Unexpected nondenitrifier nitrous oxide reductase gene diversity and abundance in soils

Abstract

Agricultural and industrial practices more than doubled the intrinsic rate of terrestrial N fixation over the past century with drastic consequences, including increased atmospheric nitrous oxide (N(2)O) concentrations. N(2)O is a potent greenhouse gas and contributor to ozone layer destruction, and its release from fixed N is almost entirely controlled by microbial activities. Mitigation of N(2)O emissions to the atmosphere has been attributed exclusively to denitrifiers possessing NosZ, the enzyme system catalyzing N(2)O to N(2) reduction. We demonstrate that diverse microbial taxa possess divergent nos clusters with genes that are related yet evolutionarily distinct from the typical nos genes of denitirifers. nos clusters with atypical nosZ occur in Bacteria and Archaea that denitrify (44% of genomes), do not possess other denitrification genes (56%), or perform dissimilatory nitrate reduction to ammonium (DNRA; (31%). Experiments with the DNRA soil bacterium Anaeromyxobacter dehalogenans demonstrated that the atypical NosZ is an effective N(2)O reductase, and PCR-based surveys suggested that atypical nosZ are abundant in terrestrial environments. Bioinformatic analyses revealed that atypical nos clusters possess distinctive regulatory and functional components (e.g., Sec vs. Tat secretion pathway in typical nos), and that previous nosZ-targeted PCR primers do not capture the atypical nosZ diversity. Collectively, our results suggest that nondenitrifying populations with a broad range of metabolisms and habitats are potentially significant contributors to N(2)O consumption. Apparently, a large, previously unrecognized group of environmental nosZ has not been accounted for, and characterizing their contributions to N(2)O consumption will advance understanding of the ecological controls on N(2)O emissions and lead to refined greenhouse gas flux models.

Fig. 1.

Bootstrapped neighbor-joining phylogeny of 136 NosZ sequences representing 133 genomes. Branches with bootstrap support <50% are indicated by open circles. The typical NosZ clade is composed of sequences with an N-terminal Tat motif and associated primarily with functionally characterized soil denitrifiers of the Proteobacteria. The atypical NosZ is distributed among Bacteria and Archaea, and has been characterized in A. dehalogenans, W. succinogenes, and G. thermodenitrificans (bold font). nosZ occurs as a single-copy gene on all genomes, except for Pseudomonas brassicacearum and Sulfurimonas denitrificans, which both have two nosZ loci. Also indicated is the presence of nitrite respiratory genes nirS/nirK (denitrification) and nrfA (DNRA ammonification). Four NosZ within the halophilic Euryarchaeota formed a discrete cluster and did not group with either the atypical or the typical NosZ. The scale bar at the top left corresponds to the mean number of amino acid substitutions per site.

Fig. 2.

Growth of A. dehalogenans strain 2CP-C with N₂O as the sole electron acceptor. The medium was amended with acetate as electron donor, and the vessels were inoculated with acetate/N₂O-grown 2CP-C cells that had consumed all N₂O. The data points represent the averages of triplicate cultures, with error bars showing the SD. In control vessels without N₂O, cell numbers did not increase. No N₂O reduction occurred in control cultures that received no inoculum. ●, N₂O; ■, dinitrogen; ▲, cells = OD_{600 nm}; ○, N₂O, no cells; △, cells = OD_{600 nm}, no N₂O.

Fig. 3.

Comparison of nos clusters carrying the atypical nosZ with the characterized nos cluster of the complete denitrifier B. japonicum strain USDA110, which harbors a typical nosZ. nos clusters of Anaeromyxobacter sp. strain Fw109-5, three A. dehalogenans strains, O. terrae, D. tiedjei, G. thermodenitrificans, four representatives of the Desulfitobacterium genus, G. aurantiaca, W. succinogenes, and Dechloromonas aromatica harbor the atypical nosZ and encode predicted iron–sulfur-binding proteins (labeled “4Fe-4S” or “Fe-S”), Rieske iron–sulfur proteins (S), b-type cytochromes (cy-b), or c-type cytochromes (cy-c). The gene encoding a protein with up to four predicted transmembrane-spanning regions (TM) is found in all atypical, but none of the typical, nos clusters. Accessory genes (nosD, nosF, nosL, and nosY) are generally conserved across nos clusters with both typical and atypical nosZ. Noncolored genes in the A. dehalogenans and O. terrae operons have no orthologs in any other known nos cluster. nosR and nosX are associated exclusively with typical nos clusters. W. succinogenes produces a unique NosZ with an additional C-terminal c-type cytochrome domain. The scale bar indicates the mean number of amino acid substitutions per site.

Fig. 4.

Correlation of Anaeromyxobacter spp. abundance with the relative detection of atypical Anaeromyxobacter nosZ genes in Dekalb and Havana spatial variogram soil core samples. Mean 16S rRNA gene copy numbers per gram of soil are plotted in each nosZ relative abundance group. Kruskal–Wallis one-way ANOVA demonstrated significant differences between negative (−, no nosZ amplicons obtained) and positive (+, nosZ amplicons obtained in direct PCR) PCR results (**P < 0.0001), and between positive and low target gene abundance [(+), nosZ amplicons obtained with nested PCR] results (*P < 0.05). No significant statistical relationship (ns) was observed between the abundance of detected Anaeromyxobacter populations and samples that tested negative for nosZ or required nested PCR for nosZ detection. Error bars represent the SD.