Unexpected Diversity and High Abundance of Putative Nitric Oxide Dismutase (Nod) Genes in Contaminated Aquifers and Wastewater Treatment Systems

Abstract

It has recently been suggested that oxygenic dismutation of NO into N 2 and O 2 may occur in the anaerobic methanotrophic "Candidatus Methylomirabilis oxyfera" and the alkane-oxidizing gammaproteobacterium HdN1. It may represent a new pathway in microbial nitrogen cycling catalyzed by a putative NO dismutase (Nod). The formed O 2 enables microbes to employ aerobic catabolic pathways in anoxic habitats, suggesting an ecophysiological niche space of substantial appeal for bioremediation and water treatment. However, it is still unknown whether this physiology is limited to "Ca. Methylomirabilis oxyfera" and HdN1 and whether it can be coupled to the oxidation of electron donors other than alkanes. Here, we report insights into an unexpected diversity and remarkable abundance of nod genes in natural and engineered water systems. Phylogenetically diverse nod genes were recovered from a range of contaminated aquifers and N-removing wastewater treatment systems. Together with nod genes from "Ca. Methylomirabilis oxyfera" and HdN1, the novel environmental nod sequences formed no fewer than 6 well-supported phylogenetic clusters, clearly distinct from canonical NO reductase (quinoldependent NO reductase [qNor] and cytochrome c-dependent NO reductase [cNor]) genes. The abundance of nod genes in the investigated samples ranged from 1.6 ϫ 10 7 to 5.2 ϫ 10 10 copies · g Ϫ1 (wet weight) of sediment or sludge biomass, accounting for up to 10% of total bacterial 16S rRNA gene counts. In essence, NO dismutation could be a much more widespread physiology than currently perceived. Understanding the controls of this emergent microbial capacity could offer new routes for nitrogen elimination or pollutant remediation in natural and engineered water systems.IMPORTANCE NO dismutation into N 2 and O 2 is a novel process catalyzed by putative NO dismutase (Nod). To date, only two bacteria, the anaerobic methaneoxidizing bacterium "Ca. Methylomirabilis oxyfera" and the alkane-oxidizing gammaproteobacterium HdN1, are known to harbor nod genes. In this study, we report efficient molecular tools that can detect and quantify a wide diversity of nod genes in environmental samples. A surprisingly high diversity and abundance of nod genes were found in contaminated aquifers as well as wastewater treatment systems. This evidence indicates that NO dismutation may be a much more widespread physiology in natural and man-made environments than currently perceived. The molecular tools presented here will facilitate further studies on these enigmatic microbes in the future.KEYWORDS nitric oxide (NO) dismutation, NO dismutase, oxygenic denitrification,

Long-Read Amplicon Sequencing of Nitric Oxide Dismutase (nod) Genes Reveal Diverse Oxygenic Denitrifiers in Agricultural Soils and Lake Sediments

“…In total, 61 nod OTUs with at least 50 sequences were classified based on 90% similarity on nucleotide level [7]. Representative sequences were used for phylogenetic analysis, revealing that almost all OTUs detected in this study were related to previously reported nod lineages [6], with most of them belonging to the nod "Aquifer cluster," 5 OTUs in the "NC10 cluster," and 1 OTU in the "Reactor cluster 1" (Fig. 2).…”

“…The sample designations are the same as in Table 1 oxygenic denitrifiers was recently established [6,7], with which, evidence was obtained suggesting that oxygenic denitrifiers could be widespread and phylogenetically diverse [6,8]. However, the high diversity inferred from nod genes provided no clue to the actual identities of these novel oxygenic denitrifiers [6]. So far, the identification of oxygenic denitrifiers and analysis of their community structures solely depend on the length and quality of obtained nod sequences.…”

“…Bootstrap support (1000 replicates) greater than 50% is indicated at the nodes. Nod clusters (same as proposed in [6]) containing soil and lake nod OTUs are shown in bold. OTUs only detected in soil are shown in blue, lake-specific OTUs in red, and OTUs comprising sequences from both soil and lake sediment are shown in green.…”

The pH dependency of N‐converting enzymatic processes, pathways and microbes: effect on net N2O production

“…This process is usually coupled with alkane oxidation by a monooxygenase (Ettwig et al, 2012). Bacteria that harbour NOD in WWTPs are diverse and are especially abundant in systems with transient occurrence of NO 2 2 /NO 2 3 or hypoxia (Zhu et al, 2017). They also have been detected in activated sludge systems and anaerobic digesters from municipal WWTP (Ho et al, 2013).…”

Microbial pathways for nitrogen loss in an upland soil

“…The abundance of anammox bacteria was quantified by targeting the hydrazine synthease beta-subunit (hzsB) gene with the primer set HSBeta396F and HSBeta742R (Wang et al, 2012). The qPCR of the n-damo putative NO dismutases (Nod) gene was performed with primers nod1446F and nod1706Rv2 (Zhu et al, 2016). The abundances of AOA and AOB were assessed by targeting the ammonia monooxygenase a-subunit (amoA) gene with the primer set archea-amoAF and archea-amoAR for archaeal amoA and amoAF and amoAR for bacterial amoA, respectively (Francis et al, 2005;Wang et al, 2011).…”