. In this study we compared the localization and possible function of two homolo- gous PSU assembly factors, Psb28-1 and Psb28-2, from the cyanobacterium Synechocystis sp. PCC 6803. We demonstrate that FLAG-tagged Psb28-2 is present in both the monomeric PSII core complex and a PSII core complex lacking the inner antenna CP43 （RC47）, whereas Psb28-1 preferentially binds to RC47. When cells are exposed to increased irradiance, both tagged Psb28 proteins additionally associate with oligo- meric forms of PSII and with PSII-PSI supercomplexes composed of trimeric photosystem I （PSI） and two PSII monomers as deduced from electron microscopy. The presence of the Psb27 accessory protein in these complexes suggests the involvement of PSI in PSII biogenesis, possibly by photoprotecting PSII through energy spillover. Under standard culture conditions, the distribution of PSII complexes is similar in the wild type and in each of the single psb28 null mutants except for loss of RC47 in the absence of Psb28-1. In comparison with the wild type, growth of mutants lacking Psb28-1 and Psb27, but not Psb28-2, was retarded under high-light conditions and, especially, intermittent high-light/dark conditions, emphasizing the physiological importance of PSII assembly factors for light acclimation.展开更多
CYCLIC electron transport around photosystem I (PS I ) is considered physiologically important not only for its coupled formation of ATP, but also for its function on protection of the photosynthetic apparatus against...CYCLIC electron transport around photosystem I (PS I ) is considered physiologically important not only for its coupled formation of ATP, but also for its function on protection of the photosynthetic apparatus against photoinhibition. However, due to the difficulty of its measurement, we know little about its operation in vivo.展开更多
In order to explore the mechanism of acute toxicity for pyrene to cyanobacterial organisms, the responses of Synechocystis sp. PCC 6803 photosystem Ⅱ （PS Ⅱ） under pyrene stress were studied. The results showed the...In order to explore the mechanism of acute toxicity for pyrene to cyanobacterial organisms, the responses of Synechocystis sp. PCC 6803 photosystem Ⅱ （PS Ⅱ） under pyrene stress were studied. The results showed there was no significant difference about the oxygen evolution under 0.125 mg/L pyrene stress when compared with control, but it was significantly lower than control at 0.625 mg/L pyrene. Polyphasic chlorophyll-a fluorescence transients in cells of Synechocystis sp. PCC 6803 exhibited a typical increase including O, J, I, and P phases. Fluorescence yield at phases J, I and P declined slightly at 0.125 and 0.625 mg/L pyrene, and significantly lower than control at 3.125 mg/L. According to the parameters deviated from JIP-test, no modification was induced by pyrene both at the donor side and at the acceptor side of PS Ⅱ, and the reaction centre of PS Ⅱ is the primary damaging target. Based on the expressing of four key genes （psbA, psbB, psbC and psbO） of PS Ⅱ, only psbA showed significant difference at 3.125 mg/L pyrene when compared with control.展开更多
The expression of the genes encoding the ferredoxin-thioredoxin system including the ferredoxin-thioredoxin reductase （FTR） genes ftrC and ftrV and the four different thioredoxin genes trxA （m-type; sir0623）, trxB...The expression of the genes encoding the ferredoxin-thioredoxin system including the ferredoxin-thioredoxin reductase （FTR） genes ftrC and ftrV and the four different thioredoxin genes trxA （m-type; sir0623）, trxB （x-type; sir1139）, trxC （sll1057） and trxQ （y-type; sir0233） of the cyanobacterium Synechocystis sp. PCC 6803 has been studied according to changes in the photosynthetic conditions. Experiments of light-dark transition indicate that the expression of all these genes except trxQ decreases in the dark in the absence of glucose in the growth medium. The use