Community analysis of betaproteobacterial ammonia-oxidizing bacteria using the <i>amoCAB</i> operon

The genes and intergenic regions of the <i>amoCAB</i> 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 <i>amoCAB</i> operon. The gene <i>amoB</i> showed the highest sequence variability of the three amo genes, suggesting that it might be a better molecular marker than the most frequently used <i>amoA</i> to resolve closely related AOB species. To test the suitability of using the <i>amoCAB</i> genes for community studies, a strategy involving nested PCR was employed. Primers to amplify the whole <i>amoCAB</i> 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 <i>amoCAB</i> 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 <i>amoA</i> gene, with about 300 bp more sequence information than the previous approaches. The coupled study of all three <i>amo</i> 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.