球形红杆菌L2的生物学特性及其应用

从淀粉废水中分离获得一株光合细菌,经形态特征,培养特征,生理生化特征及Ｇ＋Ｃｍｏｌ％含量等生物学特性分析,确定为球形红杆菌（Ｒｈｏｄｏｂａｃｔｅｒｓｐｈａｅｒｏｉｄｅｓ）Ｌ２。该菌应用于淀粉废水处理,ＣＯＤ去除率达９５．７％发酵产类胡萝卜素,产量达２９５ｍｇ／Ｌ;作为饲料添加剂进行肉鸡饲喂,增重１６．４０％。     

Identification and characterization of a GroEL homologue in Rhodobacter sphaeroides

A protein closely related to the Escherichia coli GroEL protein has been isolated from Rhodobacter sphaeroides. Native and SDS-polyacrylamide gel electrophoresis of this protein have shown that it is present in the cell as a multimeric complex of Mr 670,000 which is composed of a monomer of Mr 58,000. Antisera raised against the Mr 58,000 polypeptide have been shown to cross-react with GroEL and the alpha subunit of the pea plastid chaperonin. The N-terminal amino acid sequence of the Mr 58,000 polypeptide is identical to that of GroEL at 15 of 19 residues and is also closely related to the alpha subunit of the pea plastid chaperonin, though less so to the beta subunit.     

Chromate reduction by Rhodobacter sphaeroides

Rhodobacter sphaeroides grew in the presence of up to 43 μM chromate and reduced hexavalent chromium to the trivalent form under both aerobic and anaerobic conditions. Reduced chromium remained in the external medium. Reductase activity was present in cells of R. sphaeroides independent of whether chromate was present or not in the growth medium. The reducing activity was found in the cytoplasmic cell fraction and was dependent on NADH. The chromate-reducing enzyme was purified by anion exchange, hydroxyapatite and hydrophobic interaction chromatography, and gel filtration. The molecular weight of the enzyme was 42 kDa as determined by gel filtration. The optimum of the reaction is at pH 7.0 and 30°C. The enzyme activity showed a hyperbolic dependence on the concentrations of both substrates, NADH and chromate, with a maximum velocity at 0.15 mM NADH. A K _m of 15±1.3 μM CrO₄ ²⁻ and a V _max of 420±50 μmol min⁻¹ mg protein⁻¹ was determined for the enzyme isolated from anaerobically grown cells and 29±6.4 μM CrO₄ ²⁻ and 100±9.6 μmol CrO₄ ²⁻ min⁻¹ mg protein⁻¹ for the one from aerobically grown ones. Journal of Industrial Microbiology & Biotechnology (2000) 25, 198–203. Received 05 January 2000/ Accepted in revised form 27 May 2000     

Non-reciprocal regulation of Rhodobacter capsulatus and Rhodobacter sphaeroides recA genes expression

Abstract The Rhodobacter capsulatus recA gene has been isolated and sequenced. Its deduced amino acid sequence showed the closest identity with the Rhodobacter sphaeroides RecA protein (91% identity). However, the promoter regions of both R. capsulatus and R. sphaeroides recA genes are only 64% similar. An Escherichia coli -like LexA binding site was not present in the upstream region of the R. capsulatus recA gene. Nevertheless, the R. capsulatus recA gene is inducible by DNA damage in both hetero- and phototrophically growing conditions. The R. capsulatus recA gene is poorly induced when inserted into the chromosome of R. sphaeroides , indicating that the recA gene of both bacteria possess different control sequences despite their phylogenetically close relationship.     

Light-dependent transformation of anthranilate to indole by Rhodobacter sphaeroides OU5

Rhodobacter sphaeroides OU5 transformed anthranilate (2 mM) to an indole (0.7 mM) in a light-dependent process. Photobiotransformation was enhanced by tricarboxylic acid cycle intermediates and the indole formed was identified as 2,3 dihydroxy indole. Journal of Industrial Microbiology & Biotechnology (2000) 24, 219–221. Received 16 September 1999/ Accepted in revised form 20 December 1999     