of two electron transport inhibitors, 3-（3,4-dichlorophenyl）-1,1-dimethylurea （DCMU） and 2,5-dibromo-3-methyl-6-isopropyl-p- benzoquinone （DBMIB）, reveals a differential effect on thioredoxin genes expression being trxC and trxQ almost unaffected, whereas trxA, trxB, and the ftr genes are down-regulated. In the presence of glucose, DCMU does not affect gene expression but DBMIB still does. Analysis of the single TrxB or TrxQ and the double TrxB TrxQ Synechocystis mutant strains reveal different functions for each of these thioredoxins under different growth conditions. Finally, a Synechocystis strain was generated containing a mutated version of TrxB （TrxBC34S）, which was used to identify the potential in-vivo targets of this thioredoxin by a proteomic analysis.展开更多
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 spectro...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.展开更多
光是光合作用不可或缺的底物。然而过量的光照会对光合生物造成氧化胁迫和严重的损害。为了应对持续变化的光环境,蓝藻演化形成了灵活的电子传递网络。围绕光系统I(photosystem I,PSI)的循环电子传递(cyclic electron transport,CET)将...光是光合作用不可或缺的底物。然而过量的光照会对光合生物造成氧化胁迫和严重的损害。为了应对持续变化的光环境,蓝藻演化形成了灵活的电子传递网络。围绕光系统I(photosystem I,PSI)的循环电子传递(cyclic electron transport,CET)将电子从铁氧还蛋白Fd回流到质体醌(plastoquinone,PQ)库,产生ATP且不积累NADPH。在蓝藻和高等植物中发现了2种不同的CET途径,即NDH依赖途径和PGR5依赖途径。蓝藻中黄素二铁蛋白Flv1/Flv3参与了类梅勒(Mehler-like)反应,从PSI接受电子直接将氧气还原为水,且没有活性氧的形成。以集胞藻为试验材料,通过分析不同的CET和Flv突变株在不同光照条件下的生理特征以及其P700氧化/还原动力学,进而研究CET途径和类梅勒反应在集胞藻中的功能。结果表明NDH-1复合体对CET的贡献率超过90%,维持细胞能在持续高光环境下生长,而迅速应激的类梅勒反应在缓解瞬时高光胁迫时发挥了重要作用。因此我们认为在集胞藻中NDH-1介导的循环电子途径是稳固支持其适应高光逆境的主要机制,而类梅勒反应则是在现有主要途径严重不足时的1个备用途径。响应迅速的FLV路径是野生型和NDH-1突变株的补足。展开更多
Synthetic biology efforts have also led to the development of photosynthetic cyanobacteria as"autotrophic cell factories"for biosynthesis of various biofuels directly from CO_(2).However,the low tolerance to...Synthetic biology efforts have also led to the development of photosynthetic cyanobacteria as"autotrophic cell factories"for biosynthesis of various biofuels directly from CO_(2).However,the low tolerance to toxicity of biofuels has restricted the economic application of cyanobacterial hosts.In this study,RNAseq transcriptomics was employed to reveal stress responses to exogenous n-hexane in Synechocystis sp.PCC 6803.Functional enrichment analysis of the transcriptomic data showed that signal transduction systems were induced significantly.To further identify regulatory genes related to n-hexane tolerance,a library of transcriptional regulators(TRs)deletion mutants was then screened for their roles in nhexane tolerance.The results showed that a knockout mutant of slr0724 that encodes an Hta R suppressor protein was more tolerant to n-hexane than the wild type,indicating the involvement of slr0724 in nhexane tolerance.This study provides the foundation for better understanding the cellular responses to n-hexane in Synechocystis sp.PCC 6803,which could contribute to the further engineering of nhexane tolerance in cyanobacteria.展开更多
基金Supported by the National Natural Sciences Foundation of China (Grant No. 30500108)Hundred Talents Program of Chinese Academy of Sciences
文摘Glutaminase is widely distributed among microorganisms and mammals with important functions. Lit-tle is known regarding the biochemical properties and functions of the deamidating enzyme glutami-nase in cyanobacteria. In this study a putative glutaminase encoded by gene slr2079 in Synechocystis sp. PCC 6803 was investigated. The slr2079 was expressed as histidine-tagged fusion protein in Es-cherichia coli. The purified protein possessed glutaminase activity, validating the functional assign-ment of the genomic annotation. The apparent Km value of the recombinant protein for glutamine was 26.6 ± 0.9 mmol/L, which was comparable to that for some of other microbial glutaminases. Analysis of the purified protein revealed a two-fold increase in catalytic activity in the presence of 1 mol/L Na+. Moreover, the Km value was decreased to 12.2 ± 1.9 mmol/L in the presence of Na+. These data demon-strate that the recombinant protein Slr2079 is a glutaminase which is regulated by Na+ through in-creasing its affinity for substrate glutamine. The slr2079 gene was successfully disrupted in Synecho-cystis by targeted mutagenesis and the △slr2079 mutant strain was analyzed. No differences in cell growth and oxygen evolution rate were observed between △slr2079 and the wild type under standard growth conditions, demonstrating slr2079 is not essential in Synechocystis. Under high salt stress condition, however, △slr2079 cells grew 1.25-fold faster than wild-type cells. Moreover, the photosyn-thetic oxygen evolution rate of △slr2079 cells was higher than that of the wild-type. To further charac-terize this phenotype, a number of salt stress-related genes were analyzed by semi-quantitative RT-PCR. Expression of gdhB and prc was enhanced and expression of desD and guaA was repressed in △slr2079 compared to the wild type. In addition, expression of two key enzymes of ammonium assimi-lation in cyanobacteria, glutamine synthetase (GS) and glutamate synthase (GOGAT) was examined by semi-quantitative RT-PCR. Expression of GOGAT
基金Supported by the National Natural Science Foundation of China (Grant No. 30770175)the Natural Science Foundation of Shanghai Muncipality (Grant No. 07ZR14086)+2 种基金the Innovation Program of Shanghai Municipal Education Commission (Grant No. 08ZZ67)the Key Foundation Project of Shanghai (Grant No. 06JC14091)the Leading Academic Discipline Project of Shanghai Municipal Education Commission (Grant No. J50401)
文摘The electron-transport machinery in photosynthetic membranes is known to be very sensitive to heat. In this study, the rate of electron transport (ETR) driven by photosystem I (PSI) and photosystem II (PSII) during heat stress in the wild-type Synechocystis sp. strain PCC 6803 (WT) and its ndh gene inactiva-tion mutants △ndhB (M55) and △ndhD1/ndhD2 (D1/D2) was simultaneously assessed by using the novel Dual-PAM-100 measuring system. The rate of electron transport driven by the photosystems (ETRPSs) in the WT, M55, and D1/D2 cells incubated at 30℃ and at 55℃ for 10 min was compared. Incubation at 55 ℃ for 10 min significantly inhibited PSII-driven ETR (ETRPSII) in the WT, M55 and D1/D2 cells, and the ex-tent of inhibition in both the M55 and D1/D2 cells was greater than that in the WT cells. Further, PSI-driven ETR (ETRPSI) was stimulated in both the WT and D1/D2 cells, and this rate was increased to a greater extent in the D1/D2 than in the WT cells. However, ETRPSI was considerably inhibited in the M55 cells. Analysis of the effect of heat stress on ETRPSs with regard to the alterations in the 2 active NDH-1 complexes in the WT, M55, and D1/D2 cells indicated that the active NDH-1 supercomplex and medi-umcomplex are essential for alleviating the heat-induced inhibition of ETRPSII and for accelerating the heat-induced stimulation of ETRPSI, respectively. Further, it is believed that these effects are most likely brought about by the electron transport mediated by each of these 2 active NDH-1 complexes.
基金Supported by the National Natural Science Foundation of China(Nos.40972043,41040018,41210104058,21176145,41372108,41302079)the Higher Educational Science and Technology Program of Shandong Province(No.J10LC15)+4 种基金the China Postdoctoral Science Foundation(No.2013M540560)the Program for Scientific Research Innovation Team in Colleges and Universities of Shandong Province,and SDUST Research Fund(No.2010KYTD103)the Open Project of Key Lab of Marine Bioactive Substance and Modern Analytical Technique,State Oceanic Administration,China(No.MBSMAT-2012-03)the Scientific and Technological Program of Qingdao(No.13-1-4-232-jch)the Domestic Visiting Scholar Program for Young Core Teachers in Shandong Universities,Shandong Province,China
文摘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.
