The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations

Abstract

The naturally occurring genetic heterogeneity of autotrophic ammonia-oxidizing populations belonging to the ␤ subclass of the Proteobacteria 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 fragment 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. The resulting PCR products retrieved from rice roots, activated sludge, a freshwater sample, and an enrichment culture were used for the generation of amoA gene libraries. No false positives were detected in a set 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 activated sludge; however, only Nitrosospira-like sequences with very similar amino acid patterns were found on rice roots. Further differentiation of these molecular isolates was clearly possible on the nucleic acid level due to the accumulation of synonymous mutations, suggesting that several closely related but distinct Nitrosospira-like populations 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 the Nitrosospira cluster but could not be assigned to any of the known pure cultures. Our data suggest that amoA represents a very powerful molecular tool for analyzing indigenous ammonia-oxidizing communities due to (i) its specificity, (ii) its fine-scale resolution of closely related populations, and (iii) the fact that a functional trait rather than a phylogenetic trait is detected.

Ammonia oxidizer populations vary with nitrogen cycling across a tropical montane mean annual temperature gradient

“…Its sensitivity may have important implications for N bioavailability to primary producers as well as N mobility in soils (Hallin et al 2009). Ammonia oxidation is regulated by the gene amoA, which encodes the ammonia monooxygenase small alpha subunit (Rotthauwe et al 1997). The amoA gene is ubiquitous in terrestrial and aquatic systems (Rotthauwe et al 1997, Fierer et al 2009), and because it is phylogenetically constrained within the Betaproteobacteria and Proteoarchaeota, it is a tractable target in environmental samples (Purkhold et al 2000).…”

“…Ammonia oxidation is regulated by the gene amoA, which encodes the ammonia monooxygenase small alpha subunit (Rotthauwe et al 1997). The amoA gene is ubiquitous in terrestrial and aquatic systems (Rotthauwe et al 1997, Fierer et al 2009), and because it is phylogenetically constrained within the Betaproteobacteria and Proteoarchaeota, it is a tractable target in environmental samples (Purkhold et al 2000). Many studies have targeted amoA in soils, but most have relied on artificial warming to understand the role of temperature in AO and nitrification regulation via amoA , Horz et al 2004, Tourna et al 2008, Osborne et al 2015.…”

Relative Abundance of Ammonia Oxidizers, Denitrifiers, and Anammox Bacteria in Sediments of Hyper‐Nutrified Estuarine Tidal Flats and in Relation to Environmental Conditions

“…Ammonia oxidation is the first and rate-limiting step of nitrification in which ammonia (NH 3 ) is oxidized to nitrite (NO 2 À ) by ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). The ammonia monooxygenase gene (amoA) has been used extensively as a molecular marker for studying both types of ammonia oxidizers in environmental samples [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19]. Denitrification is an anaerobic reduction of nitrate, through nitrite and to nitrous oxide (N 2 O) and eventually dinitrogen (N 2 ).…”

Linking microbial co‐occurrences to soil ecological processes across a woodland‐grassland ecotone

“…The The abundances of bacterial, archaeal, and fungal genes were determined by quantitative real-time PCR using the Qiagen QuantiTect ™ SYBR ® Green PCR Master Mix (Qiagen Inc., Victoria, Australia), a Cavro ® Omni Robot (Tecan Group Ltd., Seestrasse, Switzerland), and an ABI 7900 real-time PCR machine (Applied Biosystems, Foster City, CA). Bacterial 16S rRNA and amoA genes were quantified using 338F and 518R (Lane et al, 1985;Muyzer, de Wall, & Uitterlinden, 1993) and amoA 1F and amoA 2R (Rotthauwe, Witzel, & Liesack, 1997) primers, respectively. Archaeal 16S rRNA and amoA genes were quantified using 771F and 957R (Ochsenreiter, Selezi, Quaiser, Bonch-Osmolovskaya, & Schleper, 2003) and Arch-amoA 104F and Arch-amoA 616R (Alves et al, 2013) primers, respectively.…”