Abstract
Ammonia-oxidizing bacteria (AOB) are important members of terrestrial, marine, and industrial microbial communities and play a fundamental role in the Nitrogen cycle within these systems. They are responsible for the first step of nitrification, ammonia oxidation to nitrite. Although AOB are widespread and essential to environmental and industrial systems, where they regularly experience fluctuations in ammonia availability, no comparative studies of the physiological response of diverse AOB species at the protein level exist. In the present study, we used 1D-LC-MS/MS proteomics to compare the metabolism and physiology of three species of ammonia AOB, Nitrosomonas europaea, Nitrosospira multiformis, and Nitrosomonas ureae, under ammonia replete and ammonia starved conditions. Additionally, we compared the expression of orthologous genes to determine the major differences in the proteome composition of the three species. We found that approximately one-third of the predicted proteome was expressed in each species and that proteins for the key metabolic processes, ammonia oxidation and carbon fixation, were among the most abundant. The red copper protein, nitrosocyanin was highly abundant in all three species hinting toward its possible role as a central metabolic enzyme in AOB. The proteomic data also allowed us to identify pyrophosphate-dependent 6-phosphofructokinase as the potential enzyme replacing the Calvin-Benson-Bassham cycle enzyme Fructose-1,6-bisphosphatase missing in N. multiformis and N. ureae. Additionally, between species, there were statistically significant differences in the expression of many abundant proteins, including those related to nitrogen metabolism (nitrite reductase), motility (flagellin), cell growth and division (FtsH), and stress response (rubrerythrin). The three species did not exhibit a starvation response at the proteome level after 24 h of ammonia starvation, however, the levels of the RuBisCO enzyme were consistently reduced after the starvation period, suggesting a decrease in capacity for biomass accumulation. This study presents the first published proteomes of N. ureae and N. multiformis, and the first comparative proteomics study of ammonia-oxidizing bacteria, which gives new insights into consistent metabolic features and differences between members of this environmentally and industrially important group.
Highlights
Nitrification is an essential process of the nitrogen cycle in terrestrial, aquatic, and wastewater systems that links reduced and oxidized pools of inorganic nitrogen (Gruber and Galloway, 2008)
The results observed in this paper provide evidence about how genomic and proteomic expression governs niche preferences by individual species
A previous proteomics study on N. europaea found a total of 876 expressed proteins (34% of predicted proteome) (PellitteriHahn et al, 2011), which is remarkably similar to the 814 expressed proteins (32% of predicted proteome) identified in this present study
Summary
Nitrification is an essential process of the nitrogen cycle in terrestrial, aquatic, and wastewater systems that links reduced and oxidized pools of inorganic nitrogen (Gruber and Galloway, 2008). Nitrification is classically considered a two-step process with the first and rate limiting step, ammonia (NH3) oxidation to nitrite (NO2−), performed by ammonia-oxidizing bacteria (AOB) and archaea, and NO2− oxidation to nitrate (NO3−) performed by nitrite-oxidizing microorganisms (Kowalchuk and Stephen, 2001; Klotz and Stein, 2011). Organisms have been discovered that can perform the complete process of NH3 oxidation to NO3− (Daims et al, 2015; Van Kessel et al, 2015). Ammonia-oxidation by AOB is catalyzed aerobically via two characterized enzymes, ammonia monooxygenase (AMO) which oxidizes NH3 to hydroxylamine (NH2OH), and hydroxylamine dehydrogenase (HAO) which oxidizes NH2OH most likely to nitric oxide (NO) (Caranto and Lancaster, 2017). A third enzyme that oxidizes NO to nitrite (NO2−) has not yet been characterized and was only recently proposed as a third requisite enzyme in the ammonia-oxidation pathway
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