Steady-State Growth under Inorganic Carbon Limitation Conditions Increases Energy Consumption for Maintenance and Enhances Nitrous Oxide Production in Nitrosomonas europaea

Nitrosomonas europaea is a chemolithoautotrophic bacterium that oxidizes ammonia (NH₃) to obtain energy for growth on carbon dioxide (CO₂) and can also produce nitrous oxide (N₂O), a greenhouse gas. We interrogated the growth, physiological, and transcriptome responses of N. europaea to conditions of replete (>5.2 mM) and limited inorganic carbon (IC) provided by either 1.0 mM or 0.2 mM sodium carbonate (Na₂CO₃) supplemented with atmospheric CO₂. IC-limited cultures oxidized 25 to 58% of available NH₃ to nitrite, depending on the dilution rate and Na₂CO₃ concentration. IC limitation resulted in a 2.3-fold increase in cellular maintenance energy requirements compared to those for NH₃-limited cultures. Rates of N₂O production increased 2.5- and 6.3-fold under the two IC-limited conditions, increasing the percentage of oxidized NH₃-N that was transformed to N₂O-N from 0.5% (replete) up to 4.4% (0.2 mM Na₂CO₃). Transcriptome analysis showed differential expression (P ≤ 0.05) of 488 genes (20% of inventory) between replete and IC-limited conditions, but few differences were detected between the two IC-limiting treatments. IC-limited conditions resulted in a decreased expression of ammonium/ammonia transporter and ammonia monooxygenase subunits and increased the expression of genes involved in C₁ metabolism, including the genes for RuBisCO (cbb gene cluster), carbonic anhydrase, folate-linked metabolism of C₁ moieties, and putative C salvage due to oxygenase activity of RuBisCO. Increased expression of nitrite reductase (gene cluster NE0924 to NE0927) correlated with increased production of N₂O. Together, these data suggest that N. europaea adapts physiologically during IC-limited steady-state growth, which leads to the uncoupling of NH₃ oxidation from growth and increased N₂O production. IMPORTANCE: Nitrification, the aerobic oxidation of ammonia to nitrate via nitrite, is an important process in the global nitrogen cycle. This process is generally dependent on ammonia-oxidizing microorganisms and nitrite-oxidizing bacteria. Most nitrifiers are chemolithoautotrophs that fix inorganic carbon (CO₂) for growth. Here, we investigate how inorganic carbon limitation modifies the physiology and transcriptome of Nitrosomonas europaea, a model ammonia-oxidizing bacterium, and report on increased production of N₂O, a potent greenhouse gas. This study, along with previous work, suggests that inorganic carbon limitation may be an important factor in controlling N₂O emissions from nitrification in soils and wastewater treatment

Kinetic characterization of the soluble butane monooxygenase from Thauera butanivorans, formerly ‘Pseudomonas butanovora’

Soluble butane monooxygenase (sBMO), a three-component di-iron monooxygenase complex expressed by the C2–C9 alkane-utilizing bacterium Thauera butanivorans, was kinetically characterized by measuring substrate specificities for C1–C5 alkanes and product inhibition profiles. sBMO has high sequence homology with soluble methane monooxygenase (sMMO) and shares a similar substrate range, including gaseous and liquid alkanes, aromatics, alkenes and halogenated xenobiotics. Results indicated that butane was the preferred substrate (defined by kcat : Km ratios). Relative rates of oxidation for C1–C5 alkanes differed minimally, implying that substrate specificity is heavily influenced by differences in substrate Km values. The low micromolar Km for linear C2–C5 alkanes and the millimolar Km for methane demonstrate that sBMO is two to three orders of magnitude more specific for physiologically relevant substrates of T. butanivorans. Methanol, the product of methane oxidation and also a substrate itself, was found to have similar Km and kcat values to those of methane. This inability to kinetically discriminate between the C1 alkane and C1 alcohol is observed as a steady-state concentration of methanol during the two-step oxidation of methane to formaldehyde by sBMO. Unlike methanol, alcohols with chain length C2–C5 do not compete effectively with their respective alkane substrates. Results from product inhibition experiments suggest that the geometry of the active site is optimized for linear molecules four to five carbons in length and is influenced by the regulatory protein component B (butane monooxygenase regulatory component; BMOB). The data suggest that alkane oxidation by sBMO is highly specialized for the turnover of C3–C5 alkanes and the release of their respective alcohol products. Additionally, sBMO is particularly efficient at preventing methane oxidation during growth on linear alkanes ≥C2, despite its high sequence homology with sMMO. These results represent, to the best of our knowledge, the first kinetic in vitro characterization of the closest known homologue of sMM

Phylogenetic congruence and ecological coherence in terrestrial Thaumarchaeota

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. Acknowledgements We would like to thank Dr Robert Griffith/CEH for providing DNA from soil samples and Dr Anthony Travis for his help with BioLinux. Sequencing was performed in NERC platform in Liverpool. CG-R was funded by a NERC fellowship NE/J019151/1. CQ was funded by a MRC fellowship (MR/M50161X/1) as part of the cloud infrastructure for microbial genomics consortium (MR/L015080/1).Peer reviewedPublisher PD

Complete genome sequence of Nitrosomonas sp. Is79, an ammonia oxidizing bacterium adapted to low ammonium concentrations

Nitrosomonas sp. Is79 is a chemolithoautotrophic ammonia-oxidizing bacterium that belongs to the family Nitrosomonadaceae within the phylum Proteobacteria. Ammonia oxidation is the first step of nitrification, an important process in the global nitrogen cycle ultimately resulting in the production of nitrate. Nitrosomonas sp. Is79 is an ammonia oxidizer of high interest because it is adapted to low ammonium and can be found in freshwater environments around the world. The 3,783,444-bp chromosome with a total of 3,553 protein coding genes and 44 RNA genes was sequenced by the DOE-Joint Genome Institute Program CSP 2006.Keywords: nitrification, Ammonia-oxidizing bacteria, Ammonia oxidation, Nitrosomonas, nitrogen cycle, oligotrophic, freshwate