集胞藻PCC 6803类铁氧还蛋白Slr1205的功能研究

类铁氧还蛋白 (ferredoxin-like, Fd-like) 在高等植物中具有调控叶绿体发育等多种重要的生理功能,但在蓝藻中的生物功能尚未被发现。通过比较集胞藻PCC 6083编码Fd-like蛋白基因的敲除突变株Δslr1205与野生型 (WT) 在不同碳源和光周期条件下的生理生化表型,分析Slr1205在集胞藻中的功能。结果显示,在高CO2浓度自养、混合营养和光异养时,Δslr1205的生长速率低于WT,而在空气中自养条件下并无差异。与此相对应,混合营养和光异养时Δslr1205比WT的呼吸速率低,与呼吸作用密切相关的NDH-1L复合体的含量少。Δslr1205在所有测试的条件下有较高的类胡萝卜素以及偏黄的表型。这些数据表明,Fd-like蛋白Slr1205的缺失造成在碳源充足条件下的生长速率下降,这可能是由于呼吸作用下调导致供能不足。研究结果为今后深入研究蓝藻Fd-like蛋白奠定了基础,为开展光合作用和呼吸作用的调节机制研究探索了新方向。     

集胞藻PCC6803中S2P同源蛋白Slr0643及Sll0862 金属蛋白酶活性的体外鉴定

【目的】金属蛋白酶S2P在细菌中通过在膜切割转录调控因子、释放δ因子参与胁迫响应是跨膜信号转导的保守机制,但蓝细菌中S2P的功能还未被鉴定,故我们考察集胞藻PCC6803中的S2P同源蛋白Slr0643及Sll0862的金属蛋白酶活性。【方法】以pET-30b(+)为载体,分别构建重组质粒pF0643和pF0862,在大肠杆菌BL21(CE3)中诱导表达并纯化Slr0643及Sll0862蛋白,以β-酪蛋白为底物检测重组蛋白的酶活性。【结果】体外酶活实验显示重组表达的Slr0643及Sll0862蛋白有内切蛋白酶活性,且其活性受金属螯合剂o-phenanthroline的抑制。体外酶活的鉴定结果为进一步研究Slr0643和Sll0862的体内酶活和生物学功能奠定了基础。【结论】集胞藻PCC6803中的S2P同源蛋白Slr0643及Sll0862具有金属蛋白酶活性。     

集胞藻PCC6803 EGY同源基因破坏突变体的构建及表型分析

摘要：拟南芥中近来发现的定位于叶绿体的膜嵌合金属蛋白酶EGY1影响叶绿体发育与脂肪酸合成,经生物信息学分析,集胞藻PCC6803 (Synechocystis sp. PCC6803)中slr0643、sll0862基因编码同源蛋白。【目的】为了鉴定这两个基因的功能,【方法】本文通过同源重组插入卡那霉素抗性基因、切断目的基因,分别构建了slr0643::km和sll0862::km两种突变体,检测突变体的生理生化表型。【结果】在30℃,20 μE/m2s自养培养下,slr0643::km与野生型相比,早期     

蓝细菌PCC6803染色体上的毒素蛋白Slr0664与抗毒素蛋白Ssr1114的相互作用

【目的】证明蓝细菌PCC6803染色体上的毒素-抗毒素系统(TA,toxin-antitoxin system)ssr1114/slr0664中毒素蛋白Slr0664与抗毒素蛋白Ssr1114之的相互作用。【方法】构建在大肠杆菌(Escherichia coli)BL21(DE3)中诱导表达H6-Ssr1114或共表达H6-Ssr1114和Slr0664的重组质粒,诱导表达后借助亲和捕捉技术在不同的条件下纯化H6-Ssr1114或共纯化重组蛋白H6-Ssr1114和Slr0664,并通过肽谱分析共纯化的重组蛋白,证明H6-Ssr1114与Slr0664之间存在相互作用。【结果】诱导Slr0664表达对细胞产生毒性作用导致生长抑制或细胞死亡,诱导H6-Ssr1114和Slr0664共表达时细胞能能正常生长,在非变性条件下可纯化共表达的重组蛋白H6-Ssr1114和Slr0664,在变性条件下仅H6-Ssr1114被纯化,肽谱分析结果表明共纯化的的重组蛋白为H6-Ssr1114和Slr0664。【结论】ssr1114/slr0664TA系统中抗毒素蛋白Ssr1114与毒素蛋白Slr0664之间存在相互作用。     

