PCR Detection of Genes Encoding Nitrite Reductase in Denitrifying Bacteria
PCR Detection of Genes Encoding Nitrite Reductase in Denitrifying Bacteria
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DOI：
10.1128/aem.65.4.1652-1657.1999
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发表时间：
1999-04
影响因子：
4.4
通讯作者：
S. Hallin;P. Lindgren
中科院分区：
文献类型：
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作者：
S. Hallin;P. Lindgren
利用反硝化途径关键酶亚硝酸盐还原酶（Nir）两种形式基因序列的一致区，设计了两组PCR引物，分别扩增cd1-和Cu-nir。通过筛选确定的反硝化菌株、废水处理厂的反硝化分离物和活性污泥提取物来评估引物。并建立了nir基因的序列关系。cdna引物设计用于扩增六个已发表序列中cd1-nir的778 ~ 799 bp区域。同样，Cu引物在8个已发表的Cu-nir序列中扩增了7个473 bp的区域。总之，两组PCR引物扩增了4属9个物种的nir基因，以及7个污泥分离物中的4个。引物不能扩增非反硝化菌株的基因。Cu引物在所有13个污泥样品中扩增出预期片段，但仅在5个样品中获得了cd1-nir片段。在与DNA探针杂交后，预期大小的PCR产物被验证为近红外基因，只有一个例外。测序的近红外片段与其他近红外序列相关，表明引物扩增的基因是正确的。Cu-nir所选择的引物位点是保守的，而针对cd1-nir保守区域的大范围引物似乎很难找到。我们还报道了Cu-nir在反硝化副球菌Pd1222中的存在。
ABSTRACT Using consensus regions in gene sequences encoding the two forms of nitrite reductase (Nir), a key enzyme in the denitrification pathway, we designed two sets of PCR primers to amplifycd1- and Cu-nir. The primers were evaluated by screening defined denitrifying strains, denitrifying isolates from wastewater treatment plants, and extracts from activated sludge. Sequence relationships ofnir genes were also established. Thecd1 primers were designed to amplify a 778 to 799-bp region of cd1-nir in the six published sequences. Likewise, the Cu primers amplified a 473-bp region in seven of the eight published Cu-nir sequences. Together, the two sets of PCR primers amplified nir genes in nine species within four genera, as well as in four of the seven sludge isolates. The primers did not amplify genes of nondenitrifying strains. The Cu primers amplified the expected fragment in all 13 sludge samples, but cd1-nir fragments were only obtained in five samples. PCR products of the expected sizes were verified as nir genes after hybridization to DNA probes, except in one case. The sequenced nir fragments were related to other nir sequences, demonstrating that the primers amplified the correct gene. The selected primer sites for Cu-nir were conserved, while broad-range primers targeting conserved regions of cd1-nir seem to be difficult to find. We also report on the existence of Cu-nir in Paracoccus denitrificans Pd1222.