Wu G, Delgado M J, Vargas C, Davies A E, Poole R K, Downie J A
John Innes Centre, Colney, Norwich, UK.
Microbiology (Reading). 1996 Dec;142 ( Pt 12):3381-8. doi: 10.1099/13500872-142-12-3381.
Following Tn5 mutagenesis of Rhizobium leguminosarum biovar viciae, two mutants in one complementation group were identified as being unable to fix nitrogen in pea nodules. Spectroscopic analysis revealed that the mutants had lowered levels of c-type cytochromes and cytochromes aa3, but increased levels of cytochrome d. Cells of the mutants were greatly reduced in their ability to oxidize the artificial electron donor N,N,N',N'-tetramethyl-p-phenylenediamine but membranes prepared from them had increased levels of succinate- and NADH-dependent respiration. NADH oxidation by the mutants was insensitive to the respiratory inhibitor antimycin A, that targets the cytochrome bc1 complex. Molecular analysis of the mutants revealed that they were affected in the cytochrome bc1 complex. One of the mutants contained Tn5 in a gene homologous to that encoding cytochrome c1, and in the other the Tn5 was in DNA homologous to that encoding the cytochrome b component of the cytochrome bc1 complex. Haem staining revealed that haem proteins of M(r)31,000 and M(r)23,000 were absent from membranes from the mutants whereas an additional soluble c-type cytochrome protein of M(r)23,000 was present. We conclude that the larger of these two haem proteins corresponds to cytochrome c1 and, in its absence, the protein of M(r)23,000 does not remain associated with the membrane. Formation of this M(r)23,000 component was specifically blocked in a third respiratory-defective mutant which contained Tn5 in a region of DNA showing homology to a Bradyrhizobium Japonicum gene previously shown to encode the membrane-bound c-type cytochrome CycM. Although the cytochrome bc1 complex is essential for symbiotic nitrogen fixation, the other membrane-bound c-type cytochrome (CycM) is not.
在用Tn5对豌豆根瘤菌蚕豆生物变种进行诱变之后，在一个互补群中鉴定出两个突变体，它们在豌豆根瘤中无法固氮。光谱分析表明，这些突变体中c型细胞色素和细胞色素aa3的水平降低，但细胞色素d的水平升高。突变体细胞氧化人工电子供体N，N，N'，N'-四甲基对苯二胺的能力大大降低，但由它们制备的膜中琥珀酸和NADH依赖性呼吸作用的水平有所提高。突变体对NADH的氧化作用对靶向细胞色素bc1复合体的呼吸抑制剂抗霉素A不敏感。对这些突变体的分子分析表明，它们在细胞色素bc1复合体中受到影响。其中一个突变体的Tn5插入到一个与编码细胞色素c1的基因同源的基因中，另一个突变体的Tn5插入到与细胞色素bc1复合体的细胞色素b组分编码DNA同源的区域。血红素染色显示，突变体的膜中不存在分子量为31,000和23,000的血红素蛋白，而存在一种额外的分子量为23,000的可溶性c型细胞色素蛋白。我们得出结论，这两种血红素蛋白中较大的一种对应于细胞色素c1，在其缺失的情况下，分子量为23,000的蛋白不再与膜结合。在第三个呼吸缺陷型突变体中，分子量为23,000的组分的形成被特异性阻断，该突变体的Tn5插入到一段与慢生根瘤菌日本种的一个基因同源的DNA区域，该基因先前已被证明编码膜结合的c型细胞色素CycM。尽管细胞色素bc1复合体对于共生固氮至关重要，但另一种膜结合的c型细胞色素（CycM）并非如此。