集胞藻PCC 6803中丝氨酸/苏氨酸激酶SpkC对高温胁迫的响应

丝氨酸/苏氨酸激酶是蓝藻感知和转导外界刺激的重要元件,但至今蓝藻中很多丝氨酸/苏氨酸激酶的功能尚属未知。【目的】研究集胞藻PCC6803中的丝氨酸/苏氨酸激酶Spk C是否参与对高温胁迫的响应。【方法】本研究采用同源重组的方法构建spC基因完全敲除突变株,检测突变株与野生株在高温胁迫下的生长状况、色素组成,并对高温胁迫下叶绿素荧光参数差异进行分析,比较光合系统Ⅱ活性差异。此外,通过测定生长速率来判断高温胁迫后藻株的恢复情况。【结果】经过42℃高温胁迫后,与野生株相比,突变株ΔspkC生长减缓,光合色素(叶绿素、类胡萝卜素和藻胆色素)的含量降低;45℃高温胁迫下突变株ΔspkC的光合系统Ⅱ活性下降幅度更大;经过5 d 42℃高温处理后,突变株生长几乎停滞,存活率较野生株明显降低。【结论】集胞藻PCC 6803中spkC基因的缺失导致突变株对高温胁迫响应出现缺陷,提示丝氨酸/苏氨酸激酶SpkC参与响应高温胁迫。     

蓝细菌Synechocystis PCC 6803染色体上的基因ssl2138和sll1092构成的毒素-抗毒素系统

摘要:【目的】证明集胞藻(Synechocystis)PCC 6803染色体上的假定基因ssl2138和sll1092构成vapBC家族的毒素-抗毒素系统(toxin-antitoxin system,TA系统)。【方法】以RT-PCR分析ssl2138和sll1092的共转录,以选择性表达系统分析其编码产物对大肠杆菌生长的影响,并通过亲和层析和质谱检测证明编码产物之间的相互作用。【结果】ssl2138和sll1092构成的二元操纵子在正常生长条件下共转录;Sll1092表达抑制大肠杆菌的生长,Ssl2138的同时表达或随后表达可拮抗Sll1092的生长抑制作用;Ssl2138与Sll1092相互作用形成复合体。【结论】位于同一操纵子中的假定基因ssl2138和sll1092构成vapBC家族的TA系统。     

集胞藻PCC6803 中relA/spoT 同源基因syn-rsh (slr1325)的鉴定

[目的]四磷酸或五磷酸鸟苷(Guanosine 3′,5′-bispyrophosphate,(p)ppGpp)是细菌在遭遇环境胁迫时细胞产生应激反应的信号分子,(p)ppGpp由其合成酶RelA或具有合成酶或水解酶双重催化功能的RelA/SpoT合成.本文证明了集胞藻PCC6803(Synechocystis sp.)中唯一编码RelA/SpoT同源蛋白(命名为Syn-RSH)的基因slr1325(syn-rsh)的功能.[方法]通过互补试验证明syn-rsh表达产物的生物学功能;以纤维素薄层层析检测不同条件下Escherichia coli(p)ppGpp合成缺陷突变株及集胞藻PCC6803细胞中的(p)ppGpp.[结果]诱导Syn-RSH表达可使(p)ppGpp合成酶和水解酶基因缺失的E.coli突变株回复野生型表型,并在细胞中积累一定水平的ppGpp;在实验室培养条件下,集胞藻PCC6803细胞中可检测到低水平的ppGpp,氨基酸饥饿可诱导ppGpp水平升高并维持在相应水平.[结论]Syn-RSH具有(p)ppGpp合成酶和水解酶的双重功能,(p)ppGpp是集胞藻PCC6803在实验室生长条件下细胞生长所必需的.     

