Studies on electron-transferring mechanism in an nitrite-reducing system containing Cu-proteins

本研究拟通过对粪产碱菌S-6反硝化过程中涉及的酶和基因进行特性分析，阐明其反硝化机理。由于细胞色素c在这一过程中通常是电子供体，因此从粪便酵母菌S-6菌株中分离纯化了一种与血红素试剂反应的蛋白质，并测定了其N端氨基酸序列。该序列与几个反硝化细菌的一氧化氮还原酶(NOR)小亚基的序列高度相似。用寡核苷酸探针法克隆含有该亚单位编码基因的DNA片段。核苷酸序列分析表明，克隆片段含有9个开放阅读框。在同源性分析的基础上，我们初步将开放阅读框命名为DNR、orf1、ORF2、Noro、norP、NORC、Norb、norQ和NorD。在Noro、NORC和norQ的上游，发现了类似于FNR识别序列的短回文序列，其基序为TTGATxxxxATCAA。考虑到FNR是在厌氧条件下表达的基因的转录激活因子，这表明这些基因的表达在无氧转录阶段受到控制，这些基因至少以三个不同的操纵子的形式转录。我们构建了几个NOR基因的表达载体，并将它们导入到大肠杆菌JM105中。经IPTG诱导后培养30h，对其膜组分进行SDS-PAGE和血红素染色。虽然CBB染色没有发现特定蛋白的积累，但通过血红素染色检测到Norb(38 KDa)和NORC(17 KDa)的产生，但在任何一种大肠杆菌细胞中都没有检测到NOR活性。这表明存在与NOR蛋白激活有关的其他基因或其产物。

We planned to elucidate the denitrification mechanism ofAlcaligenes faecalis S-6 by characterizing enzymes and genes involved in the denitrification process. Since a cytochrome c is often served as an electron donor in this process, a protein which reacted with heme-staining reagent was partially purified from A.faecalis S-6, and its N-terminal amino acid sequence was determined. The sequence was highly similar to those of small subunits of nitric oxide reductases (NOR) from several denitrifying bacteria. DNA fragments containing the gene coding for the subunit was cloned by oligonucleotide-probing method. Nucleotide sequencing revealed that the cloned fragments contained nine open reading frames. Base on the homology analysis we tentatively designated the open reading frames as dnr, orf1, orf2, norO, norP, norC, norB, norQ, and norD.Just upstream of the norO, norC, and norQ, short palin-drome sequences similar to FNR-recognition sequence with a motif of TTGATxxxxATCAA were found. Considering that FNR was a transcriptional activater for genes expressed under the anaerobic conditions, this suggested, that expression of the genes was controled in the stage of transcription in the absence of oxygen, and these genes were transcribed as at least three different operons. We constructed several plasmids for the expression of nor genes and introduced them into Escherichia coli JM105. E.coli cells carrying the plasmids were cultured for 30 h after IPTG induction, and their membrane fractions were subjected to SDS-PAGE and heme staining. Although no accumulation of specific proteins was not seen by CBB staining, production of NORB (38 kDa) and NORC (17 kDa) was detected by heme staining.However, no NOR activity was detected in any fraction of E.coli cells. This suggested the presence of some other genes or their products involved in the activation of NOR proteins.