Specific Release of Bacteriochlorophylls B800 of LH2 from Rhodobacter azotoformans Induced by Sodium Dodecyl Sulfate

采用吸收光谱法研究了十二烷基硫酸钠(SdS)诱导rHOdObACTEr AzOTOfOrMAnS外周捕光复合体lH2细菌叶绿素(bACTErIOCHlOrOPHyllS,bCHlS)的解离行为和规律.结果表明:室温下,在10MMOl？l-1TrIS-HCl(PH8.0)缓冲液中,低浓度SdS只诱导lH2中b800细菌叶绿素解离生成游离bCHlS,而b850不受影响;当浓度达到0.08%(W/V)时,能特异性地诱导b800缺失,其缺失过程和游离bCHlS的生成过程均符合单指数模型,且二者的速率常数近似相等.高浓度SdS能同时诱导b800和b850解离生成游离bCHlS,其中b800可发生缺失,而b850则不完全解离,解离过程均符合单指数模型;b800对SdS更敏感,其解离速率常数约是b850的4倍,游离bCHlS生成速率常数明显低于b800解离速率常数,而与b850解离速率常数相接近.The release behaviors of bacteriochlorophylls of peripheral light-harvesting complex LH2 from Rhodobacter azotoformans induced by sodium dodecyl sulfate (SDS) were investigated using absorption spectroscopy.The results indicated that bacteriochlorophylls of B800 band are released from their binding sites and transformed into free bacteriochlorophylls by incubating LH2 sample in 10 mmol?L-1 Tris-HCl (pH 8.0) buffer containing SDS of low concentration at room temperature.However, the bacteriochlorophylls of B850 band are not released.The dynamics of B800 release and free BChl formation induced by 0.08% (w/V) SDS can be well fitted by the monoexponential model.The rate constant of B800 release is nearly equal to that of free BChls formation.The release of both B800 and B850 of LH2 can be induced by high concentration SDS, simultaneously.The bacteriochlorophylls of B800 band can be completely transformed into free BChls, but not for B850.Although both of their release processes show monoexponential models in 1% SDS solution, the release rate constant of B850 is remarkably lower than that of B800 and close to that of free BChls formation.国家自然科学基金(No.30970068);国家科技基础条件平台建设(No.2005DKA21209);厦门大学近海海洋环境科学国家重点实验室高级访问学者基金(No.MELRS0907);山西省回国留学人员科研(No.200713)资助项

Isolation and characterization of pigment-protein complexes from Rhodobacter azotoformans

【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。ObjectiveIn order to reveal the relationships of compositions and content of pigment in pigment-protein complexes(PPC ) and hydrogen photoevolution from anoxygenic phototrophic bacteria.Methods We isolated and identified pigment-protein complexes using a separation strategy of subsequent fractionated ammonium-sulfate precipitation,ion exchange column chromatography,absorption spectra and sodium dodecylsulfate polyacrylamide gel electrophoresis(SDS-PAGE ) from hydrogen-producing Rhodobacter azotoformans R7.We investigated the characterizations of the peripheral light-harvesting complex(LH2 ) with an unusual absorption spectrum by surface enhanced laser desorption / ionization time of flight mass spectrometry(SELDI-Tof-MS ),high performance liquid chromatography-mass spectrometry(HPLC-MS) and fluorescence spectra.ResultsWe acquired three types of PPC, the reaction center and core light-harvesting complex(RC-LH1 ) and two kinds of LH2,from strain R7 incubated anaerobically in the light.The two LH2 showed the different absorption spectra,one of them displayed unusual absorption spectrum with the maximum absorption band at 423 nm.The unusual LH2 consisted of two kinds of protein subunits with the molecular weight of 5556.8 Da and 5697.8 Da and carotenoid of spheroidene series with the molecular weight of 562 Da.It was also capable of transferring energy from carotenoid to bacterialcholorophyll and from B800 bacterialcholorophyll to B850 bacterialcholorophyll.Conclusions Rhodobacter azotoformans R7 with hydrogen-producing capacity could photosynthesize two types of LH2 under anaerobically in the light,one of them presented novel spectral property.国家自然科学基金(30970068);国家科技基础条件平台建设项目(2005DKA21209);近海海洋环境科学国家重点实验室(厦门大学)高级访问学者基金(MELRS0907)---