文摘Heterologous expression of the isoprene synthase gene in the cyanobacterium Synechocystis PCC 6803 conferred upon these microorganisms the property of photosynthetic isoprene （C5H8） hYdrocarbons production. Continuous production of isoprene from CO2 and H2O was achieved in the light, occurring via the endogenous methylerythritolphosphate （MEP） pathway, in tandem with the growth of Synechocystis. This work addressed the issue of photosynthetic carbon partitioning between isoprene and biomass in Synechocystis. Evidence is presented to show heterologous genomic integration and cellular expression of the mevalonic acid （MVA） pathway genes in Synechocystis endowing a non-native pathway for carbon flux amplification to isopentenyl-diphosphate （IPP） and dimethylallyl-diphosphate （DMAPP） precursors of isoprene. Heterologous expression of the isoprene synthase in combination with the MVA pathway enzymes resulted in photosynthetic isoprene yield improvement by approximately 2.5-fold, compared with that measured in cyanobacteria transformed with the isoprene synthase gene only. These results suggest that the MVA pathway introduces a bypass in the flux of endogenous cellular substrate in Synechocystis to IPP and DMAPP, overcoming flux limitations of the native MEP pathway. The work employed a novel chromosomal integration and expression of synthetic gene operons in Synechocystis, comprising up to four genes under the control of a single promoter, and expressing three operons simultaneously. This is the first time an entire biosynthetic pathway with seven recombinant enzymes has been heterologously expressed in a photosynthetic microorganism. It constitutes contribution to the genetic engineering toolkit of photosynthetic microorganisms and a paradigm in the pursuit of photosynthetic approaches for the renewable generation of high-impact products.
文摘Biogenesis of photosynthetic pigment/protein complexes is a highly regulated process that requires various assisting factors. Here, we report on the molecular analysis of the Pitt gene （sir1644） from the cyanobacterium Synechocystis sp. PCC 6803 （Synechocystis 6803） that encodes a membrane-bound tetratricopeptide repeat （TPR） protein of formerly unknown function. Targeted inactivation of Pitt affected photosynthetic performance and light-dependent chlorophyll synthesis. Yeast two-hybrid analyses and native PAGE strongly suggest a complex formation between Pitt and the light-dependent protochlorophyllide oxidoreductase （POR）. Consistently, POR levels are approximately threefold reduced in the pitt insertion mutant. The membrane sublocalization of Pitt was found to be dependent on the presence of the periplasmic photosystem Ⅱ （PSⅡ） biogenesis factor PratA, supporting the idea that Pitt is involved in the early steps of photosynthetic pigment/protein complex formation.
文摘Formation of the multi-subunit oxygen-evolving photosystem II （PSII） complex involves a number of auxiliary protein factors. In this study we compared the localization and possible function of two homolo- gous PSU assembly factors, Psb28-1 and Psb28-2, from the cyanobacterium Synechocystis sp. PCC 6803. We demonstrate that FLAG-tagged Psb28-2 is present in both the monomeric PSII core complex and a PSII core complex lacking the inner antenna CP43 （RC47）, whereas Psb28-1 preferentially binds to RC47. When cells are exposed to increased irradiance, both tagged Psb28 proteins additionally associate with oligo- meric forms of PSII and with PSII-PSI supercomplexes composed of trimeric photosystem I （PSI） and two PSII monomers as deduced from electron microscopy. The presence of the Psb27 accessory protein in these complexes suggests the involvement of PSI in PSII biogenesis, possibly by photoprotecting PSII through energy spillover. Under standard culture conditions, the distribution of PSII complexes is similar in the wild type and in each of the single psb28 null mutants except for loss of RC47 in the absence of Psb28-1. In comparison with the wild type, growth of mutants lacking Psb28-1 and Psb27, but not Psb28-2, was retarded under high-light conditions and, especially, intermittent high-light/dark conditions, emphasizing the physiological importance of PSII assembly factors for light acclimation.
文摘CYCLIC electron transport around photosystem I (PS I ) is considered physiologically important not only for its coupled formation of ATP, but also for its function on protection of the photosynthetic apparatus against photoinhibition. However, due to the difficulty of its measurement, we know little about its operation in vivo.
基金supported by the National Key Scienceand Technology Program of China (No. 2008ZX07105-006)the National Natural Science Foundation of China(No. 30821140542) the Hundred Talents Program of the Chinese Academy of Sciences (No. 0823031501)
文摘In order to explore the mechanism of acute toxicity for pyrene to cyanobacterial organisms, the responses of Synechocystis sp. PCC 6803 photosystem Ⅱ （PS Ⅱ） under pyrene stress were studied. The results showed there was no significant difference about the oxygen evolution under 0.125 mg/L pyrene stress when compared with control, but it was significantly lower than control at 0.625 mg/L pyrene. Polyphasic chlorophyll-a fluorescence transients in cells of Synechocystis sp. PCC 6803 exhibited a typical increase including O, J, I, and P phases. Fluorescence yield at phases J, I and P declined slightly at 0.125 and 0.625 mg/L pyrene, and significantly lower than control at 3.125 mg/L. According to the parameters deviated from JIP-test, no modification was induced by pyrene both at the donor side and at the acceptor side of PS Ⅱ, and the reaction centre of PS Ⅱ is the primary damaging target. Based on the expressing of four key genes （psbA, psbB, psbC and psbO） of PS Ⅱ, only psbA showed significant difference at 3.125 mg/L pyrene when compared with control.
