不产氧光合细菌光合色素的光氧调控机制

[目的]为不产氧光合细菌光合色素研究提供可行的较系统规范的研究方法和数据,揭示固氮红细菌(Rhodobacter azotoformans 134K20)光合色素光氧适应性机制.[方法]采用光谱法和色谱法对光和氧调控下的类胡萝卜素和细菌叶绿素合成代谢进行了研究.[结果]134K20菌株光照好氧时细胞得率最高.光照厌氧时主要合成3黄、1红、1紫、2绿、2蓝9种色素,黄色素大量表达.有氧时红色素大量表达,且启动2种新的红色素和1种新的紫色素表达,而黄色和蓝绿色素则受氧抑制.黑暗好氧主要合成2黄、3红、2紫、1绿、1蓝9种色素,但不同于光照厌氧.光照好氧时黄色素减少到1种,紫色素含量增加,其余同黑暗好氧.[结论]固氮红细菌(Rhodobacter azotoformans 134K20)是通过PpsR调节途径来调节光合基因表达的.黄色和红色素属于类胡萝卜素.黄色素1属于球形烯系列,其余两种黄色素是新的类胡萝卜素组分.红色素为新的球形烯酮组分,3种红色素极性、峰形和峰位差别显著,正己烷能显示其精细结构.紫色为极性较大的细菌脱镁叶绿素,绿色和蓝色为4种极性不同的细菌叶绿素a中间产物.乙醚甲醇法适合类胡萝卜素的提取,丙酮甲醇冰冻研磨法能快速有效完全提取光合色素.溶剂效应可有效鉴别细菌叶绿素a中间产物.     

紫细菌反应中心中的超快能量传递过程

利用飞秒泵探测技术研究了紫细菌光合反应中心RS601中的超快能量传递过程,通过选择激反应中心中的不同色素,观察到了以不同色素为起点发生在飞秒时域的超快能量传递过程,从细菌去镁叶绿素H到辅助细胞叶绿素B的能量传递发生在约130fs时间尺度,而通过激发色素B则观察到了从B到原始电子供体P的约240fs的超快能量传递,另外,P激发态的超快弛豫过程则说明其上、下激子能级间存在超快的内转换过程,通过对不同色素激发态的能量弛豫过程的分析,说明由原初电子供体H的电子传递过在几个皮秒时间内完成,其中辅助细菌叶绿素B为该电子传递过程中间态。     

光合细菌H3菌株的分离及其生物学特性研究

H3菌株系由盐田微生物垫中分离获得的光合细菌株。革兰氏阴性杆菌,0.5-0.7×15-2.5μm,在培养条件改变时呈多形态,其膨大细胞可达4.0-7.0μm.单根极生鞭毛,二均分裂。菌落及液体培养物呈深紫红色。菌体蛋白质含量约50%,各种必需氨基酸齐全,并含有丰富的细菌叶绿素a和类胡萝卜素。H3菌株在光照和黑暗条件下均可生长,能利用多种有机和无机碳、氮源,并能固N2.在含0.5-7.0%NaCI培养基中均可生长。但光照、盐浓度、温度、pH和通气等条件对其生长量有明显的影响。作者对其分类地位和应用潜力作了讨论。       

固氮红细菌(Rhodobacter azotoforman)色素蛋白复合体的分离纯化与特性

【目的】为揭示不产氧光合细菌产氢菌株色素蛋白复合体(PPC)色素组成和含量与光合放氢的关系奠定基础。【方法】以PPC特征光谱为检测指标,采用硫酸铵分级分离、DEAE-纤维素层析、吸收光谱和SDS-PAGE等方法进行了固氮红细菌(Rhodobacter azotoformans,R.azotoformans)R7产氢菌株PPC的分离纯化、纯度分析和鉴定;采用表面增强激光解吸电离离子飞行时间质谱、HPLC-MS和荧光光谱法对其中一种PPC进行了组成分析和能量传递活性测定。【结果】从R7菌株获得了3种纯化的PPC,1种为反应中心与中心捕光色素蛋白复合体(RC-LH1),2种为外周捕光色素蛋白复合体(LH2),其中一种LH2的吸收光谱具有异常的423nm强吸收峰,其蛋白的两种亚基的分子量分别为5356.8Da和5697.8Da,类胡萝卜素属球形烯系,分子量为562Da,激发光能够从类胡萝卜素向细菌叶绿素以及细菌叶绿素向细菌叶绿素传递。【结论】固氮红细菌产氢菌株含有2种不同光谱特性的LH2,其中一种具有新光谱特性。     