浑球红假单胞菌（Rhodobacter sphaeroides）谷氨酸合酶的纯化及性质

浑球红假单胞菌菌株601经超声击碎,粗提液通过Triton处理,硫酸铵沉淀,DE—52和DEAE—sephadex A—50柱层析及 Seqhadex G—200凝胶过滤等步骤,将谷氨酸合酶(GOGAT)分离纯化,在聚丙烯酰胺凝胶电泳上呈现一条带。GOGAT表观分子量约为138 kD。该酶最大光吸收在278,375,450 nm和475 nm处,表明GOGAT可能是一种黄素蛋白。纯化的GOGAT对其底物 Gln,α—酮戊二酸和NADPH的表观K_m值分别为830,150和6μmol/L。反应产物Gln和NADP,几种氨基酸对GOGAT活力有不同程度的抑制作用,Gln类似物DON对GOGAT活力有强烈的抑制作用。     

Optimization of activation conditions of Rhodobacter sphaeroides in hydrogen generation process

AIMS: To examine the effects of the culture age, illuminance intensity and changes in these parameters during activation on hydrogen generation process carried out by purple nonsulfur Rhodobacter sphaeroides bacteria. METHODS AND RESULTS: The following parameters were determined in all experiments: the amount of hydrogen evolved (measured using gas chromatography), biomass increase as dry mass, pH values and consumption of organic substance as chemical oxygen demand (COD). The medium used in the process of activation and hydrogen generation contained malic acid (15 mmol) and sodium glutamate (2 mmol). The optimum age of bacteria was 12-24 h and the best intensity of illuminance was found to be 5 cd sr m-2 on activation and 9 cd sr m-2 on hydrogen generation. These conditions provided hydrogen evolution of 1.39 l l-1 of the medium with the highest specific hydrogen production of 0.146 l H2 l-1 medium h-1 g-1 inoculum. An increase in the illuminance intensity resulted in a slight inhibition of the process. CONCLUSIONS: The activation stage of bacteria has a significant effect on the parameters of hydrogen photogeneration. The optimization of the activation stages allowed a shortening of the time of hydrogen generation and of the period after which hydrogen evolution starts. SIGNIFICANCE AND IMPACT OF THE STUDY: An innovative method of bacteria activation before the initiation of the hydrogen generation process has been used to optimize this process. The shortening of the process duration as well as the twice higher hydrogen yield can help in the designing of other systems (including also those operating under solar irradiation) in which R. sphaeroides bacteria are to be applied.     

含硒类球红细菌的研究

为了确定类球红细菌转硒培养的最佳条件 ,研究了无机硒的加入浓度、时间以及分批补料培养对菌体生长和转硒效率的影响。实验表明 ,无机硒的浓度低于 1× 10 -5mol/L时 ,对类球红细菌的生长基本没有影响 ,并能将6 3.9%的无机硒转化为有机硒。转硒的最佳时间是在接种后 12h左右 ,此时转硒效率最高。实验还表明 ,分批补料培养可以提高菌体浓度 ,可使转硒效率和绝对量增加。体内试验表明 ,用 5mL/kgbw和 10mL/kgbw剂量的含硒类球红细菌灌养小鼠 ,可以使其全血GSH Px酶活性提高 2 0 .9%和 2 5 .5 % ,使其血清丙二醛 (MDA)含量降低2 1.0 %和 2 3.2 %。     

类球红细菌发酵生产类胡萝卜素条件优化

本文对类球红细菌3757产类胡萝卜素进行了发酵条件优化,结果得到了较优的培养基组成:葡萄糖2%,苹果酸钠0.5%,酵母浸粉1.3%,硫酸铵0.9%,磷酸氢二钾0.09%,磷酸二氢钾0.06%,生长因子溶液1%,p H 8.0;其中,生长因子溶液配方:维生素B1 0.1%,烟酰胺(VPP)0.1%,生物素0.0016%。较优培养条件为:接种量5%,转速200 r/min,种龄24 h,发酵温度32℃,发酵时间40 h。优化后类胡萝卜素产率较优化前提高了76.2%。     

Photobiotransformation of indole to its value-added derivatives by Rhodobacter sphaeroides OU5

A purple non-sulfur anoxygenic phototrophic bacterium, Rhodobacter sphaeroides OU5 was able to photobiotransform indole in the presence of various organic substrates to its value-added derivatives tryptophan, tryptamine, indole lactic acid and indigo, which are of high commercial value. The product formed varied with the precursors provided in the medium. Received 21 August 1997/ Accepted in revised form 10 January 1998     