文摘The expression of the genes encoding the ferredoxin-thioredoxin system including the ferredoxin-thioredoxin reductase （FTR） genes ftrC and ftrV and the four different thioredoxin genes trxA （m-type; sir0623）, trxB （x-type; sir1139）, trxC （sll1057） and trxQ （y-type; sir0233） of the cyanobacterium Synechocystis sp. PCC 6803 has been studied according to changes in the photosynthetic conditions. Experiments of light-dark transition indicate that the expression of all these genes except trxQ decreases in the dark in the absence of glucose in the growth medium. The use of two electron transport inhibitors, 3-（3,4-dichlorophenyl）-1,1-dimethylurea （DCMU） and 2,5-dibromo-3-methyl-6-isopropyl-p- benzoquinone （DBMIB）, reveals a differential effect on thioredoxin genes expression being trxC and trxQ almost unaffected, whereas trxA, trxB, and the ftr genes are down-regulated. In the presence of glucose, DCMU does not affect gene expression but DBMIB still does. Analysis of the single TrxB or TrxQ and the double TrxB TrxQ Synechocystis mutant strains reveal different functions for each of these thioredoxins under different growth conditions. Finally, a Synechocystis strain was generated containing a mutated version of TrxB （TrxBC34S）, which was used to identify the potential in-vivo targets of this thioredoxin by a proteomic analysis.
文摘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.
基金Elite Youth Program of Chinese Academy of Agricultural Sciences and the Agricultural Science and Technology Innovation Program。
文摘光是光合作用不可或缺的底物。然而过量的光照会对光合生物造成氧化胁迫和严重的损害。为了应对持续变化的光环境,蓝藻演化形成了灵活的电子传递网络。围绕光系统I(photosystem I,PSI)的循环电子传递(cyclic electron transport,CET)将电子从铁氧还蛋白Fd回流到质体醌(plastoquinone,PQ)库,产生ATP且不积累NADPH。在蓝藻和高等植物中发现了2种不同的CET途径,即NDH依赖途径和PGR5依赖途径。蓝藻中黄素二铁蛋白Flv1/Flv3参与了类梅勒(Mehler-like)反应,从PSI接受电子直接将氧气还原为水,且没有活性氧的形成。以集胞藻为试验材料,通过分析不同的CET和Flv突变株在不同光照条件下的生理特征以及其P700氧化/还原动力学,进而研究CET途径和类梅勒反应在集胞藻中的功能。结果表明NDH-1复合体对CET的贡献率超过90%,维持细胞能在持续高光环境下生长,而迅速应激的类梅勒反应在缓解瞬时高光胁迫时发挥了重要作用。因此我们认为在集胞藻中NDH-1介导的循环电子途径是稳固支持其适应高光逆境的主要机制,而类梅勒反应则是在现有主要途径严重不足时的1个备用途径。响应迅速的FLV路径是野生型和NDH-1突变株的补足。
基金supported by grants from the National Key Research and Development Program of China(2020YFA0906800,2021YFA0909700,2018YFA0903600 and 2019YFA0904600)。
文摘Synthetic biology efforts have also led to the development of photosynthetic cyanobacteria as"autotrophic cell factories"for biosynthesis of various biofuels directly from CO_(2).However,the low tolerance to toxicity of biofuels has restricted the economic application of cyanobacterial hosts.In this study,RNAseq transcriptomics was employed to reveal stress responses to exogenous n-hexane in Synechocystis sp.PCC 6803.Functional enrichment analysis of the transcriptomic data showed that signal transduction systems were induced significantly.To further identify regulatory genes related to n-hexane tolerance,a library of transcriptional regulators(TRs)deletion mutants was then screened for their roles in nhexane tolerance.The results showed that a knockout mutant of slr0724 that encodes an Hta R suppressor protein was more tolerant to n-hexane than the wild type,indicating the involvement of slr0724 in nhexane tolerance.This study provides the foundation for better understanding the cellular responses to n-hexane in Synechocystis sp.PCC 6803,which could contribute to the further engineering of nhexane tolerance in cyanobacteria.
