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。     

浑球红假单胞菌(Rhodopseudomonas sphaeroides)色素基因的突变对生长速率及光合固氮的影响

浑球红假单胞菌Rps.sphaeroides 6128经甲基磺酸乙酯诱变处理,分离获得23株色素突变种。不具有细菌叶绿素a和类胡萝卜素的无色突变株不能光养生长,蓝绿突变株305不含带色的类胡萝卜素,但能光养生长,其世代时间比亲本株长5倍左右,而且,没有还原乙炔和放氢的固氮酶活性。绿色突变株309缺失球形烯和球形烯酮。当光照强度从3000lx增加到4000lx时,绿色突变株与亲本株生长速率之差由5.3小时缩短为0.3小时,其光合固氮和光合放氢的活性分别为亲本株的30％和45％。各菌株ATP的含量因所含色素成份不同而异。在指数生长期,蓝绿突变株305的ATP含量只有亲本株的8％,绿色突变株309的ATP含量为亲本株的32％,各色素变种的固氮能力与它们菌体ATP的含量相关。类胡萝卜素在为光合固氮提供能源中起着重要的作用。     

浑球红假单胞菌GOGAT缺失株中依赖Z—酮戊二酸的固氮酶诱导

2—酮戊二酸加于限量氮培养基中后,引起泽球红假单胞菌谷氨酸合成酶(GOGAT)缺失菌株(asm~-,Nif~-)固氮酶的诱导表达,诱导生成的固氮酶的活性随着培养时间的延长而下降。此时如果加入谷氨酸,固氮酶活性又重新出现,而谷氨酸通常是阻遏GOGAT缺失菌株固氮酶表达的。诱导出的固氮酶与野生菌株固氮酶有相同的调节方式,但前后两次出现的固氮酶活性在氨的敏感性上有差异。此外,在GOGAT缺失菌株内含有较高的谷氨酰胺库,同时谷氨酸胺合成酶(GS)的腺苷酸化状态也较野生型菌株高。     

浑球红假单胞菌氨同化途径的研究

本文测定了浑球红假单胞菌(Rhodobacter sphaeroides)菌株601谷氨酰胺合成酶(GS)、谷氨酸合酶(GOGAT)、谷氨酸脱氢酶(GDH)和丙氨酸脱氢酶(ADH)的活性。低氨时,GS/GOGAT活力高,GDH活力低,高氨时,GS/GOGAT活力低,GDH活力高。在以分子氮或低浓度氨为氮源的培养条件下,加入GS抑制刑MSX(L—methionine—DL—sulphoximine),细菌生长受到抑制。但是,生长在以谷氨酸为氮源的细菌则不受影响。上述结果表明,浑球红假单胞菌菌株601氨同化是通过GS/GOGAT途径和GDH途径。     

浑球红假单胞菌及其谷氨酸合成酶突变株氢酶活性和合成

浑球红假单胞菌野生型菌株的氢酶表达被有机碳、氮底物所抑制。在光照和黑暗时,氧浓度变化对氢酶的作用不同,但高氧浓度都阻遏氢酶的表达。微量Ni~(2+)能专一性地促进氢酶活性,固氮酶的产氢也可以调节氢酶的表达水平。该野生菌株的GOGAT突变株缺乏固氮酶和氢酶活性,在加入谷氨酰胺合成酶抑制剂MSX后,固氮酶和氢酶以相关联的方式合成出来,固氮酶产生的氢看来诱导了氢酶的合成。然而在固氮酶不表达的情况下,外源氢也可诱导氢酶的合成。     

缺乏固氮活性的浑球红假单孢菌谷氨酸合成酶突变株

光合细菌浑球红假单孢菌601菌株,经过NlG诱变获得谷氨酸缺陷型204菌株。生化分析表明,突变株204缺乏谷氨酸合成酶活性(GoGAT),谷氨酰胺合成酶话性比亲株低,用放氢和乙蛱还原法未测出固氮酶活性。此外,突变株204不能在多种氮源上生长,例如：氨、尿素、组氨酸、丝氨酸、精氨酸和腺嘌呤,表现出氮素代谢上多效缺陷的表型。从含氨基础培养基或充氮气的低限培养基上分离回复子,自发回复突变频率均为2×10一·回复子的固氮酶和GS活性恢复到亲株的水平,同时重新获得利用上述各种氮源的能力。胞内游离氨基酸库分析表明,突变株的谷氨酰胺含量是亲株的16倍,外源谷氨酰胺加入培养基也抑制固氮活性。从上述结果推论,浑球红假单胞菌具有对固氮酶调节高度敏感的反馈系统,它随代谢中间产物而变化,胞内谷氨酰胺的含量是固氮酶活性反馈调节的关键成份。     

丙酮酸对浑球红假单胞菌突变株(GOGAT~-Nif~-)固氮酶活性的调节

固氮无效的浑球红假单胞菌GOGAT突变株经丙酮酸诱导产生固氮酶活性。固氮酶比活随丙酮酸浓度增加而提高,同时依赖于蛋白质合成的菌体生长。丙酮酸产生固氮酶时氮源Glu的浓度高达60 mmol/L。液相色谱分析表明,丙酮酸诱导固氮酶活性的形成与胞内Gln耗竭有关而与Asn无关。用丙酮酸代替苹果酸诱导,突变株向胞外分泌的游离氨大大减少。丙酮酸诱导时变种谷氨酰胺酶比活较高,它可能参与胞内Gln的分解。     

Derepressive effect of NH on hydrogen production by deleting the glnA1 gene in Rhodobacter sphaeroides

Purple non‐sulfur (PNS) bacteria produce hydrogen by photofermentation of organic acids in wastewater. However, NH in wastewater may inhibit hydrogen synthesis by repressing the expression and activity of nitrogenase, the enzyme catalyzing hydrogen production in PNS bacteria. In this study, the Rhodobacter sphaeroides 6016 glnA gene encoding glutamine synthetase (GS) was knocked out by homologous recombination, and the effects on hydrogen production and nitrogenase activity were examined. Using 3 mM glutamine as the nitrogen source, hydrogen production (1,245–1,588 mL hydrogen/L culture) and nitrogenase activity were detected in the mutant in the presence of relatively high NH concentrations (15–40 mM), whereas neither was detected in the wild‐type strain under the same conditions. Further analysis indicated that high NH concentrations greatly inhibited the expression of nifA and nitrogenase gene in the wild‐type strain but not in the glnA1^? mutant. These observations suggest that GS is essential to NH repression of nitrogenase and that deletion of glnA1 results in the complete derepression of nitrogenase by preventing NH assimilation in vivo, thus relieving the inhibition of nifA and nitrogenase gene expression. Knocking out glnA1 therefore provides an efficient approach to removing the inhibitory effects of ammonium ions in R. sphaeroides and possibly in other hydrogen‐producing PNS bacteria. Biotechnol. Bioeng. 2010;106: 564–572. © 2010 Wiley Periodicals, Inc.     

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.     

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.     

浑球红细菌谷氨酰胺合成酶基因(glnA)及PⅡ蛋白基因(glnB)的序列分析

测定了浑球红细菌的glnA和／glnB基因DNA序列,共2707nt。其中glnA基因编码区为1401nt,编码467个氨基酸;glnB基因编码区为336nt,编码112个氨基醚。DNA序列的G＋C百分含量为65％,它们的密码子第三位GC利用率高达89．1％。在氨基酸序列上,GS酶和PⅡ蛋白与其它不同属的细菌间有较好的同源性,尤其是固氮类细菌间同源性较高。