利用蓝藻生物报告体检测水体中可利用铁的研究

鱼腥藻PCC 7120 中的alr2581 基因编码的蛋白质在缺铁胁迫时显著上调。将该基因的启动子Palr2581和费氏弧菌的luxAB 基因融合, 通过同源单交换, 整合到鱼腥藻PCC 7120 的基因组上, 构建了可以感知环境中铁的生物报告体Palr2581-luxAB。该藻株在不含铁的BG11 中培养时, 启动子Palr2581 的转录活性增强, LuxAB酶活显著升高。通过测定Palr2581-luxAB 藻株在不同铁浓度Fraquil 培养基中的LuxAB 酶活, 得到了铁浓度pFe(-lgFe3+)与 LuxAB 酶活的剂量反应曲线。结果显示, 12h 时, LuxAB 酶活随培养基中Fe3+浓度增加呈S形递减关系, 其中在pFe=20.7—21.2 范围内有很好的线性关系。根据这一特性, 我们利用Palr2581-luxAB 作为铁的生物报告体, 测定了武汉市东湖水体中可利用的铁浓度为10-20.56 mol/L。研究显示, 通过这一方法可以较方便地监测各种淡水中可利用的铁浓度。       

ALKALINE PHOSPHATASE ACTIVITY AS A FUNCTION OF INTERNAL PHOSPHORUS CONCENTRATION IN FRESHWATER PHYTOPLANKTON1

Single‐cell alkaline phosphatase (AP) activity is being increasingly used to characterize phosphorus (P) status of individual species of phytoplankton. As phytoplankton growth rates depend more directly on the internal rather than external P concentrations, we determine the AP activity in the two species of freshwater phytoplankton, Scenedesmus quadricauda (Turpin) Bréb. and Asterionella formosa Hassall, as a function of internal P concentration. AP activity strongly correlated with cellular P, increasing almost linearly with decreasing cellular P in both species. The dynamics of initial responses of AP activity to P limitation, as well as the final levels of AP activity, when cellular P approached minimum quotas, differed in two species. After P addition, cellular P concentrations increased rapidly, but AP activity remained high for several days. The lag in AP activity down‐regulation following an increase in cellular P made it difficult to infer current P status of cells under dynamic P conditions.     

人铜锌超氧化物歧化酶基因改良及在聚球藻中表达

应用PCR定点突变技术把质粒pESOD中人铜锌超氧化物歧化酶基因(hCu,Zn-SOD)的Cys111密码子突变为Ala111密码子,再构建重组子,通过随机同源重组将突变后的hCu,Zn-SOD整合入聚球藻Synechococcussp.PCC7942,并实现表达。表达产物用SDS-PAGE、Western blot、酶活等方法测定均为阳性反应;热稳定性测定显示,hCu,Zn-SOD在80℃保温30min后仍具有95%的活力,耐热能力比天然hCu,Zn-SOD有了较大的提高。蛋白扫描结果显示目的蛋白占可溶性蛋白的3.61%。     

GROWTH AND PHOSPHORUS UPTAKE BY THE TOXIC DINOFLAGELLATE ALEXANDRIUM CATENELLA (DINOPHYCEAE) IN RESPONSE TO PHOSPHATE LIMITATION1

Alexandrium catenella (Whedon et Kof.) Balech has exhibited seasonal recurrent blooms in the Thau lagoon (South of France) since first reported in 1995. Its appearance followed a strong decrease (90%) in phosphate (PO₄^3?) concentrations in this environment over the 1970–1995 period. To determine if this dinoflagellate species has a competitive advantage in PO₄^3?‐limited conditions in terms of nutrient acquisition, semicontinuous cultures were carried out to characterize phosphorus (P) uptake by A. catenella cells along a P‐limitation gradient using different dilution rates (DRs). Use of both inorganic and organic P was investigated from measurements of ³³PO₄^3? uptake and alkaline phosphatase activity (APA), respectively. P status was estimated from cellular P and carbon contents (Q_P and Q_C). Shifts in trends of Q_P/Q_C and Q_P per cell (Q_P·cell?1) along the DR gradient allowed the definition of successive P‐stress thresholds for A. catenella cells. The maximal uptake rate of ³³PO₄^3? increased strongly with the decrease in DR and the decrease in Q_P/Q_C, displaying physiological acclimations to PO₄^3? limitation. Concerning maximal APA per cell, the observation of an all‐or‐nothing pattern along the dilution gradient suggests that synthesis of AP was induced and maximized at the cellular scale as soon as PO₄^3? limitation set in. APA variations revealed that the synthesis of AP was repressed over a PO₄^3? threshold between 0.4 and 1 μM. As lower PO₄^3? concentrations are regularly observed during A. catenella blooms in Thau lagoon, a significant portion of P uptake by A. catenella cells in the field may come from organic compounds.     

