Nitric oxide scavengers differentially inhibit ammonia oxidation in ammonia-oxidizing archaea and bacteria

Abstract

Differential inhibitors are important for measuring the relative contributions of microbial groups, such as ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA), to biogeochemical processes in environmental samples. In particular, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) represents a nitric oxide scavenger used for the specific inhibition of AOA, implicating nitric oxide as an intermediate of thaumarchaeotal ammonia oxidation. This study investigated four alternative nitric oxide scavengers for their ability to differentially inhibit AOA and AOB in comparison to PTIO. Caffeic acid, curcumin, methylene blue hydrate and trolox were tested onNitrosopumilus maritimus, two unpublished AOA representatives (AOA-6f and AOA-G6) as well as the AOB representative Nitrosomonas europaea All four scavengers inhibited ammonia oxidation by AOA at lower concentrations than for AOB. In particular, differential inhibition of AOA and AOB by caffeic acid (100 μM) and methylene blue hydrate (3 μM) was comparable to carboxy-PTIO (100 μM) in pure and enrichment culture incubations. However, when added to aquarium sponge biofilm microcosms, both scavengers were unable to inhibit ammonia oxidation consistently, likely due to degradation of the inhibitors themselves. This study provides evidence that a variety of nitric oxide scavengers result in differential inhibition of ammonia oxidation in AOA and AOB, and provides support to the proposed role of nitric oxide as a key intermediate in the thaumarchaeotal ammonia oxidation pathway.

“Candidatus Nitrosotenuis aquarius,” an Ammonia-Oxidizing Archaeon from a Freshwater Aquarium Biofilter

“…Nitrosotenuis aquarius" enrichment cultures, and PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide) was inhibitory at low concentrations (Fig. S6), consistent with previous studies (16,76,77), whereas octyne was not inhibitory at the low concentrations (e.g., 10 M) used previously for specific inhibition of AOB (78). When incubated in aquarium water supplemented with 0.5 mM NH 4 Cl, aquarium filter biomass depleted all ammonia within approximately 2 days in the absence of inhibitors (Fig.…”

Cultivation and Genomic Analysis of “Candidatus Nitrosocaldus islandicus,” an Obligately Thermophilic, Ammonia-Oxidizing Thaumarchaeon from a Hot Spring Biofilm in Graendalur Valley, Iceland

“…Interestingly, ammonia oxidation by “ Ca. N. islandicus” was completely inhibited after the addition of ≥33 μM of the NO-scavenger PTIO (Supplementary Figure S3 ), a concentration that is lower or in the same range as previously reported to be inhibitory for other AOA ( Shen et al, 2013 ; Jung et al, 2014 ; Martens-Habbena et al, 2015 ; Sauder et al, 2016 ). This finding suggests that NO is required for ammonia oxidation in “ Ca.…”

Proteomics and comparative genomics of Nitrososphaera viennensis reveal the core genome and adaptations of archaeal ammonia oxidizers

“…NirK is broadly distributed in AOA with the exceptions of the symbiont C. symbiosum (39), for which evidence for capability of chemolithoautotrophic growth is still missing, and Nitrosocaldus yellowstonii (12), for which the full genome is not published. Nevertheless, NirK (or a noncanonical NirK) has been suggested to assist in the process of ammonia oxidation (14,21,26), because NO (the product of the NirK reaction) is essential for ammonia oxidation in AOA (18,21,62). In support of this hypothesis, NirK is found highly expressed on the mRNA level in cultivated AOA and environmental samples (63)(64)(65) and was identified in the N. viennensis proteome (NVIE_000530).…”