Screening of High-yield Cellulase Mutants of Aspergillus glaucus

以一株具有较高纤维素酶活性的野生菌株-灰绿曲霉XC9为出发菌株,经过紫外线、氯化锂、甲基磺酸乙酯(EMS)等物理化学诱变剂多重诱变处理,获得了抗葡萄糖阻遏的突变株E2-26、E5-65、U6-31和EU7-22,其CMC酶活与出发菌株相比分别提高至1.7、1.4、1.3、2.0倍.突变株经PDA斜面接种传代5次后产酶性能仍然保持稳定.变株的菌丝、孢子颜色也发生了较大的改变.对突变株EU7-22的形态结构进行了观察,并与出发菌株XC9作了发酵性状的比较,发现其产酶性能有了明显提高.Cellulase producing strain,Aspergillus glaucus XC9,was selected as the initial strain for mutation.Four mutant strains(E2-26,E5-65,U6-31,EU7-22)were obtained after it was treated multiplicatively with UV-ray,ethyl methane sulphonate(EMS)and LiCl.The CMC activity of mutants increased to 1.7,1.4,1.3 and 2.0 times comparing with the initial strain respectively.The spore colours of mutants also varied.The morphological and fermentative properties of mutant strain EU7-22 were studied in this paper.The producing capability of cellulase of mutant strain was remarkably enhanced.国家“863”计划项目(2001AA515040);; 福建省重点科技项目(2005I016);; 福建省青年科技人才创新项目(2005J003);; 厦门市科技项目(3502Z20041070)资

Construction and Characterization of B850-Only LH2 Energy Transfer System in Purple Bacteria

构建b800缺失lH2对于阐明光合作用中光能传递的分子机制与捕光复合体组装机制具有重要意义。采用吸收光谱、荧光光谱、分子筛层析、超滤和SdS-PAgE等方法研究了紫细菌两个典型种外周捕光复合体(lH2)约800nM特征光谱(b800)细菌叶绿素(bCHl)缺失能量传递模型的构建方法及性质。结果表明:在PH 8.0TrIS-HCl(10MMOl·l-1)缓冲液中,0.08%SdS能够使来自rHOdObACTEr AzOTOfOrMAnS的lH2b800bCHl特异性解离,解离体系中加入10%(φ)甲醇,通过超滤脱除游离bCHl,构建了b800缺失lH2,但该缺失模型不够稳定。在PH 1.9缓冲液中,来自rHOdOPSEudOMOnAS PAluSTrIS的lH2b800bCHl能够特异性解离,通过层析得到两个组分。一个组分的b800bCHl不能通过层析脱除,能够重新自组装成lH2。另一个组分为b800缺失lH2,该缺失模型稳定。两种lH2均存在2类以上b800bCHl结合位点,并得到了两类rHOdOPSEudOMOnAS PAluSTrIS b800bCHl解离的lH2,但未发现类似紫色硫细菌中的b800吸收光谱劈裂现象。b800缺失lH2均未呈现约800nM特征荧光光谱。采用两种方法构建了两个物种b800缺失lH2能量传递模型。利用bCHl与缺失b800lH2结合能力不同的特性,将rHOdOPSEudOMOnAS PAluSTrIS中的lH2分成两个类型,实现了异质性亚基lH2的分离。To seek microscopic molecular mechanism of energy transfer and complex reconstitution in the photosynthesis,the conditions for construction of B850-only peripheral light-harvesting complex(LH2)and their properties were investigated using absorption,fluorescence spectroscopy,molecular sieve chromatography,ultrafiltration and sodium dodecylsulfate polyacrylamide gel electrophoresis(SDS-PAGE)from the purple bacteria.The results indicated that bacteriochlorophylls(BChl)of B800 incubated in 10mmo·L-1 Tris-HCl(pH 8.0)buffer are selectively released from their binding sites of LH2 of Rhodobacter azotoformans(A-LH2)by 0.08%(W/V)SDS.B850-only A-LH2 was constructed after removing free BChl mixing with 10% methyl alcohol by ultrafiltration.B850 BChl was released after A-LH2 was incubated for 240 min in dark at room temperature(RT).While BChl of B800 incubated in pH 1.9buffer were selectively released from their binding sites of LH2 of Rhodopseudomonas palustris(P-LH2).The authors acquired two components using molecular sieve chromatography.Free BChl of one component was not removed and self-assembled to P-LH2.The other removed free BChl and B850-only P-LH2 was constructed.B850 unchanged after P-LH2 was incubated.P-LH2αandβsubunits have different molecular weights,but those of A-LH2 are in the contrary.It is concluded that B850-only P-LH2 is more stable than A-LH2.The enigmatic split of the B800 absorption band was not observed in these LH2,but we acquired two kinds of B800-released LH2 from Rhodopseudomonas palustris.The authors' results may provide a new light to separate homogeneous Apoprotein LH2.国家海洋公益性行业科研专项项目(201505026); 国家自然科学基金项目(31070054;31270106); 福建省自然科学基金项目(2012J01136); 近海海洋环境科学国家重点实验室(厦门大学)高级访问学者基金项目(MELRS0907)资