聚球藻7942混养培养中碳代谢与能量利用

为了考察聚球藻7942在混养条件下的能量利用效率,分别以葡萄糖和乙酸为碳源开展了聚球藻7942的混养培养研究,并在此基础上利用代谢通量分析方法对聚球藻7942混养条件下的碳代谢和能量利用进行了探讨。结果表明:葡萄糖和乙酸均能促进藻细胞生长,且乙酸促进藻细胞生长的作用更为明显;葡萄糖利用可明显增加藻细胞糖酵解途径中碳代谢流量,而乙酸利用则导致糖酵解途径中碳代谢流量减小,两种有机碳源均增加了柠檬酸循环中碳代谢流量;有机碳源导致藻细胞光化学效率下降,而葡萄糖较之乙酸降低藻细胞光化学效率更为明显。虽然混养条件下光能的贡献率要小于光自养,但基于能量的细胞得率和能量转换率均高于光自养,光自养和以葡萄糖、乙酸为碳源的混养中基于ATP生成的能量转换效率分别为6.81%、7.43%和8.77%。     

Genetically engineered herbicide resistance in cyanobacterium Synechococcus sp. by bialaphos resistance gene from Streptomyces hygroscopicus

A novel cyanobacterial vector, pTT201, containing the bar gene encoding resistance to herbicides, bialaphos and phosphinothricin, was constructed. In Synechococcus sp. strain PCC7942-SPc, the bar gene was successfully expressed. Plasmid pTT201 increased a minimum inhibitory concentration for bialaphos 16-fold over Synechococcus sp. strain PCC7942-SPc without pTT201. The combination of the bialaphos as a selective agent and the transformation by bar gene serves as a photostable selection system for Synechococcus.     

聚球藻7942光自养培养的碳代谢和能量代谢

代谢通量分析是研究微藻光自养培养过程中CO2和光能利用的一个非常有用的工具。本研究建立了聚球藻7942光自养培养代谢网络,并通过代谢通量方法分析了不同入射光强下的碳代谢流分布和能量代谢。研究结果表明,CO2固定是代谢能量和还原力消耗的主要途径,分别约占总消耗能量的85%和70%。研究还发现在一定光强范围,基于ATP生成的细胞得率和最大细胞得率基本维持不变,分别约为2.80g/molATP和2.95g/molATP,但基于总吸收光能的细胞得率和对应的光能转换效率则随着光强的增加而降低。     

NITRATE UTILIZATION BY PHYTOPLANKTON IN LAKE SUPERIOR IS IMPAIRED BY LOW NUTRIENT (P,Fe) AVAILABILITY AND SEASONAL LIGHT LIMITATION — A CYANOBACTERIAL BIOREPORTER STUDY1

