Transcriptomic Response of Nitrosomonas europaea Transitioned from Ammonia- to Oxygen-Limited Steady-State Growth

Abstract

Ammonia-oxidizing microorganisms perform the first step of nitrification, the oxidation of ammonia to nitrite. The bacterium Nitrosomonas europaea is the best-characterized ammonia oxidizer to date. Exposure to hypoxic conditions has a profound effect on the physiology of N. europaea, e.g., by inducing nitrifier denitrification, resulting in increased nitric and nitrous oxide production. This metabolic shift is of major significance in agricultural soils, as it contributes to fertilizer loss and global climate change. Previous studies investigating the effect of oxygen limitation on N. europaea have focused on the transcriptional regulation of genes involved in nitrification and nitrifier denitrification. Here, we combine steady-state cultivation with whole-genome transcriptomics to investigate the overall effect of oxygen limitation on N. europaea. Under oxygen-limited conditions, growth yield was reduced and ammonia-to-nitrite conversion was not stoichiometric, suggesting the production of nitrogenous gases. However, the transcription of the principal nitric oxide reductase (cNOR) did not change significantly during oxygen-limited growth, while the transcription of the nitrite reductase-encoding gene (nirK) was significantly lower. In contrast, both heme-copper-containing cytochrome c oxidases encoded by N. europaea were upregulated during oxygen-limited growth. Particularly striking was the significant increase in transcription of the B-type heme-copper oxidase, proposed to function as a nitric oxide reductase (sNOR) in ammonia-oxidizing bacteria. In the context of previous physiological studies, as well as the evolutionary placement of N. europaea’s sNOR with regard to other heme-copper oxidases, these results suggest sNOR may function as a high-affinity terminal oxidase in N. europaea and other ammonia-oxidizing bacteria. IMPORTANCE Nitrification is a ubiquitous microbially mediated process in the environment and an essential process in engineered systems such as wastewater and drinking water treatment plants. However, nitrification also contributes to fertilizer loss from agricultural environments, increasing the eutrophication of downstream aquatic ecosystems, and produces the greenhouse gas nitrous oxide. As ammonia-oxidizing bacteria are the most dominant ammonia-oxidizing microbes in fertilized agricultural soils, understanding their responses to a variety of environmental conditions is essential for curbing the negative environmental effects of nitrification. Notably, oxygen limitation has been reported to significantly increase nitric oxide and nitrous oxide production during nitrification. Here, we investigate the physiology of the best-characterized ammonia-oxidizing bacterium, Nitrosomonas europaea, growing under oxygen-limited conditions.

Small Sample Stress: Probing Oxygen-Deprived Ammonia-Oxidizing Bacteria with Raman Spectroscopy In Vivo

“…The elevated cytochrome c activity of oxygen-deprived N. eutropha and Nitrosospira fit well with the results reported by Sedlacek et al [9] and Pellitteri-Hahn et al [16], whose works indicated a significant increase in the cytochrome c content of stressed cells, but contradicted Wei et al [22], who reported mRNA levels for cytochrome c in growing cells to exceed those of energy-starved cells by a factor of 10. Sedlacek et al [9] used transcriptomics on stressed N. europaea to elucidate the stress The different oxygen profiles corresponding to aerated (+O 2 ) and oxygen-deprived (−O 2 ) culture conditions in Figures 3a and 4a were the result of an interplay of two sequential processes, namely oxygen transfer at the air-liquid interphase and dissolved oxygen consumption in the liquid medium [48]. In the incubation with shaking (+O 2 ), the oxygen transfer at the air-liquid interphase was higher compared to the non-shaking condition, which led to a higher oxygen concentration in the medium throughout the entire growth experiment.…”

“…Microbial nitrification, i.e., the metabolization of ammonia to nitrite, performed by AOB under aerobic conditions is crucial for the biological treatment of wastewater [2]. However, in cases of oxygen limitation, additional nitrogenous gases such as nitric oxide (NO) and nitrous oxide (NO 2 ) are produced [6,9,10]. In soil management, this negatively affects the efficiency of fertilizer, leading to nitrogen loss for the crops, an increased release of greenhouse gases from the soil, and eutrophication of water bodies downstream [11][12][13].…”

“…Recently, the response of AOB to either ammonia or dissolved oxygen deprivation has been studied extensively based on transcriptomics [9,15], proteomics [16], and genome analysis [10], finding that N. europaea, N. eutropha, and Nitrosospira all encode multiple copies of ammonia monooxygenase (AMO), hydroxylamine dehydrogenase (HAO), and nitride oxide reductase (NOR) as central components of their ammonia oxidation pathways. The electron transport along these pathways is predominantly performed by several cytochrome c proteins, some of which hold more than one heme c in their protic matrix [9,10,[17][18][19][20][21]. For a scheme of the electron transport and the ammonia oxidation pathways for N. europaea, including the associated cytochrome c proteins, see Sedlacek et al [9] (Figure 1).…”

“…The electron transport along these pathways is predominantly performed by several cytochrome c proteins, some of which hold more than one heme c in their protic matrix [9,10,[17][18][19][20][21]. For a scheme of the electron transport and the ammonia oxidation pathways for N. europaea, including the associated cytochrome c proteins, see Sedlacek et al [9] (Figure 1).…”

“…Yu and Chandran [15] reported for N. europaea batch cultures an increase in exponential phase mRNA concentrations of both AMO and HAO genes with decreasing dissolved oxygen concentrations, suggesting a mechanism to metabolize ammonia and hydroxylamine more effectively under dissolved oxygen limitation. Sedlacek et al [9] investigated the overall effect of oxygen limitation on N. europaea using steady-state cultivation with whole-genome transcriptomics. They reported that the heme-copper-containing cytochrome-c oxidases encoded by N. europaea were upregulated during oxygen-limited growth.…”