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 identify 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 monooxygenase (amoA), defined lengths of terminal restriction fragments (= operational taxonomic units, OTUs) of amoA were predicted to correlate in TaqI-based T-RFLP analysis with phylogenetically defined subgroups of ammonia oxidizers. 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. Two amoA sequence clusters designated previously as the lineages 'PluBsee' and 'Schöhsee' [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 differentiated by AluI-based T-RFLP analysis. A survey of various environmental samples and enrichment cultures by T-RFLP analysis and by comparative analysis of cloned amoA sequences confirmed the predicted correlations between distinct OTUs and phylogenetic information. Our data suggest that amoA-based T-RFLP analysis is a reliable tool to rapidly assess the complexity of ammonia-oxidizing communities in environmental samples with respect to the presence of major subgroups, i.e. nitrosospiras versus nitrosomonads.
Horz
H P
HP
Max-Planck-Institut für terrestrische Mikrobiologie, Marburg, Germany.
Rotthauwe
J H
JH
Lukow
T
T
Liesack
W
W
D000641:Ammonia / Q000378:metabolism*; D020562:Betaproteobacteria / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004269:DNA, Bacterial / Q000032:analysis; D004783:Environmental Microbiology; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial; D000641:Ammonia; D010088:Oxidoreductases; C045582:ammonia monooxygenase