一株极端环境光合细菌的生理特性研究

从山西运城盐厂解化池分离获得一株嗜盐嗜碱细菌,编号为Y。其纯培养物经形态学、生理生化特性和DNA G+C含量等特征分析,结果表明,该菌株可在盐度ρ(NaCl)/gL-1为160和pH9.0碱性条件下生长。单细胞为杆状,大小为0.4-0.8×0.9-1.5μm。二分裂繁殖。革兰氏阴性。光合内膜为片层堆积并与细胞质膜相连,但并不与细胞质膜平行。细胞含有细菌叶绿素a,液体培养物呈玫瑰红色。Y菌株不仅可将硫化物氧化为元素硫沉积于细胞外,也可光同化多种有机物,并具有固氮和产H2特性。DNA中 G+C含量为61.6%。根据以上鉴定特征及相关资料,Y菌株应归于嗜盐红螺菌属(Halorhodospira)。但其生长所依赖的盐度、pH、细胞内色素成分及鞭毛着生方式与该属正式承认的3个种有明显的不同。该菌株独特的生理生化特性,对于极端环境微生物的资源开发和利用有重要的理论意义和应用价值。     

紫细菌光合色素指纹图谱的建立与色素分析

【目的】探索快速高效的色素样品制备方法;为建立紫细菌全色素TLC和HPLC标准指纹图谱和数据库提供研究方法和思路;为色素代谢与调控等研究提供快速的色素分析方法。【方法】选择沼泽红假单胞菌(Rhodopesudomonas palustris CQV97)为材料,采用改良丙酮甲醇提取法、TLC重复展层法、图像灰度分析法、吸收光谱法、HPLC和MS法进行色素分析。【结果】甲醇或丙酮可选择性地提取样品的细菌叶绿素和类胡萝卜素,通过对丙酮甲醇法的改良,使色素提取总量提高了13.5%。建立了CQV97菌株全色素的TLC和HPLC指纹图谱,二者均含有11种色素组分。图像灰度分析法估算了TLC指纹图谱各色素组分的表观相对含量。以TLC图谱的各色素组分为外标物,明确了HPLC图谱中各色素组分的保留时间(Rt)与TLC图谱中各色素组分的迁移率(Rf)之间的对应关系。结合色素代谢途径、光谱分析和MS,定性分析了指纹图谱中11种色素组分。TLC或HPLC分析结果表明,理论样品与对照样品色素组分和含量一致,而实际样品与对照样品色素组分一致,但含量不同,重复测定3次,样品中色素相对含量的RSD均小于5%。【结论】改良的丙酮甲醇法可以快速高效地提取光合细菌的色素。采用TLC重复展层法和HPLC法建立的全色素指纹图谱色素组成一致,重复稳定性好,各具特色。TLC和HPLC两种指纹图谱分析方法均能进行光合色素的快速分析,适合于紫细菌色素合成途径中主要积累色素的组分和含量变化规律研究。     

