Nitric oxide reductase (norB) genes from pure cultures and environmental samples
Nitric oxide reductase (norB) genes from pure cultures and environmental samples
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DOI：
10.1128/aem.69.6.3476-3483.2003
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发表时间：
2003-06-01
影响因子：
4.4
通讯作者：
Tiedjë, JM
中科院分区：
文献类型：
--
作者：
Braker, G;Tiedjë, JM
利用PCR技术从一氧化氮还原酶（norB）基因中分离出一氧化氮还原酶（norB）基因，并将其作为细菌的功能性标记基因。norB数据库序列分为两个非常不同的分支。一个编码喹啉氧化单亚基类（qNorB），而另一类是细胞色素bc型复合物（cNorB）。后者氧化细胞色素c，该基因位于norC附近。虽然这两种norB类型的发生在purifying菌株，qnorB类型也被发现在各种nonpurifying菌株，表明解毒NO的功能。分支特异性简并引物组检测到两个norB类型在我们的purifier文化。通过norB扩增子的序列分析和未能从非扩增菌株中扩增norB证实了特异性。这些引物组还特异性地扩增来自淡水和海洋沉积物的norB。扩增的norB序列的成对比较表明qnorB和cnorB的氨基酸同一性最低水平分别为43.9%和38%。系统发育分析证实存在两类norB基因，根据各自的引物集进行聚类。在qnorB簇内，来自分离株和少数环境克隆的大多数基因形成了一个单独的亚簇。大多数环境qnorB克隆起源于这两个栖息地聚类成两个不同的亚群的新序列，据推测尚未栽培的生物。cnorB克隆位于来自已知生物的基因亚群内的独立分支上，表明起源于相似的生物。
A PCR-based approach was developed to recover nitric oxide (NO) reductase (norB) genes as a functional marker gene for denitrifying bacteria. norB database sequences grouped in two very distinct branches. One encodes the quinol-oxidizing single-subunit class (qNorB), while the other class is a cytochrome bc-type complex (cNorB). The latter oxidizes cytochrome c, and the gene is localized adjacent to norC. While both norB types occur in denitrifying strains, the qnorB type was also found in a variety of nondenitrifying strains, suggesting a function in detoxifying NO. Branch-specific degenerate primer sets detected the two norB types in our denitrifier cultures. Specificity was confirmed by sequence analysis of the norB amplicons and failure to amplify norB from nondenitrifying strains. These primer sets also specifically amplified norB from freshwater and marine sediments. Pairwise comparison of amplified norB sequences indicated minimum levels of amino acid identity of 43.9% for qnorB and 38% for cnorB. Phylogenetic analysis confirmed the existence of two classes of norB genes, which clustered according to the respective primer set. Within the qnorB cluster, the majority of genes from isolates and a few environmental clones formed a separate subcluster. Most environmental qnorB clones originating from both habitats clustered into two distinct subclusters of novel sequences from presumably as yet uncultivated organisms. cnorB clones were located on separate branches within subelusters of genes from known organisms, suggesting an origin from similar organisms.