We previously developed a luminescent Synechococystis sp. strain PCC 6803 cyanobacterial bioreporter that is used as a real‐time whole‐cell sensor to assess nitrate assimilatory capacity in freshwaters. Applying the bioreporter assay to Lake Superior, a system whose nitrate levels have increased 6‐fold since 1900, we investigated factors that constrain nitrate utilization in this oligotrophic system. Clean sampling methods were used to collect water from Lake Superior during spring and summer 2004, and nitrate utilization was measured by monitoring bioreporter luminescence. Bioreporter response was monitored during experiments in which the lake water was amended with nutrients and incubated under light regimes simulating integrated spring and summer mixing depths. These studies demonstrated that nitrate utilization was enhanced at most stations following addition of phosphorus (P). Moreover, at many stations, addition of iron (Fe) enhanced the P effect. Strength‐of‐effect statistical analysis provided the individual contribution of P and Fe toward stimulating bioreporter response. In general, distance from shore and season were not good predictors of nitrate assimilatory capacity. Manipulation of light flux during bioreporter experiments also showed that light intensities experienced during spring mixing are likely insufficient to saturate the rate of nitrate utilization. Overall, these data suggest that P‐limited algae are deficient in their ability to assimilate nitrate in Lake Superior. Furthermore, we suggest that a secondary limitation for Fe may occur that further constrains nitrate drawdown. Lastly, during spring, light fluxes are sufficiently low to prevent maximal nitrate utilization, even in the absence of nutrient limitation.     

Phosphate-uptake behaviour of a mutant of Synechococcus sp. PCC 7942 lacking one protein of the high-affinity phosphate-uptake system

The phosphate-uptake behaviour of a sphX mutant of the cyanobacterium Synechococcus leopoliensis (Raciborski) Komarek, strain PCC 7942 has been studied. This mutant lacks the high-affinity phosphate-binding protein that is abundantly expressed under phosphate-deficient growth conditions. The following observations have been made: (i) The mutant is still capable of utilizing phosphate at nanomolar external concentrations. (ii) Elimination of the sphX gene leads to an increase in the Michaelis constant and the maximum velocity of the initial influx of ³²P-phosphate. (iii) The capacity of the wild type to adapt within a few minutes to a transitory increase in the external phosphate concentration in an energetically efficient way (G. Falkner et al. 1994, C R Acad Sci Paris, Life sciences 317: 535–541) is lost in the mutant. As a result, the mutant can no longer attain pulse-adapted states that reflect in a characteristic way preceding exposures to higher phosphate concentrations. Received: 6 February 1998 / Accepted: 8 May 1998     

A method to probe the cytoplasmic osmolality and osmotic water and solute fluxes across the cell membrane of cyanobacteria with chlorophyll a fluorescence: Experiments with Synechococcus sp. PCC7942

We present a method with which osmotic properties of the cytoplasm of cyanobacterial cells and the osmotic permeability of plasma membranes to water and solutes can be assessed from measurements of chlorophyll a fluorescence. When the electron transport of photosystem II is inhibited, the quantum yield of chlorophyll a fluorescence in cyanobacterial cells varied between a low yield limit that was attained after acclimation to darkness (state 2) and a high yield limit that was attained after acclimation to light (state 1). It was shown recently that the difference between chlorophyll a fluorescence of light‐acclimated and of dark‐acclimated cells relates quantitatively to the internal osmolality of cyanobacteria (G. C. Papageorgiou and A. Alygizaki‐Zorba. 1997. Biochim. Biophys. Acta 1335: 1‐4). In the present work we employed rapid mixing of Synechococcus sp. PCC7942 (strain PAMCOD) suspensions with solutions of defined osmolality in order to measure cell osmolality and turgor threshold, as well as water and solute fluxes across cell membranes. Concentration upshocks with sorbitol, glycine betaine, Na⁺ and K⁺ salts caused rapid (t_1/2 < 10 ms) depression of fluorescence that was correlated to osmotic water outflow from the cells. The fluorescence remained depressed in all cases except for NaCl. With NaCl, the depression was transient and fluorescence recovered with an apparent time constant of 200 ms. The fluorescence rise correlates to inflows of NaCl and water.     

Establishment of a Highly Efficient Ammonia Secreting Mutant of Synechococcus sp. PCC 7942 and the Glutamine Synthetase Activity,Photosynthesis and Growth in Its Immobilized Cells

A recombinate plasmid pDC-ATGS was constructed, which contained the antisense fragment of glnA gene from Anabaena sp. PCC 7120 and transformed the unicellular cyanobactefium Synechococcus sp. PCC 7942. The foreign DNA was inserted into the site of glnA locus of the chromosome through the homologous recombination. By using neomyisin, a highly efficient ammonia secretion mutant was selected. After immobilized, the cells of the mutant within polyurethane (PU) foams, glutamine synthetase (GS) and NIt4+ secretory activity of GS, and its growth and photosynthesis were measured. It was shown that NH4+ secretion of the immobilized mutant was enhanced 156 folds which was much higher than that of free-living cells of the wild type. The activity of GS was decreased by 73.6%. Growth of the mutant was the same as that of the wild type. The activity of photosystem Ⅱ in the immobilized mutant cells increased by 44% with 77 K fluorescence spectrum measurement.     

