Ye R W, Arunakumari A, Averill B A, Tiedje J M
Department of Microbiology and Public Health, Michigan State University, East Lansing 48824-1325.
J Bacteriol. 1992 Apr;174(8):2560-4. doi: 10.1128/jb.174.8.2560-2564.1992.
Five Tn5 mutants of Pseudomonas fluorescens AK-15 deficient in dissimilatory reduction of nitrite were isolated and characterized. Two insertions occurred inside the nitrite reductase structural gene (nirS) and resulted in no detectable nitrite reductase protein on a Western immunoblot. One mutant had Tn5 inserted inside nirC, the third gene in the same operon, and produced a defective nitrite reductase protein. Two other mutants had insertions outside of this nir operon and also produced defective proteins. All of the Nir- mutants characterized showed not only loss of nitrite reductase activity but also a significant decrease in nitric oxide reductase activity. When cells were incubated with 15NO in H2(18)O, about 25% of the oxygen found in nitrous oxide exchanged with H2O. The extent of exchange remained constant throughout the reaction, indicating the incorporation of 18O from H2(18)O reached equilibrium rapidly. In all nitrite reduction-deficient mutants, less than 4% of the 18O exchange was found, suggesting that the hydration and dehydration step was altered. These results indicate that the factors involved in dissimilatory reduction of nitrite influenced the subsequent NO reduction in this organism.
分离并鉴定了荧光假单胞菌AK-15的五个亚硝酸异化还原缺陷型Tn5突变体。两个插入发生在亚硝酸还原酶结构基因（nirS）内部，在Western免疫印迹上未检测到亚硝酸还原酶蛋白。一个突变体的Tn5插入到同一操纵子的第三个基因nirC内部，并产生了有缺陷的亚硝酸还原酶蛋白。另外两个突变体的插入位于该nir操纵子之外，也产生了有缺陷的蛋白。所有表征的Nir-突变体不仅显示出亚硝酸还原酶活性丧失，而且一氧化氮还原酶活性也显著降低。当细胞在H2(18)O中与15NO一起孵育时，一氧化二氮中约25%的氧与H2O发生交换。整个反应过程中交换程度保持恒定，表明来自H2(18)O的18O掺入迅速达到平衡。在所有亚硝酸还原缺陷型突变体中，发现的18O交换不到4%，这表明水合和脱水步骤发生了改变。这些结果表明，参与亚硝酸异化还原的因素影响了该生物体中随后的NO还原。