Identification and characterization of a purple sulfur bacterium from mangrove with rhodopin as predominant carotenoid

【目的】挖掘我国海洋紫色硫细菌物种资源、深入理解紫色硫细菌在红树林生态系统中的作用。【方法】采用琼脂振荡稀释法、显微技术、紫外可见吸收光谱法、TlC、HPlC和MS法。【结果】从福建泉州洛阳桥红树林潮间带泥水样分离获得一株细胞内含多个硫粒的细菌菌株,光合内膜呈囊状、含细菌叶绿素A和螺菌黄质系类胡萝卜素,结合16S rrnA基因序列分析和系统发育分析,表明该菌株属于海洋着色菌属(MArICHrOMATIuM)。该菌株细胞球杆状;最适PH范围5.7-6.7;最适盐度范围2%-3.5%;温度范围20℃-35℃;能耐受3.6 MMOl/l硫化物;主要积累玫红品类胡萝卜素,而不是螺菌黄质;3种细菌叶绿素组分中,一种为bCHl AP,另2种未见报道;不需要生长因子;可光同化固定CO2、能很好利用多种有机酸盐、多价态氮化物和硫化物,尤其能利用柠檬酸、葡萄糖、蔗糖、果糖和丙醇;对氯霉素、头孢唑林、苯、对羟基联苯、恩诺沙星、啶虫脒、HgCl2和CdCl2的IC50值分别为70、100、20、20、3、170、5 Mg/l和25 Mg/l。【结论】该菌株是一株轻度耐酸、高含玫红品类胡萝卜素的紫色硫细菌,被鉴定为MArICHrOMATIuM grACIlE新菌株,编号yl28。该菌株具有广泛利用多种碳源、氮源和硫源物质的能力,对抗生素氯霉素和头孢唑啉、农药啶虫脒、重金属汞和镉具有较强耐受性,对抗生素恩诺沙星较敏感。[Objective]To exploit resources of purple sulfur bacteria in China and further investigate its response mechanism to ecological environment of mangrove.[Methods] Repeated agar shake dilution method,microscope techniques,UV-Vis absorption spectra,thin layer chromatography,HPLC and MS were used.[Results] We isolated a purple sulfur bacterium,designated as strain YL28,from a intertidal sediment sample collected from inshore mangrove near Luoyang Bridge of Quanzhou city,Fujian Province of China.Cells were ovoid to rod shaped,0.5 μm-1 μm × 2 μm-3 μm.Color of cell suspensions was reddish-brown.It possessed vesicular intracytoplasmic membrane structures,contained rhodopin and phytylated bacteriochlorophyll a as well as the other two novel bacteriochlorophyll a intermediates.The optimum growth was at 2%-3.5% NaCl,pH 5.7-6.7 and 20℃-35℃.Photoautotrophically growth anaerobically in the light with sulphide,sulphur,thiosulfate,sulfite as electron donor.Globules of S0 distributed inside the cells.Photoheterotrophic growth with various organic substrates,especially citrate,glucose,sucrose,fructose and propanol in the presence of sulfide.Nitrogen sources: ammonium salts,N2,urea,glutamate,nitrate and nitrite.Vitamins were not required.Qualitative assessment of IC50 values of chloromycetin,cefazolin,benzene,hydroxy biphenyl,enrofloxacin,acetamiprid,mercuric chloride and cadmium chloride were 70,100,20,20,3,170,5 mg/L and 25 mg/L,respectively.[Conclusion]Based on phenotype characteristics and 16S rRNA gene sequence similarity of 99% to M.gracile,strain YL28 was identified as novel isolate of M.gracile despite its different physiological characteristics with respect to the species of M.gracile.The organism is possessed of slightly acid tolerance,higher amount of carotenoid of rhodopin and tolerance towards certain antibiotics,pesticide as well as heavy metals.国家自然科学基金(31070054);福建省自然科学基金(2010J01209);中国科学院城市环境与健康重点实验室基金(KLUEH201005)---