Ye R W, Averill B A, Tiedje J M
Department of Microbiology and Public Health, Michigan State University, East Lansing 48824.
J Bacteriol. 1992 Oct;174(20):6653-8. doi: 10.1128/jb.174.20.6653-6658.1992.
Tn5 was used to generate mutants that were deficient in the dissimilatory reduction of nitrite for Pseudomonas sp. strain G-179, which contains a copper nitrite reductase. Three types of mutants were isolated. The first type showed a lack of growth on nitrate, nitrite, and nitrous oxide. The second type grew on nitrate and nitrous oxide but not on nitrite (Nir-). The two mutants of this type accumulated nitrite, showed no nitrite reductase activity, and had no detectable nitrite reductase protein bands in a Western blot (immunoblot). Tn5 insertions in these two mutants were clustered in the same region and were within the structural gene for nitrite reductase. The third type of mutant grew on nitrate but not on nitrite or nitrous oxide (N2O). The mutant of this type accumulated significant amounts of nitrite, NO, and N2O during anaerobic growth on nitrate and showed a slower growth rate than the wild type. Diethyldithiocarbamic acid, which inhibited nitrite reductase activity in the wild type, did not affect NO reductase activity, indicating that nitrite reductase did not participate in NO reduction. NO reductase activity in Nir- mutants was lower than that in the wild type when the strains were grown on nitrate but was the same as that in the wild type when the strains were grown on nitrous oxide. These results suggest that the reduction of NO and N2O was carried out by two distinct processes and that mutations affecting nitrite reduction resulted in reduced NO reductase activity following anaerobic growth with nitrate.
Tn5被用于产生在异化还原亚硝酸盐方面存在缺陷的突变体，该突变体来自含有铜亚硝酸盐还原酶的假单胞菌属菌株G-179。分离出了三种类型的突变体。第一种类型在硝酸盐、亚硝酸盐和一氧化二氮上均无法生长。第二种类型在硝酸盐和一氧化二氮上能够生长，但在亚硝酸盐上不能生长（Nir-）。这种类型的两个突变体积累了亚硝酸盐，没有亚硝酸盐还原酶活性，并且在蛋白质免疫印迹（免疫印迹法）中没有可检测到的亚硝酸盐还原酶蛋白条带。这两个突变体中的Tn5插入集中在同一区域，且位于亚硝酸盐还原酶的结构基因内。第三种类型的突变体在硝酸盐上能够生长，但在亚硝酸盐或一氧化二氮上不能生长（N2O）。这种类型的突变体在硝酸盐厌氧生长过程中积累了大量的亚硝酸盐、一氧化氮和一氧化二氮，并且生长速度比野生型慢。二乙基二硫代氨基甲酸盐抑制野生型中的亚硝酸盐还原酶活性，但不影响一氧化氮还原酶活性，这表明亚硝酸盐还原酶不参与一氧化氮的还原。当菌株在硝酸盐上生长时，Nir-突变体中的一氧化氮还原酶活性低于野生型，但当菌株在一氧化二氮上生长时，其活性与野生型相同。这些结果表明，一氧化氮和一氧化二氮的还原是通过两个不同的过程进行的，并且影响亚硝酸盐还原的突变导致在硝酸盐厌氧生长后一氧化氮还原酶活性降低。
