THE AMMONIA MONOOXYGENASE STRUCTURAL GENE AMOA AS A FUNCTIONAL MARKER- MOLECULAR FINE-SCALE ANALYSIS OF NATURAL AMMONIA-OXIDIZING POPULATIONS

The naturally occurring genetic heterogeneity of autotrophic ammonia-o xidizing populations belonging to the beta subclass of the Proteobacte ria was studied by using a newly developed PCR-based assay targeting a partial stretch of the gene which encodes the active-site polypeptide of ammonia monooxygenase (amoA). The PCR yielded a specific 491-bp fr agment with all of the nitrifiers tested, but not with the homologous stretch of the particulate methane monooxygenase, a key enzyme of the methane oxidizing bacteria. The assay also specifically detected amoA in DNA extracted from various aquatic and terrestrial environments. Th e resulting PCR products retrieved from rice roots, activated sludge, a freshwater sample, and an enrichment culture were used for the gener ation of amoA gene libraries. No false positives were detected in a se t of 47 randomly selected clone sequences that were analyzed further. The majority of the environmental sequences retrieved from rice roots and activated sludge grouped within the phylogenetic radiation defined by cultured strains of the genera Nitrosomonas and Nitrosospira. The comparative analysis identified members of both of these genera in act ivated sludge; however, only Nitrosospira-like sequences with very sim ilar amino acid patterns were found on rice roots. Further differentia tion of these molecular isolates was clearly possible on the nucleic a cid level due to the accumulation of synonymous mutations, suggesting that several closely related but distinct Nitrosospira-like population s are the main colonizers of the rhizosphere of rice. Each of the amoA gene libraries obtained from the freshwater sample and the enrichment culture was dominated by a novel lineage that shared a branch with th e Nitrosospira cluster but could not be assigned to any of the known p ure cultures. Our data suggest that amoA represents a very powerful mo lecular tool for analyzing indigenous ammonia-oxidizing communities du e to (i) its specificity, (ii) its fine-scale resolution of closely re lated populations, and (iii) the fact that a functional trait rather t han a phylogenetic trait is detected.