Junier Pilar, Kim Ok-Sun, Junier Thomas, Ahn Tae-Seok, Imhoff Johannes F, Witzel Karl-Paul
Ecole Polytechnique Fédérale de Lausanne (EPFL ENAC ISTE EML), CE 1 644 (Centre Est), Station 6, 1015 Lausanne, Switzerland.
Appl Microbiol Biotechnol. 2009 May;83(1):175-88. doi: 10.1007/s00253-009-1923-x. Epub 2009 Mar 10.
The genes and intergenic regions of the amoCAB operon were analyzed to establish their potential as molecular markers for analyzing ammonia-oxidizing betaproteobacterial (beta-AOB) communities. Initially, sequence similarity for related taxa, evolutionary rates from linear regressions, and the presence of conserved and variable regions were analyzed for all available sequences of the complete amoCAB operon. The gene amoB showed the highest sequence variability of the three amo genes, suggesting that it might be a better molecular marker than the most frequently used amoA to resolve closely related AOB species. To test the suitability of using the amoCAB genes for community studies, a strategy involving nested PCR was employed. Primers to amplify the whole amoCAB operon and each individual gene were tested. The specificity of the products generated was analyzed by denaturing gradient gel electrophoresis, cloning, and sequencing. The fragments obtained showed different grades of sequence identity to amoCAB sequences in the GenBank database. The nested PCR approach provides a possibility to increase the sensitivity of detection of amo genes in samples with low abundance of AOB. It also allows the amplification of the almost complete amoA gene, with about 300 bp more sequence information than the previous approaches. The coupled study of all three amo genes and the intergenic spacer regions that are under different selection pressure might allow a more detailed analysis of the evolutionary processes, which are responsible for the differentiation of AOB communities in different habitats.
对amoCAB操纵子的基因和基因间区域进行了分析，以确定其作为分析氨氧化β-变形菌（β-AOB）群落分子标记的潜力。最初，针对完整amoCAB操纵子的所有可用序列，分析了相关分类群的序列相似性、线性回归得出的进化速率以及保守区和可变区的存在情况。amoB基因在三个amo基因中表现出最高的序列变异性，这表明它可能是比最常用的amoA更好的分子标记，用于区分密切相关的AOB物种。为了测试使用amoCAB基因进行群落研究的适用性，采用了一种涉及巢式PCR的策略。测试了用于扩增整个amoCAB操纵子和每个单独基因的引物。通过变性梯度凝胶电泳、克隆和测序分析了所产生产物的特异性。获得的片段与GenBank数据库中的amoCAB序列显示出不同程度的序列同一性。巢式PCR方法提供了一种可能性，可提高在AOB丰度较低的样品中检测amo基因的灵敏度。它还能够扩增几乎完整的amoA基因，比以前的方法多约300 bp的序列信息。对处于不同选择压力下的所有三个amo基因和基因间间隔区进行联合研究，可能会对进化过程进行更详细的分析，这些进化过程导致了不同生境中AOB群落的分化。