蓝细菌6803中糖基转移酶Sll1466对胁迫响应及其对糖基化、磷酸化反应的调控作用

糖基转移酶存在于原核和真核生物中,参与低聚糖和多糖的生物合成,在生物转化过程中起着重要的作用．本研究中我们去除了Synechocystis PCC 6803中的糖基转移酶基因sll1466．在不同培养基的光合自养条件下,Sll1466缺失突变体较野生型的细胞内部结构变化明显(超薄电镜观察),突变体在缺碳培养基条件下羧酶体含量比野生型高,并且在0.5 mol/L NaCl条件下的肝醣含量也明显高于野生型．突变体在不同光密度生长条件下的吸收光谱与野生型差异明显．分子水平上,突变体较野生型显现出如下3个方面的差异：a．2个碳水化合物选择性OprB孔蛋白在类囊体膜上发生糖基化;b．核杆连接蛋白CpcG1(Slr2051)在类囊体膜的上清中发生磷酸化;c．与上述表型差异相关的基因的转录水平亦呈现相同的变化趋势．这些结果预示着Sll1466在调控蓝细菌6803生理、代谢及能量转化等方面有着重要作用．     

集胞藻PCC 6803中脂肪酸激活酶Slr1609互作蛋白的鉴定

集胞藻中slr1609是编码脂肪酸激活酶的基因,对与其相关的重要功能伴侣蛋白进行研究,可以完善对脂肪酸合成模块的认识,为进一步通过合成生物学技术改造蓝细菌提供理论支持。本研究在集胞藻PCC 6803中建立了蛋白质复合体分析及鉴定技术:利用氯霉素抗性基因筛选,构建带有3×FLAG标签的Slr1609突变株,通过RT-PCR优化重组蛋白表达条件;同时对突变株进行了Western blotting鉴定,以及利用Native-PAGE验证了蛋白质复合体的存在。最后,LC-MS/MS质谱鉴定获得了Slr1609蛋白复合体中的可能伴侣蛋白。     

集胞藻6803光合自养生长突变株的筛选与鉴定

集胞藻(Synechocystis sp.)PCC6803(以下称集胞藻6803)是一种单细胞蓝藻,既可进行自养生长,也可在光合系统失活的情况下利用葡萄糖进行异养生长[1],具有天然的DNA转化系统,为筛选光合自养生长突变株和基因功能的鉴定提供了便利.其全基因组序列已于1996年公布[2].     

Slr1293 in Synechocystis sp. strain PCC 6803 Is the C-3',4' desaturase (CrtD) involved in myxoxanthophyll biosynthesis

When grown at high light intensity, more than a quarter of the total carotenoids in the unicellular cyanobacterium Synechocystis consists of myxoxanthophyll, a polar carotenoid glycoside. The biosynthetic pathway of myxoxanthophyll is unknown but is presumed to involve a number of enzymes, including a C-3',4' desaturase required to add one double bond to generate 11 conjugated double bonds in the monocyclic myxoxanthophyll. A candidate for this desaturase is Slr1293, which was identified by genome similarity searching. To determine whether Slr1293 is a desaturase recognizing neurosporene and lycopene, slr1293 was expressed in Escherichia coli strains accumulating neurosporene or lycopene. Confirming such a desaturase function for Slr1293, these E. coli strains accumulated 3',4'-didehydroneurosporene and 3',4'-didehydrolycopene, respectively. Indeed, deletion of slr1293 in Synechocystis provides further evidence that Slr1293 is a desaturase recognizing neurosporene: In the slr1293 deletion mutant, neurosporene was found to accumulate and was further processed to produce neurosporene glycoside. Neurosporene hereby becomes a primary candidate to be the branch point molecule between carotene and myxoxanthophyll biosynthesis in this cyanobacterium. The slr1293 gene was concluded to encode a C-3',4' desaturase that is essential for myxoxanthophyll biosynthesis, and thus it was designated as crtD. Furthermore, as Slr1293 appears to recognize neurosporene and to catalyze the first committed step on the myxoxanthophyll biosynthesis pathway, Slr1293 plays a pivotal role in directing a portion of the precursor pool for carotenoid biosynthesis toward myxoxanthophyll biosynthesis in Synechocystis sp. strain PCC 6803.     

