Community analysis of betaproteobacterial ammonia-oxidizing bacteria using the <Emphasis Type="Italic">amoCAB</Emphasis> operon |
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Authors: | Pilar Junier Ok-Sun Kim Thomas Junier Tae-Seok Ahn Johannes F Imhoff Karl-Paul Witzel |
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Institution: | 1.Ecole Polytechnique Fédérale de Lausanne (EPFL ENAC ISTE EML),Lausanne,Switzerland;2.Max-Planck Institute for Evolutionary Biology,Pl?n,Germany;3.School of Biological Sciences and Institute of Microbiology,Seoul National University,Seoul,Republic of Korea;4.University of Geneva,Geneva,Switzerland;5.Department of Environmental Science,Kangwon National University,Chuncheon,South Korea;6.Leibniz-Institute of Marine Sciences,University of Kiel,Kiel,Germany |
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Abstract: | 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.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
Pilar Junier and Ok-Sun Kim contributed equally to this work. |
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Keywords: | Ammonia-oxidizing betaproteobacteria PCR primers amoC amoA amoB |
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