产乙醇大肠杆菌的构建及其活性检测方法的改进

adhB和pdc是运动发酵单胞菌产乙醇途径的关键基因,分别编码乙醇脱氢酶和丙酮酸脱羧酶,将添加有聚球藻PCC7942rbcLS基因RBS序列的adhB和pdc基因插入pUC18载体,经双重菌液PCR检验和酶切检验得到分别含有pUC-adhB、pUC-pdc和pUC-adhB-pdc载体的3个重组菌株。活性检测实验表明聚球藻PCC7942的rbcLS基因的RBS序列能有效介导运动发酵单胞菌的adhB和pdc基因在大肠杆菌中表达,摇瓶发酵实验表明重组大肠杆菌的产乙醇能力较出发菌株大幅提升。鉴于乙醛指示平板法存在着对希夫试剂的要求较高、易产生较强的背景色等缺点,对定性检测丙酮酸脱羧酶和乙醇脱氢酶表达菌株的方法做了改进,即:将菌液诱导表达,然后分别添加对应于两种酶的底物,让酶与底物反应0.5至1小时,之后再加希夫试剂进行显色反应,结果表明改进后的方法比乙醛指示平板法更加简便、快速、可靠。     

AS-1 cyanophage infection inhibits the photosynthetic electron flow of photosystem II in Synechococcus sp. PCC 6301, a cyanobacterium

In Synechococcus sp. cells AS-1 cyanophage infection gradually inhibits the photosystem II mediated photosynthetic electron flow whereas the activity of photosystem I is apparently unaffected by the cyanophage infection. Transient fluorescence induction and flash-induced delayed luminescence decay studies revealed that the inhibition may occur at the level of the secondary acceptor, Q_B of photosystem II. In addition, the breakdown of D₁-protein is inhibited, comparable to DCMU-induced protection of D₁-protein turnover, in AS-1-infected cells.     

ALKALINE PHOSPHATASE IN FRESHWATER CLADOPHORA‐EPIPHYTE ASSEMBLAGES: REGULATION IN RESPONSE TO PHOSPHORUS SUPPLY AND LOCALIZATION1

Extracellular alkaline phosphatase enzyme activity (APA) is important for algal phosphorus (P) acquisition in P‐limited freshwater ecosystems and is often used as an indicator of P deficiency. APA allows access to organic P (monophosphate esters), but the regulation of APA in response to availability of both PO₄³⁻ and organic P is poorly characterized. This study aimed to examine the regulation of APA in freshwater Cladophora‐epiphyte assemblages in response to PO₄³⁻ and a hydrolyzable organic P source, and for the first time to apply enzyme linked fluorescence (ELF) to localize APA within freshwater macroalgal‐epiphyte assemblages. In response to elevated PO₄³⁻ concentrations, a component of net APA was suppressed, but there was also a constitutive APA, which was maintained even after prolonged exposure to nearly 1,000 μM PO₄³⁻ and saturation of internal P pools. When supplied with organic glycerol P as the sole P source, the algae maintained APA in excess of needs for supplying PO₄³⁻ for uptake, resulting in PO₄³⁻ release into the medium. Constitutive APA may be adaptive to growth under chronic P limitation in oligotrophic freshwater habitats. Excess APA and release of PO₄³⁻ could benefit different algal and bacterial partners within assemblages. APA in both Cladophora sp. and epiphytic algae was localized with ELF only when ethanol fixation was omitted. In algal subsamples exposed to different P treatments, there was no correlation between bulk APA (using 4‐methylumbelliferyl phosphate [MUP] substrate) and % cell labeling with ELF, suggesting that ELF labeling of APA was at best semiquantitative in the algal assemblages.     

