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.

Nitrobacter winogradskyi Nb-255 is a nitrite-oxidizing bacterium that can grow solely on nitrite (NO2(-)) as a source of energy and nitrogen. In most natural situations, NO2(-) oxidation is coupled closely to ammonium (NH4(+)) oxidation by bacteria and archaea and, conceptually, N. winogradskyi can save energy using NH4(+) to meet its N-biosynthetic requirements. Interestingly, NH4(+) delayed the growth of N. winogradskyi when at concentrations higher than 35 mM, but grew well at concentrations below 25 mM NH4(+) while adjusting the expression of 24% of its genes. Notable genes that changed in expression included those with roles in nitrogen and carbon assimilation. Contrary to expectations, higher expression of glutamate synthase (GOGAT), instead of glutamate dehydrogenase, was detected at higher NH4(+) concentration. Genes in assimilatory NO2(-) metabolism and the degradation of glycogen and biofilm/motility were downregulated when N. winogradskyi was grown in the presence of NH4(+). Nitrobacter winogradskyi grown in medium with 25 mM NH4(+) upregulated genes in post-translational modification, protein turnover, biogenesis and chaperons. The data suggest that N. winogradskyi physiology is modified in the presence of NH4(+) and is likely to be modified during coupled nitrification with NH3 oxidizers.

word count: 250 34 Text word count: 4885 35 36 3 Abstract 37Ammonia-oxidizing microorganisms perform the first step of nitrification, the oxidation of 38 ammonia to nitrite. The bacterium Nitrosomonas europaea is the best characterized ammonia oxidizer to 39 date. Exposure to hypoxic conditions has a profound effect on the physiology of N. europaea, e.g. by 40 inducing nitrifier denitrification, resulting in increased nitric and nitrous oxide production. This metabolic 41 shift is of major significance in agricultural soils, as it contributes to fertilizer loss and global climate 42 change. Previous studies investigating the effect of oxygen limitation on N. europaea have focused on 43 the transcriptional regulation of genes involved in nitrification and nitrifier denitrification. Here, we 44 combine steady-state cultivation with whole genome transcriptomics to investigate the overall effect of 45 oxygen limitation on N. europaea. Under oxygen-limited conditions, growth yield was reduced and 46 ammonia to nitrite conversion was not stoichiometric, suggesting the production of nitrogenous gases. 47However, the transcription of the principal nitric oxide reductase (cNOR) did not change significantly 48 during oxygen-limited growth, while the transcription of the nitrite reductase-encoding gene (nirK) was 49 significantly lower. In contrast, both heme-copper containing cytochrome c oxidases encoded by N. 50 europaea were upregulated during oxygen-limited growth. Particularly striking was the significant 51 increase in transcription of the B-type heme-copper oxidase, proposed to function as a nitric oxide 52 reductase (sNOR) in ammonia-oxidizing bacteria. In the context of previous physiological studies, as well 53 as the evolutionary placement of N. europaea's sNOR with regards to other heme-copper oxidases, 54 these results suggest sNOR may function as a high-affinity terminal oxidase in N. europaea and other 55 AOB. 56 4 Importance 57 Nitrification is a ubiquitous, microbially mediated process in the environment and an essential 58 process in engineered systems such as wastewater and drinking water treatment plants. However, 59 nitrification also contributes to fertilizer loss from agricultural environments increasing the eutrophication 60 of downstream aquatic ecosystems and produces the greenhouse gas nitrous oxide. As ammonia-61 oxidizing bacteria are the most dominant ammonia-oxidizing microbes in fertilized agricultural soils, 62 understanding their response to a variety of environmental conditions is essential for curbing the 63 negative environmental effects of nitrification. Notably, oxygen limitation has been reported to 64 significantly increase nitric oxide and nitrous oxide production during nitrification. Here we investigate the 65 physiology of the best characterized ammonia-oxidizing bacterium, Nitrosomonas europaea, growing 66 under oxygen-limited conditions. 67 5 1 Introduction 68Nitrification is a microbially mediated, aerobic process involving the successive oxidation of 69 ammonia (NH 3 ) and n...