Casciotti K L, Ward B B
Department of Geosciences, Princeton University, Princeton, NJ 08544, USA.
Appl Environ Microbiol. 2001 May;67(5):2213-21. doi: 10.1128/AEM.67.5.2213-2221.2001.
The presence of a copper-containing dissimilatory nitrite reductase gene (nirK) was discovered in several isolates of beta-subdivision ammonia-oxidizing bacteria using PCR and DNA sequencing. PCR primers Cunir3 and Cunir4 were designed based on published nirK sequences from denitrifying bacteria and used to amplify a 540-bp fragment of the nirK gene from Nitrosomonas marina and five additional isolates of ammonia-oxidizing bacteria. Amplification products of the expected size were cloned and sequenced. Alignment of the nucleic acid and deduced amino acid (AA) sequences shows significant similarity (62 to 75% DNA, 58 to 76% AA) between nitrite reductases present in these nitrifiers and the copper-containing nitrite reductase found in classic heterotrophic denitrifiers. While the presence of a nitrite reductase in Nitrosomonas europaea is known from early biochemical work, preliminary sequence data from its genome indicate a rather low similarity to the denitrifier nirKs. Phylogenetic analysis of the partial nitrifier nirK sequences indicates that the topology of the nirK tree corresponds to the 16S rRNA and amoA trees. While the role of nitrite reduction in the metabolism of nitrifying bacteria is still uncertain, these data show that the nirK gene is present in closely related nitrifying isolates from many oceanographic regions and suggest that nirK sequences retrieved from the environment may include sequences from ammonia-oxidizing bacteria.
利用聚合酶链反应（PCR）和DNA测序技术，在多个β-亚纲氨氧化细菌分离株中发现了含铜异化亚硝酸盐还原酶基因（nirK）。基于已发表的反硝化细菌nirK序列设计了PCR引物Cunir3和Cunir4，并用于扩增来自滨海亚硝化单胞菌及另外5株氨氧化细菌分离株的nirK基因540 bp片段。对预期大小的扩增产物进行克隆和测序。核酸和推导的氨基酸（AA）序列比对显示，这些硝化细菌中的亚硝酸盐还原酶与经典异养反硝化细菌中发现的含铜亚硝酸盐还原酶之间存在显著相似性（DNA相似性为62%至75%，AA相似性为58%至76%）。虽然早期生化研究已表明欧洲亚硝化单胞菌中存在亚硝酸盐还原酶，但其基因组的初步序列数据显示与反硝化细菌的nirK基因相似度较低。对部分硝化细菌nirK序列的系统发育分析表明，nirK基因树的拓扑结构与16S rRNA基因树和amoA基因树一致。虽然亚硝酸盐还原在硝化细菌代谢中的作用仍不确定，但这些数据表明nirK基因存在于来自许多海洋学区域的密切相关硝化细菌分离株中，并且提示从环境中检索到的nirK序列可能包括来自氨氧化细菌的序列。
