光合菌Rhodobacter sphaeroides 601 hupT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1332bp,编码一分子量约为４８.23kD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氨酸蛋白激酶。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,所克隆的R.sphaeroides 601的hupT基因在吸氢酶的表达中起负调节作用。     

光合菌SDH2 hupT基因的突变与吸氢酶表达

利用三亲本杂交将自杀质粒ｐＳＥ８引入光合细菌Ｒｏｄｏｂａｃｔｅｒ ｓｐ．ＳＤＨ２０菌株,经过质粒上插入了ｋａｎ＾Ｒ基因的ｈｕｐＴ基因片段与受体基因组同源双交换,构建成ｈｕｐＴ插入突变株ＳＤＨＴ１和ＳＤＨＴ２。     

浑球红细菌Rhodobacter sphaeroides吸氢酶（hup）基因簇的分离 …

以Ｒｈｏｄｏｂａｃｔｅｒ ｃａｐｓｕｌａｔｕｓ的ｈｕｐＳ‘Ｌ基因ＤＮＡ片段为探讨,通过Ｓｏｕｔｈｅｒｎ杂交,从构建的Ｒｈｏｄｏｂａｃｔｅｒ ｓｐｈａｅｒｏｉｄｅｓ ６０１基因库中调取ｈｕｐ基因。阳性克隆Ｃｏｓｍｉｄ１可与Ｈｕｐ突变株ＪＰ９１,ＲＣＣ８以及ＢＳＥ８互补,而Ｃｏｓｍｉｄ３和Ｃｏｓｍｉｄ９只能与ＢＳＥ８互补。试验所产生的接合转移子均恢复了吸氢酶的活性和自养生长能力。     

浑球红细菌Rhodobacter sphaeroides吸氨酶(hup)基因簇的分离与鉴定

以Rhodobactercapsulatus的hopS’L基因DNA片段为探针,通过Southem杂交,从构建的Rhodobactersphaeroides601基因库中调取hup基因。阳性克隆Cosmid1可与Hup突变株JP91（HupS－）、RCC8（HupR-）以及BSE8（HupT-）互补,而Cosmid3和Cosmid9只能与BSE8互补;试验所产生的接合转移子均恢复了吸氢酶的活性和自养生长能力、将Cosmidl的3．5kbPstⅠ和4．5kbBamHⅠ片段分别亚克隆到pWY11和pWY10中。pWY11和pWY10的部分DNA序列与R．capsulatus中的相关基因具有很高的同源性,从而证明了所得到的Cosmid1确实含有R．sphaeroides的基因簇。     

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。     

不产氧光合细菌Rhodobacter sphaeroides产氢影响因子研究

对不产氧光合细菌球形红细菌Rhodobacter sphaeroides产氢的影响因子进行了初步研究。结果表明,处于不同生长期的球形红细菌接种后的产氢速率略有差异,稳定期的菌株的产氢能力相对较低。苹果酸钠、乳酸钠、丙酮酸钠和葡萄糖都是球形红细菌产氢的良好碳源,这表明球形红细菌具有利用食品工业等高浓度废水为底物产氢的可能性。以葡萄糖和谷氨酸钠为C源和N源产氢时,适宜的葡萄糖浓度在25~50mmol/L之间,谷氨酸钠浓度在2~10mmol/L之间。球形红细菌产氢的适宜pH值在7.0~8.0范围内,酸性环境明显不利于该菌的催化放氢,适宜的温度在30~35℃范围内。光照强度在5000~7000lx之间适合于产氢。球形红细菌的固氮酶活性和放氢活性之间呈正相关性。吸氢酶虽然可在无固氮酶和无放氢活性的状态下独立表达,但多数情况下仍受氢气浓度的调节。以氮气为氮源时,固氮酶活性和放氢活性较低,铵的浓度高于0.5mmol/L时,固氮酶活性完全受到抑制,进而抑制产氢。     

巴西固氮螺菌中吸氢酶基因同源性的分子检测

通过TTC(2 ,3,5 氯化三苯基氮唑 )实验筛选出 12株能产生吸氢酶蛋白 (Hup+ )的巴西固氮螺菌 (Azospir rilumbrasilense)菌株。用Qiagen柱分离提纯含豌豆根瘤菌的hup基因片段的质粒 pHVT10 9和pHVT115 ,并用地高辛标记法标记pHVT115 ,与 12株Hup+ 巴西固氮螺菌的总DNA进行斑点杂交 ,结果显示pHVT115所含的吸氢酶基因 (hup基因 )片段与巴西固氮螺菌的大部分菌株的hup基因同源性不强。这一结果表明hup基因在固氮生物中存在着遗传多样性 ,异源hup基因探针不一定都适宜于探测hup基因。     