类球红细菌SOD发酵条件优化

本文对类球红细菌3757产SOD进行了发酵条件优化,结果得到了较优的培养基组成(g/L):苹果酸3,胰蛋白胨4,磷酸氢二钾0.9,磷酸二氢钾O.6,硫酸镁0.2,无水氯化钙0.075,硫酸亚铁0.012,EDATA 0.02,微量元素溶液10 mL,生长因子溶液10 mL,pH 7.5。其中,微量元素溶液配方(g/L):硼酸2.8,硫酸锰1.6,钼酸钠0.76,硫酸锌0.24,硫酸铜0.04;生长因子溶液配方(g/L):维生素B_1 1,烟酰胺(VPP)1,生物素0.016,对氨基苯甲酸1。较优培养条件为:接种量5%,转速150 r/min,种龄24 h,发酵温度32℃,发酵时间24 h。优化后酶活力较优化前提高了88.0%。     

Water clusters in the reaction centre of Rhodobacter sphaeroides

The structure of the reaction centre from Rhodobacter sphaeroides has been refined up to 2.4 Å resolution. Several clusters of firmly bound water molecules were found proximal to the primary and secondary quinones. They represent putative pathways for proton transfer.     

光合菌Rhodobacter sphaeroides 601hupT基因的克隆与功能分析

从紫色非硫光合细菌Rhodobacter sphaeroides 601的吸氢酶(hup)基因簇中,克隆了hupT基因,并对该基因进行了测序,分析了由其推测的氨基酸序列的同源性.hupT基因全长1 332 bp,编码一分子量约为48 23 kD的蛋白.将hupT基因引入大肠杆菌进行了体外表达.纯化基因产物HupT,并进行HupT的自身磷酸化分析.结果表明,HupT属于双组份调节系统中的组氧酸蛋白激酶.将hupT基因导入光合菌Rhodobacter capsulatus吸氢酶负调节基因突变株BSE8后.野生型吸氢酶的表型得以恢复,说明所克隆的R.sphaeroides 601中的hupT基因在吸氢酶的表达中起负调节作用.     

氨和谷氨酰胺对浑球红假单胞菌（Rhodobacter sphaeroides）固氮酶...

浑球红假单胞菌菌株601具有迅速对外源氨作出“关闭”固氮酶活性的反应。氨对固氮酶的抑制作用,可被谷氨酰胺合成酶(GS)抑制剂MSX所解除。反之,加入Glu代谢抑制剂DON,可延长氨抑制的持续时间。Gln对固氮酶也有抑制作用。在脱腺苷化GS的透性细胞中,加入Gln可抑制固氮酶活性,同时,GS腺苷化状态提高。然而,氨则对透性细胞的固氮酶活性和GS腺苷化状态没有影响。     

Effects of growth conditions on production of methyl selenides in cultures of Rhodobacter sphaeroides

Rhodobacter sphaeroides 2.4.1 exposed to selenate or selenite produced volatile selenium compounds. Total amounts of dimethyl selenide, dimethyl diselenide, dimethyl sulfide and dimethyl disulfide in culture medium and headspace were determined. The highest selenate volatilization occurred in the late stationary phase of growth. However, cultures deprived of light in the stationary phase of growth produced much less of the volatile organo-selenium compounds. Lower culture pHs increased the rate of selenium volatilization. Low sulfate concentration limited biomass production and selenium volatilization; high sulfate concentrations had an enhancing effect on the release of organo-selenium compounds. Cultures of R. sphaeroides reacted very differently to amendments with increasing amounts of selenate and selenite. Only small amounts of selenite were volatilized; meanwhile high amounts of methylated selenides were found in selenate-poisoned cultures. Received 03 February 1997/ Accepted in revised form 16 May 1997     

Analysis of the expression of the Rhodobacter sphaeroides lexA gene

The regulation of the Rhodobacter sphaeroides lexA gene has been analyzed using both gel-mobility experiments and lacZ gene fusions. PCR-mediated mutagenesis demonstrated that the second GAAC motif in the sequence GAACN₇GAACN₇GAAC located upstream of the R. sphaeroides lexA gene is absolutely necessary for its DNA damage-mediated induction. Moreover, mutagenesis of either the first or the third GAAC motif in this sequence reduced, but did not abolish, the inducibility of the R. sphaeroides lexA gene. A R. sphaeroides lexA-defective (Def) mutant has also been constructed by replacing the active lexA gene with an inactivated gene copy constructed in vitro. Crude extracts of the R. sphaeroides lexA(Def) strain are unable to form any protein-DNA complex when added to the wild-type lexA promoter of R. sphaeroides. Likewise, the R. sphaeroides lexA(Def) cells constitutively express the recA and lexA genes. All these data clearly indicate that the lexA gene product is the negative regulator of the R. sphaeroides SOS response. Furthermore, the morphology, growth and viability of R. sphaeroides lexA(Def) cultures do not show any significant change relative to those of the wild-type strain. Hence, R. sphaeroides is so far the only bacterial species whose viability is known not to be affected by the presence of a lexA(Def) mutation. Received: 31 January 2000 / Accepted: 3 April 2000     