铁对产铁载体的沼泽红假单胞菌光合色素与铁载体合成的影响

【目的】探求铁对1株能产生铁载体的不产氧光合细菌(Anoxygenic Phototrophic Bacteria,APB)光合色素和铁载体合成的影响。【方法】通用CAS法检测铁载体产生,Arnow、Csaky和Shenker法检测铁载体类型;吸收光谱法和HPLC法分析光合色素的组分和含量。【结果】Rhodopseudomonas palustris CQV97能够产生异羟肟酸型铁载体,未添加FeCl3时,铁载体含量最高,铁载体的产生与生长并非关联型。随FeCl3浓度升高,菌体生长潜伏期缩短,生长速率、最终生物量以及细菌叶绿素(Bacteriochlorophyll,BChl)a和类胡萝卜素(Carotenoid,Car)含量均提高,而检测到的游离铁载体含量降低;菌体积累的BChl a的组成和相对含量未见明显变化,但主要的Car组分由Spirilloxanthin转化为Rhodopin,菌体中积累Car组分的平均共轭体系降低,Car组成的改变与色素提取液的Car特征性光谱蓝移现象相吻合。【结论】首次报道APB能够产生铁载体,CQV97菌株能够产生异羟肟酸型铁载体。阐明了铁对CQV97生长、铁载体产生和光合色素合成的影响规律,这些研究结果为深入开展APB铁载体分离纯化以及生物功能研究奠定了基础。     

光合细菌叶绿素代谢研究进展

细菌叶绿素和捕光蛋白及类胡萝卜素一起组成色素蛋白复合物,进而构成完整的捕光单位进行光合作用.简述了细菌叶绿素合成途径及其中关键的酶,并从分子水平上介绍了细菌叶绿素合成相关基因的表达调控的研究进展.     

弱细颤藻脂溶性化合物分析

对人工培养的弱细颤藻 Oscillatoria tenuis 中有机化合物的初步分析结果表明,脂溶性有机化合物占藻细胞干重5.8%,其中色素化合物含量较高,烃类化合物含量较低。烷烃化合物碳原子数分布范围为 C_(14)—C_(19),以正十七烷含量最高。色素化合物主要为脱镁叶绿素α,β-胡萝卜素和橙红色的未知色素化合物等。     

溶藻细菌BS03(Microbulbifer sp.)对塔玛亚历山大藻生长及抗氧化系统的影响

【目的】研究溶藻细菌BS03(Microbulbifer sp.)胁迫下塔玛亚历山大藻细胞光合作用、抗氧化酶系统和半胱氨酸蛋白酶3(Caspase-3)变化,探讨溶藻细菌BS03对塔玛亚历山大藻的溶藻机制。【方法】通过0.5%、1.0%、1.5%、2.0%不同终浓度BS03上清液处理藻细胞后12、24、36、48h取样,测定溶藻过程藻细胞光合色素、叶绿素荧光效率、抗氧化酶系统、Caspase酶活性变化。【结果】(1)BS03上清液处理藻细胞后,藻细胞叶绿素a含量和叶绿素荧光Fv/Fm比值随BS03上清液处理时间延长和浓度的增加呈逐渐下降趋势;低浓度处理组藻细胞类胡萝卜素含量上升到一峰值,高于对照组后逐渐回落,而高浓度处理组类胡萝素含量呈下降趋势,低于对照组;(2)藻细胞抗氧化酶保护系统(SOD和CAT)活性随着BS03上清液处理浓度增加而升高,但随着处理时间的延长呈现先上升后下降趋势。藻细胞膜脂过氧化产物MDA积累量随着BS03上清液处理时间延长和处理浓度的增加而显著提高;(3)处理组藻细胞Caspase-3活性显著高于对照组,呈现出类似程序性死亡特征。【结论】BS03的抑藻机理可能是通过抑制藻细胞光合作用,降低抗氧化酶活性、加大膜脂过氧化起到对塔玛亚历山大藻的溶解作用,并呈现出类程序性死亡特征。     

Further evidence for dissipative energy migration via triplet states in photosynthesis. The protective mechanism of carotenoids in Rhodopseudomonas spheroides chromatophores.

