Identification of major subgroups of ammonia-oxidizing bacteria in environmental samples by T-RFLP analysis of amoA PCR products

A cloning-independent method based on T-RFLP (terminal restriction fragment length polymorphism) analysis of amoA PCR products was developed to identi fy major subgroups of autotrophic ammonia oxidizers of the beta-subclass of the class Proteobacteria in total community DNA. Based on a database of 28 partial gene sequences encoding the active-site polypeptide of ammonia mon ooxygenase (amoA), defined lengths of terminal restriction fragments (= ope rational taxonomic units, OTUs) of amoA were predicted to correlate in TaqI -based T-RFLP analysis with phylogenetically defined subgroups of ammonia o xidizers. Members of the genus Nitrosospira showed a specific OTU of 283 bp in length, while a fragment size of 219 bp was indicative of Nitrosomonas- like sequence types including N. europaea, N. eutropha, and N. halophila. T wo amoA sequence clusters designated previously as the lineages 'Plu ss see ' and 'Schohsee' [Rotthauwe, J.-H., Witzel, K.-P., Liesack, W., 1997. Appl. Environ. Microbiol. 63, 4704-4712] shared a TaqI-based OTU with a fragment size of 48 bp, but sequence types of these two lineages could be different iated by AluI-based T-RFLP analysis. A survey of various environmental samp les and enrichment cultures by T-RFLP analysis and by comparative analysis of cloned amoA sequences confirmed the predicted correlations between disti nct OTUs and phylogenetic information. Our data suggest that amoA-based T-R FLP analysis is a reliable tool to rapidly assess the complexity of ammonia -oxidizing communities in environmental samples with respect to the presenc e of major subgroups, i.e. nitrosospiras versus nitrosomonads. (C) 2000 Pub lished by Elsevier Science B.V. All rights reserved.