玉米淀粉分支酶基因启动子的克隆与功能分析

以玉米品种“吉糯1号”的基因组DNA为模板,通过PCR扩增得到玉米淀粉分支酶基因的启动子序列,克隆到pMD18-TVector上,经测序,该启动子大小为934bp。与已报道的序列比较仅有14个核苷酸发生改变,同源性为98.5%。用该启动子取代植物表达载体pBI121的35S启动子,与GUS基因编码区连接,构建成融合质粒pSBE-GUS。经农杆菌介导法转化烟草,获得了转基因植株。GUS活性检测结果表明,由该启动子序列引导的GUS基因能在种子中表达,而在其他组织中表达微弱或未表达,证实该启动子具有种子特异性表达的功能。     

与人体ε-珠蛋白基因5''''旁侧正调控元件(ε-PREI)专一结合的调控因子的初步研究

人体ε－珠蛋白基因５′旁侧ＤＮＡ序列对该基因时空表达与调控起着十分重要的作用。本文运用凝胶电泳阻抑法,ＤＮａｓｅＩ足印法和Ｓｏｕｔｈｗｅｓｔｅｒｎｂｌｏｔ分析法发现在胚胎型红白血病细胞株Ｋ５６２细胞中有一个特异的核蛋白因子（简称ε－ＳＳＰ,其分子量大约为８０ｋＤ）,它能专一地与人体ε－珠蛋白基因５′旁侧一个红细胞专一和发育时期特异的正调控元件（ε－ＰＲＥＩＩ,－４４６ｂｐ到－４１９ｂｐ）相结合。竞争实验表明该因子与ε－ＰＲＥＩＩ的结合能被人体ε－珠蛋白基因启动子区ＤＮＡ片段（－１７７ｂｐ到＋１ｂｐ）所竞争;同时也能被人体β－类珠蛋白基因远侧端调控元件ＬＣＲ中的ＤＮａｓｅＩ超敏感点Ｉ核心区ＤＮＡ片段（－１０９６５ｂｐ到－１０６８１ｂｐ）与超敏感点ＩＩ核心区ＤＮＡ片段（－１４９９３ｂｐ到－１４７１８ｂｐ）所竞争。我们的结果提示了ε－ＳＳＰ不仅是一个与红细胞专一性和发育时期特异性相关的反式调控因子,而且它可能介导远侧端调控元件（ＬＣＲ）与近侧端调控元件启动子之间的相互作用,共同调节ε－珠蛋白基因在胚胎期的表达。     

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.     

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     

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.     

球形红细菌厌氧降解2,4-二硝基甲苯

【目的】研究不同环境条件对2,4-二硝基甲苯(2,4-DNT)生物降解的影响。【方法】采用光合细菌球形红细菌在温度为30 °C的光照培养箱中厌氧降解2,4-DNT,并用高效液相色谱仪测定其浓度。【结果】去除2,4-DNT的最佳条件是初始浓度40 mg/L、初始pH 7.0和接种量15%。另外,2,4-DNT在菌体延滞期被细胞吸收,然后在指数期作为碳源被降解。2,4-DNT的去除率在72 h达到98.8%。从液相色谱图中观察到有2种中间代谢产物,但在120 h内产物被逐渐降解。2,4-DNT的去除动力学符合一级速率模型。【结论】不同条件下2,4-DNT的去除率表明球形红细菌能有效降解2,4-DNT。     

Phosphotransfer in Rhodobacter sphaeroides chemotaxis

The two-component sensing system controlling bacterial chemotaxis is one of the best studied in biology. Rhodobacter sphaeroides has a complex chemosensory pathway comprising two histidine protein kinases (CheAs) and eight downstream response regulators (six CheYs and two CheBs) rather than the single copies of each as in Escherichia coli. We used in vitro analysis of phosphotransfer to start to determine why R.sphaeroides has these multiple homologues. CheA(1) and CheA(2) contain all the key motifs identified in the histidine protein kinase family, except for conservative substitutions (F-L and F-I) within the F box of CheA(2), and both are capable of ATP-dependent autophosphorylation. While the K(m) values for ATP of CheA(1) and CheA(2) were similar to that of E.coli, the k(cat) value was three times lower, but similar to that measured for the related Sinorhizobium meliloti CheA. However, the two CheAs differed both in their ability to phosphorylate the various response regulators and the rates of phosphotransfer. CheA(2) phosphorylated all of the CheYs and both CheBs, whilst CheA(1) did not phosphorylate either CheB and phosphorylated only the response regulators encoded within its own genetic locus (CheY(1), CheY(2), and CheY(5)) and CheY(3). The dephosphorylation rates of the R.sphaeroides CheBs were much slower than the E.coli CheB. The dephosphorylation rate of CheY(6), encoded by the third chemosensory locus, was ten times faster than that of the E.coli CheY. However, the dephosphorylation rates of the remaining R.sphaeroides CheYs were comparable to that of E.coli CheY.