The protection action of carotenoids against irreversible photodestruction was discovered in photosynthetic bacteria by Stanieda and coworkers. In green plant material it was found by Wolff and Witt (1969) Z. Naturforsch, 24b, 1031-1037 and (1972) Proc. 2nd. Int. Congr. Photosynthesis Res. Stresa (Forti, G., Avron, M. and Melandri, A., eds.), Vol. 2, pp. 931-936, Dr. W. Junk, N. V. Publ. The Hague) that the formation of special carotenoid triplet states (via very rapid energy transfer from excited chlorophylls) and their fast radiationless decay in tau1/2 approximately 3 microns is at least one mechanism for the protective action of carotenoids to irreversible photooxidation of the chlorophylls. Hence, it is anticipated that the same mechanism might be realized also in bacteria. The present study gives evidence for such a "triplet valve" to be established also in bacteria. This conclusion was derived from the following observations: 1. The light-induced difference spectrum shows a bleaching of a carotenoid at three characteristic wavelength between 400 and 500 nm. A positive peak around 533 nm indicates the formation of a carotenoid triplet state. 2. The absorption changes can be induced by red light which excites only bacteriochlorophyll. This indicates an energy transfer from bacteriochlorophyll to carotenoids. 3. The light-induced carotenoid triplets decay radiationless in 3 microns in air-saturated aqueous suspensions of the chromatophores. 4. The carotenoid triplet formation occurs only at actinic flash intensities where the photosynthesis becomes saturated. 5. Addition of dithionite, which blocks photosynthesis, markedly increases the extent of carotenoid triplet formation. The different types of exciton migration within the photosynthetic unit are discussed, especially the routes leading to the dissipation of excess excitation energy.     

Effects of Carotenoid Inhibition on the Photosynthetic RC–LH1 Complex in Purple Sulphur Bacterium Thiorhodospira sibirica

Core complexes (LH1–RC) were isolated using preparative gel electrophoresis from photosynthetic membranes of the purple bacterium, Thiorhodospira sibirica, grown in the absence or presence of the carotenoid biosynthesis inhibitor, diphenylamine. The biosynthesis of carotenoids is affected by diphenylamine both quantitavely and qualitatively: after inhibition, the level of carotenoids in core complexes reaches only 10% of the normal content, as analyzed by HPLC and absorption spectroscopy. The normally grown bacterium biosynthesizes spirilloxanthin, rhodopin, anhydrorhodovibrin and lycopene, whereas after inhibition only neurosporene, ζ-carotene and their derivatives are found in the complexes. There is no concomitant accumulation of appreciable amounts of colorless carotenoid precursors. Interestingly, the main absorption band of the core light harvesting complex isolated from carotenoid-inhibited cells, shows a red shift to 889 nm, instead of a blue shift observed in many carotenoid-deficient species of purple photosynthetic bacteria. The stability of isolated core complexes against n-octyl-β-D-glucopyranoside clearly depends on the presence of carotenoids. Subcomplexes resulting from the detergent treatment, were characterized by non-denaturating gel electrophoresis combined with in situ absorption spectroscopy. Core complexes with the native carotenoid complement dissociate into three subcomplexes: (a) LH1 complexes partially depleted of carotenoids, with an unusual spectrum in the NIR region (λ_max = 791, 818, 847 and 875 nm), (b) reaction centers associated with fragments of LH1, (c) small amounts of a carotenoidless B820 subcomplex. The core complex from the carotenoid-deficient bacterium is much less stable and yields only the two sub-complexes (b) and (c). We conclude that carotenoids contribute critically to stability and interactions of the core complexes with detergents.     

Carotenoid S(1) state in a recombinant light-harvesting complex of Photosystem II.

The carotenoid species lutein, violaxanthin, and zeaxanthin are crucial in the xanthophyll-dependent nonphotochemical quenching occurring in photosynthetic systems of higher plants, since they are involved in dissipation of excess energy and thus protect the photosynthetic machinery from irreversible inhibition. Nonetheless, important properties of the xanthophyll cycle carotenoids, such as the energy of their S(1) electronic states, are difficult to study and were only recently determined in organic solvents [Polívka, T. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 4914. Frank, H. A. (2000) Biochemistry 39, 2831]. In the present study, we have determined the S(1) energies of three carotenoid species, violaxanthin, lutein, and zeaxanthin, in their LHCII (peripheral light-harvesting complex of photosystem II) protein environment by constructing recombinant Lhcb1 (Lhc = light-harvesting complex) proteins containing single carotenoid species. Within experimental error the S(1) energy is the same for all three carotenoids in the monomeric LHCII, 13,900 +/- 300 cm(-1) (720 +/- 15 nm), thus well below the Q(y)() transitions of chlorophylls. In addition, we have found that, although the S(1) lifetimes of violaxanthin, lutein, and zeaxanthin differ substantially in solution, when incorporated into the LHCII protein, their S(1) states have in fact the same lifetime of about 11 ps. Despite the similar spectroscopic properties of the carotenoids bound to the LHCII, we observed a maximal fluorescence quenching when zeaxanthin was present in the LHCII complex. On the basis of these observations, we suggest that, rather than different photochemical properties of individual carotenoid species, changes in the protein conformation induced by binding of carotenoids with distinct molecular structures are involved in the quenching phenomena associated with Lhc proteins.     