光合细菌Rhodobacter sphaeroides glnBlacZ 融合子的构建与表达

亚克隆了Ｒｈｏｄｏｂａｃｔｅｒ ｓｐｈａｅｒｏｉｄｅｓ ｇｌｎＢ启动子,以ｐＭＰ２２０ 为载体构建成ｇｌｎＢｌａｃＺ融合子。将ｇｌｎＢｌａｃＺ、ｎｉｆＨｌａｃＺ、ｎｉｆＡｌａｃＺ分别导入Ｒ． ｓｐｈａｅｒｏｉｄｅｓ 谷氨酸合酶突变株ｇｌｔＢ－ 、ｇｌｔＤ－ 和野生型菌株中,分析了突变对固氮基因转录表达的影响。试验证明,在ｇｌｔＢＤ 突变株中ｎｉｆＨ 的表达受阻遏,ｎｉｆＡ 表达水平很低。这证明ｇｌｔ 基因的突变引起固氮酶结构基因和固氮正调节基因的转录被阻遏,而ｇｌｎＢ 基因的表达几乎不受影响。试验还测定了环境中结合态氮和有机酸等信号分子对ｇｌｎＢ 和ｎｉｆＨ 表达的影响,发现加入氨或谷氨酰胺后,ｎｉｆＨ 的表达受到明显的阻遏作用,ｇｌｎＢｌａｃＺ的β半乳糖苷酶活性虽下降３０ ％ 左右但不随结合态氮浓度升高而变化,仍维持在一个较高的水平。α酮戊二酸和丙酮酸对ｎｉｆＨ 的表达有部分去阻遏作用而对ｇｌｎＢ的表达无诱导作用     

浑球红细菌（Rhodobacter sphaeroides）中氢化酶正调节基因hupR的克隆及功能分析

从光合细菌Rhodobacter sphaeroides基因文库中分离出含有氢化酶基因簇（hup）的粘粒cosmid 1后,亚克隆了R.sphaeroides的氢化酶调节基因hupR,测定了hupR的核苷酸序列,并完成了氢化酶基因簇的部分物理图谱。实验结果表明,hupR基因全长1476bp,编码的HupR基因分子量约为54.031kD(EMBL接受号：A243734)。与R.capsulatus中HupR相比,同源性高达73％。同源性比较结果表明,它属于双组分调节系统中受体蛋白。hupR基因在E.coli中进行了体外表达,纯化后测定得到的HupR蛋白 分子量大小与hupR基因推测的分子量大小一致。通过双交换,将卡那霉素抗性基因插入hupR基因,获得丧失氢化酶活性的hupR^-的突变株,KR5和KR7。hupS∷lacZ融合基因在野生型中的转录表达量是在该突变株中的7－9倍。将hupR基因置于弱启动子pfru下游,构建了质粒pNRC3,并将其导入hupR^-的突变株,可使突变株重新获得氢化酶活性。以上结果说明,HupR蛋白对氢化酶的转录表达起着正调节作用。在HupR蛋白的磷酸化区域进行定点和缺失突变。不影响HupR激活氢化酶基因的表达,推测HupR蛋白是在非磷酸化的状态下起调节作用的。     

The solution structure of the PufX polypeptide from Rhodobacter sphaeroides

PufX organises the photosynthetic reaction centre–light harvesting complex 1 (RC–LH1) core complex of Rhodobacter sphaeroides and facilitates quinol/quinone exchange between the RC and cytochrome bc₁ complexes. The structure of PufX in organic solvent reveals two hydrophobic helices flanked by unstructured termini and connected by a helical bend. The proposed location of basic residues and tryptophans at the membrane interface orients the C-terminal helix along the membrane normal, with the GXXXG motifs in positions unsuitable as direct drivers of dimerisation of the RC–LH1 complex. The N-terminal helix is predicted to extend 40 Å along the membrane interface.