Biochemical and functional characterization of BLUF-type flavin-binding proteins of two species of cyanobacteria

BLUF (a sensor of Blue-Light Using FAD) is a novel putative photoreceptor domain that is found in many bacteria and some eukaryotic algae. As found on genome analysis, certain cyanobacteria have BLUF proteins with a short C-terminal extension. As typical examples, Tll0078 from thermophilic Thermosynechococcus elongatus BP-1 and Slr1694 from mesophilic Synechocystis sp. PCC 6803 were comparatively studied. FAD of both proteins was hardly reduced by exogenous reductants or mediators except methylviologen but showed a typical spectral shift to a longer wavelength upon excitation with blue light. In particular, freshly prepared Tll0078 protein showed slow but reversible aggregation, indicative of light-induced conformational changes in the protein structure. Tll0078 is far more stable as to heat treatment than Slr1694, as judged from flavin fluorescence. The slr1694-disruptant showed phototactic motility away from the light source (negative phototaxis), while the wild type Synechocystis showed positive phototaxis toward the source. Yeast two-hybrid screening with slr1694 showed self-interaction of Slr1694 (PixD) with itself and interaction with a novel PatA-like response regulator, Slr1693 (PixE). These results were discussed in relation to the signaling mechanism of the "short" BLUF proteins in the regulation of cyanobacterial phototaxis.     

Tocopherols protect Synechocystis sp. strain PCC 6803 from lipid peroxidation

Tocopherols (vitamin E) are lipid-soluble antioxidants synthesized only by photosynthetic eukaryotes and some cyanobacteria, and have been assumed to play important roles in protecting photosynthetic membranes from oxidative stress. To test this hypothesis, tocopherol-deficient mutants of Synechocystis sp. strain PCC 6803 (slr1736 and slr1737 mutants) were challenged with a series of reactive oxygen species-generating and lipid peroxidation-inducing chemicals in combination with high-light (HL) intensity stress. The tocopherol-deficient mutants and wild type were indistinguishable in their growth responses to HL in the presence and absence of superoxide and singlet oxygen-generating chemicals. However, the mutants showed enhanced sensitivity to linoleic or linolenic acid treatments in combination with HL, consistent with tocopherols playing a crucial role in protecting Synechocystis sp. strain PCC 6803 cells from lipid peroxidation. The tocopherol-deficient mutants were also more susceptible to HL treatment in the presence of sublethal levels of norflurazon, an inhibitor of carotenoid synthesis, suggesting carotenoids and tocopherols functionally interact or have complementary or overlapping roles in protecting Synechocystis sp. strain PCC 6803 from lipid peroxidation and HL stress.     

Inactivation of the open reading frame slr0399 in Synechocystis sp. PCC 6803 functionally complements mutations near the Q(A) niche of photosystem II. A possible role of Slr0399 as a chaperone for quinone binding.