Carotenoids in photosynthesis: Protection of D1 degradation in the light

Photosynthesis has been determined with mutants of Anacystis which form different amounts of carotenoids. With these cultures a highly significant correlation between photosynthetic oxygen evolution and the amounts of synthesized carotenoids was observed. In addition, the influence of carotenoids on light-dependent degradation of thylakoid proteins was investigated with Scenedesmus cultures grown in darkness in the presence of norflurazon, an inhibitor of carotenoid biosynthesis. Pre-illumination of cells resulted in decrease of photosynthetic activity accompanied by loss of the D1 protein. This effect is dependent on the length of illumination, and the light intensity, and increased when carotenoid content was lowered during previous growth of the norflurazon-treated cultures.Abbreviations BSA bovine serum albumin - D1 32 kDa Q_B-binding protein - EDTA ethylenediaminetetraacetic acid - LHCII light-harvesting complex II - PMSF phenylmethylsulfonyl fluoride - PS photosystem - tricine N-[tris(hydroxymethyl)methyl] glycine     

Stark effect spectroscopy of carotenoids in photosynthetic antenna and reaction center complexes

The effects of electric fields on the absorption spectra of the carotenoids spheroidene and spheroidenone in photosynthetic antenna and reaction center complexes (wild-type and several mutants) from purple non-sulfur bacteria are compared with those for the isolated pigments in organic glasses. In general, the field effects are substantially larger for the carotenoid in the protein complexes than for the extracted pigments and larger for spheroidenone than spheroidene. Furthermore, the electrochromic effects for carotenoids in all complexes are much larger than those for the Qx transitions of the bacteriochlorophyll and bacteriopheophytin pigments which absorb in the 450-700 nm spectral region. The underlying mechanism responsible for the Stark effect spectra in the complexes is found to be dominated by a change in permanent dipole moment of the carotenoid upon excitation. The magnitude of this dipole moment change is found to be considerably larger in the B800-850 complex compared to the reaction center for spheroidene; it is approximately equivalent in the two complexes for spheroidenone. These results are discussed in terms of the effects of differences in the carotenoid functional groups, isomers and perturbations on the electronic structure from interactions with the organized environment in the proteins. these data provide a quantitative basis for the analysis of carotenoid bandshifts which are used to measure transmembrane potential, and they highlight some of the pitfalls in making such measurements on complex membranes containing multiple populations of carotenoids. The results for spheroidenone should be useful for studies of mutant proteins, since mutant strains are often grown semi-aerobically to minimize reversion.     

The gene crtI mediates the conversion of phytoene into colored carotenoids in Rhodopseudomonas capsulata

Carotenoids are membrane pigments present in all photosynthetic organisms, providing essential photoprotective functions. The first carotenoid formed in the pathway is phytoene, a colorless compound which is then converted into colored carotenoids by a series of dehydrogenation reactions. In the photosynthetic bacterium Rhodopseudomonas capsulata mutations that affect carotenoid biosynthesis before colored carotenoids are formed have a "blue-green" phenotype as opposed to the "red" of wild type cells. We have extracted carotenoids from several blue-green mutants and found that two strains (BPY69 and BPY102) accumulate phytoene and no colored carotenoids. These mutants failed to dehydrogenate phytoene in an in vitro assay. However, dehydrogenation of this compound can be achieved in vitro by adding a cell-free extract from another blue-green mutant blocked earlier in the pathway. Genetic complementation and deletion mapping indicate that the gene crtI is responsible for the conversion of phytoene into colored carotenoids in these mutants.     