A Synechococcus sp. PCC 7942 mutant with a higher tolerance towards bentazone

In this article we describe the partial characterization of a Synechococcus sp. PCC 7942 mutant Mu1 with an enhanced resistance towards the herbicide bentazone (3-isopropyl-1H-2,1,3-benzothiadiazine-4(3H)-one 2,2-dioxide). The mutant was derived from a random mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine (NSG) and exhibited superior growth rates, pigment content and overall photosynthetic activities under regular growth conditions compared to wild type. Whereas Synechococcus PCC 7942 wild type showed significant photoinhibition, especially in the presence of lincomycin, Mu1 was much more robust. A comparative analysis of the content of several photosynthesis-associated proteins revealed that Mu1 had an increased expression of PsbO on mRNA and protein level and that PsbO is tightly bound to Photosystem II, relative to wild type. This result was substantiated by mass spectrometer measurements of photosynthetic water oxidation revealing a higher stability and integrity of the water oxidizing complex in Mu1 cells grown under regular or calcium deficient conditions. Therefore, our results give rise to the possibility that the overexpression of PsbO in mutant Mu1 confers resistance to reactive oxygen species (ROS) formed as a consequence of bentazone binding to the acceptor side of PS II. In addition, we observed a significantly higher tolerance towards bentazone in iron depleted wild type cells, conditions under which the IdiA protein becomes expressed in highly elevated amounts. As we have previously shown, IdiA preferentially protects the acceptor site of PS II against oxidative stress, especially under iron limitation. Thus, it is likely that IdiA due to its topology interferes with bentazone binding or protects PS II against ROS generated in the presence of bentazone. This revised version was published online in June 2006 with corrections to the Cover Date.     

Glycinebetaine alleviates the inhibitory effect of moderate heat stress on the repair of photosystem II during photoinhibition

Transformation with the bacterial gene codA for choline oxidase allows Synechococcus sp. PCC 7942 cells to accumulate glycinebetaine when choline is supplemented exogenously. First, we observed two types of protective effect of glycinebetaine against heat-induced inactivation of photosystem II (PSII) in darkness; the codA transgene shifted the temperature range of inactivation of the oxygen-evolving complex from 40-52 °C (with half inactivation at 46 °C) to 46-60 °C (with half inactivation at 54 °C) and that of the photochemical reaction center from 44-55 °C (with half inactivation at 51 °C) to 52-63 °C (with half inactivation at 58 °C). However, in light, PSII was more sensitive to heat stress; when moderate heat stress, such as 40 °C, was combined with light stress, PSII was rapidly inactivated, although these stresses, when applied separately, did not inactivate either the oxygen-evolving complex or the photochemical reaction center. Further our studies demonstrated that the moderate heat stress inhibited the repair of PSII during photoinhibition at the site of synthesis de novo of the D1 protein but did not accelerate the photodamage directly. The codA transgene and, thus, the accumulation of glycinebetaine alleviated such an inhibitory effect of moderate heat stress on the repair of PSII by accelerating the synthesis of the D1 protein. We propose a hypothetical scheme for the cyanobacterial photosynthesis that moderate heat stress inhibits the translation machinery and glycinebetaine protects it against the heat-induced inactivation.     

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.     

Nucleotide Sequence of Plasmid pAQ1 of Marine Cyanobacterium Synechococcus sp. PCC7002

We have determined the complete nucleotide sequence of pAQ1,the smallest plasmid of the unicellular marine cyanobacteriumSynechococcus sp. PCC7002. The plasmid consists of 4,809 bpand has at least four open reading frames that potentially encodepolypeptides of 50 or more amino acids. We found that a palindromicelement, the core sequence of which is G(G/A)CGATCGCC, is over-representednot only in plasmid pAQ1 but also in the accumulated cyanobacterialgenomic sequences from Synechococcus sp. PCC6301, PCC7002, PCC7942,vulcanus and Synechocystis sp. PCC6803 within GenBank and EMBLdatabases. It suggests that this sequence might mediate generearrangement, thus increasing genetic diversity, since recombinationevents are frequent in cyanobacteria.