The Synechocystis sp. PCC 6803 triple mutant D2R8 with V247M/A249T/M329I mutations in the D2 subunit of the photosystem II is impaired in Q(A) function, has an apparently mobile Q(A), and is unable to grow photoautotrophically. Several photoautotrophic pseudorevertants of this mutant have been isolated, each of which retained the original psbDI mutations of D2R8. Using a newly developed mapping technique, the site of the secondary mutations has been located in the open reading frame slr0399. Two different nucleotide substitutions and a deletion of about 60% of slr0399 were each shown to restore photoautotrophy in different pseudorevertants of the mutant D2R8, suggesting that inactivation of Slr0399 leads to photoautotrophic growth in D2R8. Indeed, a targeted deletion of slr0399 restores photoautotrophy in D2R8 and in other psbDI mutants impaired in Q(A) function. Slr0399 is similar to the hypothetical protein Ycf39, which is encoded in the cyanelle genome of Cyanophora paradoxa; in the chloroplast genomes of diatoms, dinoflagellates, and red algae; and in the nuclear genome of Arabidopsis thaliana. Slr0399 and Ycf39 have a NAD(P)H binding motif near their N terminus and have some similarity to isoflavone reductase-like proteins and to a subunit of the eukaryotic NADH dehydrogenase complex I. Deletion of slr0399 in wild type Synechocystis sp. PCC 6803 has no significant phenotypic effects other than a decrease in thermotolerance under both photoautotrophic and photomixotrophic conditions. We suggest that Slr0399 is a chaperone-like protein that aids in, but is not essential for, quinone insertion and protein folding around Q(A) in photosystem II. Moreover, as the effects of Slr0399 are not limited to photosystem II, this protein may also be involved in assembly of quinones in other photosynthetic and respiratory complexes.     

Identification of the genes encoding enzymes involved in the early biosynthetic pathway of pteridines in Synechocystis sp. PCC 6803

The biosynthetic pathway for the pteridine moiety of cyanopterine, as well as tetrahydrobiopterine, has been investigated in Synechocystis sp. PCC 6803. Open reading frames slr0426, slr1626, slr0078 and sll0330 of the organism putatively encoding GTP cyclohydrolase I, dihydroneopterine aldolase, 6-pyruvoyltetrahydropterine synthase and sepiapterine reductase, respectively, have been cloned into T7-based vectors for expression in Escherichia coli. The recombinant proteins have been purified to homogeneity and demonstrated to possess expected genuine activities except that of sll0330. Our result is the first direct evidence for the functional assignment of the open reading frames in Synechocystis sp. PCC 6803. Furthermore, the 6-pyruvoyltetrahydropterine synthase gene is demonstrated for the first time in prokaryotes. Based on the result, biosynthesis of cyanopterine is discussed.     

Proteomic study of the impact of Hik33 mutation in Synechocystis sp. PCC 6803 under normal and salt stress conditions

Cyanobacteria are the only prokaryotes possessing plasma, thylakoid, and outer membranes. The plasma membrane of a cyanobacterial cell is essential for the biogenesis of cyanobacterial photosystems and serves as a barrier against environmental stress. We previously identified dozens of salt-responsive proteins in the plasma membrane of Synechocystis sp. PCC 6803. Five histidine kinases (Hiks) including Hik33 were also proposed to be involved in the perception of salt stress in Synechocystis. In this study, we analyzed proteomic profiles of the plasma membrane from a hik33-knockout mutant (ΔHik33) under normal and salt-stress conditions. Using 2D-DIGE followed by mass spectrometry analysis, we identified 26 differentially expressed proteins in ΔHik33 mutant cells. Major changes, due to the Hik33 mutation, included the substrate-binding proteins of ABC transporters, such as GgtB and FutA1, regulatory proteins including MorR and Rre13, as well as several hypothetical proteins. Under salt-stress conditions, the Hik33 mutation reduced levels of 7 additional proteins, such as NrtA, nitrate/sulfonate/bicarbonate-binding protein and LexA, and enhanced levels of 9 additional proteins including SphX. These observations suggest a substantial rearrangement in the plasma membrane proteome of Synechocystis due to the loss of hik33. Furthermore, a comprehensive molecular network was revealed in ΔHik33 mutant coping with salt stress.