How carotenoids function in photosynthetic bacteria

Carotenoids are essential for the survival of photosynthetic organisms. They function as light-harvesting molecules and provide photoprotection. In this review, the molecular features which determine the efficiencies of the various photophysical and photochemical processes of carotenoids are discussed. The behavior of carotenoids in photosynthetic bacterial reaction centers and light-harvesting complexes is correlated with data from experiments carried out on carotenoids and model systems in vitro. The status of the carotenoid structural determinations in vivo is reviewed.     

Photosynthetic Action Spectra of Scots Pine Needles of Different Ages from Seedlings Grown under Different Nursery Conditions

Photosynthetic action spectra of pine needles have been measured with an automatic recording method. A thin longitudinal cut from the flat surface of the needle was placed on top of an oxygen sensor and illuminated with monochromatic light in the range 400–700 nm. Good correspondence between absorption and action spectra was obtained in the range 550–700 nm. In the blue part of the spectrum photosynthetic efficiency was low compared with absorption. This low effect in blue light is probably due to screening absorption by photosynthetically inactive carotenoids. One-year-old needles showed a higher effect in the blue part of the spectrum than did current year needles. Needles from seedlings grown in the open had a lower photosynthetic effect in the blue part than needles from seedlings grown in a greenhouse. Pigment analyses support the idea that these differences are due to differences in the ratio chlorophylls/total carotenoids and/or differences in the composition of the carotenoid pigments.     

Characterization of unusual hydroxy- and ketocarotenoids in<Emphasis Type="Italic"> Rubrivivax gelatinosus</Emphasis>: involvement of enzyme CrtF or CrtA

Carotenoids are widely spread terpenoids found in photosynthetic organisms and a number of non-photosynthetic fungi and bacteria. The photosynthetic non-sulfur purple bacterium Rubrivivax gelatinosus produces carotenoids by both the spheroidene and the normal spirilloxanthin pathways. The characteristics of two carotenogenesis enzymes, spheroidene monooxygenase CrtA and O-methyltransferase CrtF, were investigated. Disruption of the corresponding genes by insertional mutagenesis affected carotenoid species in both pathways, and the genetic evidence indicated that both genes are involved in the two pathways. In these mutants, several unusual hydroxy- and ketocarotenoids were identified by spectroscopic and chemical methods. Moreover, the carotenoid analyses demonstrated that a large number of different carotenoid intermediates are accepted as substrates by the CrtA enzyme. The combined manipulation of crtF and crtA allowed new carotenoids to be produced and broadened the diversity of structurally different carotenoids synthesized by Rvi. gelatinosus. Methylated carotenoids, such as spheroidene and spirilloxanthin, are known to function as accessory pigments in the light-harvesting and reaction-center complexes of purple bacteria; the demethylated carotenoids described here were able to fulfill the same functions in the mutants.     

斜生栅藻中虾青素的积累过程及其光合活性变化

分析了斜生栅藻（Scenedesmus obliquus）在光温（30℃,180 μmol/m2·s）胁迫条件下积累虾青素的过程,观察了该过程中细胞形态及细胞光合生理的变化。胁迫条件下,细胞在48h内生成并积累了包括海胆酮、角黄素、金盏花黄素和金盏花红素在内的多种次生类胡萝卜素,并合成了虾青素及其酯。该过程中,细胞形态由两端尖细变得不规则、膨大,原来由4、8个细胞组成的定形群体变为游离的单个细胞或2个细胞组成的群体。藻细胞光合速率在24h内先下降后上升,而后又呈现下降趋势,从34.29 μmol O2/mg Chla/h迅速下降为5.21 μmol O2/mg Chla/h;呼吸速率在前24h内升高至60.37 μmol O2/mg Chla/h,而后缓慢下降到38.40 μmol O2/mg Chla/h;光合系统Ⅱ的活性随着胁迫时间的延续而逐步下降,较初始值降低了63.9％。结果表明,斜生栅藻细胞在高光照条件下可以合成虾青素,并通过调节光合速率、呼吸速率以及光合系统Ⅱ的效率来应对胁迫。